CELLULAR ONCOLOGY
`
`Volume 28, Numbers 5,6, 2006
`
`CONTENTS
`
`Reviews
`
`
`
`UNIVERSITY OF NORTH CAROLINA
`
`FEB - 5 200/
`
`
`HEALTH SCIENCES LIBRARY
`
`M.S.Q. Kortenhorst, M.A. Carducci and S. Shabbeer
`Acetylation and histone deacetylaseinhibitors in cancer
`
`J.-H. Mikesch, K. Schier, A. Roetger, R. Simon, H. Buerger and B. Brandt
`The expression and action of decay-accelerating factor (CD55) in human malignancies and
`cancer therapy
`
`E.R. Nijhuis, N. Reesink-Peters, G.B.A. Wisman, H.W. Nijman, J. van Zanden, H. Volders,
`H. Hollema, A.J.H. Suurmeijer, E. Schuuring and A.G.J. van der Zee
`An overview of innovative techniques to improve cervical cancer screening
`
`Original contributions
`
`S. Derks, C. Postma, P.T.M. Moerkerk, S.M. van den Bosch, B. Carvalho, M.A.J.A. Hermsen,
`W. Giaretti, J.G. Herman, M.P. Weijenberg, A.P. de Bruine, G.A. Meijer and M. van Engeland
`Promoter methylation precedes chromosomalalterations in colorectal cancer development
`
`G.E.Lind, K. Kleivi, G.I. Meling, M.R. Teixeira, E. Thiis-Evensen, T.O. Rognum and
`R.A. Lothe
`ADAMTS1, CRABP7, and NR3C7 identified as epigenetically deregulated genes in
`colorectal tumorigenesis
`
`B.M. Ghadimi, M. Grade, C. Ménkemeyer, B. Kulle, J. Gaedcke, B. Gunawan, C. Langer,
`T. Liersch and H. Becker
`Distinct chromosomalprofiles in metastasizing and non-metastasizing colorectal carcinomas
`
`B. Carvalho, T.E. Buffart, R.M. Reis, T. Mons, C. Moutinho, P. Silva, N.C.T. van Grieken,
`H. Grabsch, C.J.H. van de Velde, B. Yistra, G.A. Meijer and F. Carneiro
`Mixed gastric carcinomas show similar chromosomalaberrations in both their diffuse and
`glandular components
`
`E.A.M. Janssen, PJ. van Diest, H. Sailand, E. Gudlaugson, A. Nysted, FJ. Voorhorst,
`J.B. Vermorken, J.-A. Soreide and J.RA. Baak
`Successpredictors of adjuvant chemotherapy in node-negative breast cancer patients under
`55 years
`
`G.S. Henderson, PJ. van Diest, H. Burger, J. Russo and V. Raman
`Expression pattern of a homeotic gene, HOXAS, in normal breast and in breast tumors
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`Cellular Oncology 28 (2006) 247-257
`IOSPress
`
`247
`
`Promoter methylation precedes chromosomal
`alterations in colorectal cancer development
`
`Sarah Derks *, Cindy Postma > Peter T.M. Moerkerk*, Sandra M. van den Bosch®, Beatriz Carvalho°,
`Mario A.J.A. Hermsen*, Walter Giaretti‘*, James G. Herman®, Matty P. Weijenberg ‘,
`Adriaan P. de Bruine*, Gerrit A. Meijer” and Manon van Engeland**
`@ Department of Pathology, Research Institute GROW, University Maastricht, The Netherlands
`> DepartmentofPathology, VU University Medical Center, Amsterdam, The Netherlands
`© Present address: Dept. of Otolaryngology, Instituto Universitario de Oncologia del Principado de Asturias
`(IUOPA), Hospital Central de Asturias, Oviedo, Spain
`d Department of Oncogenesis, Biophysics and Cytometry, National Institutefor Cancer Research, Genoa, Italy
`© Department of Tumor Biology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore,
`Maryland, USA
`€ Department of Epidemiology, Nutrition and Toxicology, Research Institute NUTRIM,University Maastricht, The
`Netherlands
`
`Abstract. Background: Colorectal cancers are characterized by genetic and epigenetic alterations. This study aimed to explore
`the timing of promoter methylation and relationship with mutations and chromosomalalterations in colorectal carcinogenesis.
`Methods: Inaseries of 47 nonprogressed adenomas, 41 progressed adenomas(malignant polyps), 38 colorectal carcinomas and
`18 paired normaltissues, we evaluated promoter methylation status of hMLH1/, O°MGMT,APC, pigte: pig RASSFIA,
`GATA-4, GATA-5, and CHFR using methylation-specific PCR. Mutation status of TP53, APC and KRAS were studied by p53
`immunohistochemistry and sequencing of the APC and KRAS mutationcluster regions. Chromosomalalterations were evaluated
`by comparative genomic hybridization. Results: Our data demonstrate that nonprogressed adenomas, progressed adenomas and
`carcinomas showsimilar frequencies of promoter methylation for the majority of the genes. Normaltissues showedsignificantly
`lower frequencies of promoter methylation of APC, pio, GATA-4, and GATA-5 (P-values: 0.02, 0.02, 1.1 x 10~> and
`(0.008 respectively). P53 immunopositivity and chromosomal abnormalities occur predominantly in carcinomas (P values: 1.1 x
`1075 and 4.1 x 107!°). Conclusions: Since promoter methylation was already present in nonprogressed adenomas without
`chromosomalalterations, we concludethat promoter methylation can be regardedas an early event preceding TP53 mutation and
`chromosomal abnormalities in colorectal cancer development.
`Keywords: Colorectal cancer, promoter methylation, genetic alterations, chromosomalinstability
`
`|. Introduction
`
`Colorectal cancer development is characterized by
`he growth of a benign precursorlesion of which even-
`‘ually a small percentage will progress into a carci-
`noma[28]. The genetic alterations underlying the ade-
`noma to carcinoma transition have been extensively
`studied over the past two decades. Pioneering research
`of Vogelstein and co-workers has proposed a progres-
`
`
`Corresponding author: Manon van Engeland, Dept. of Pathology,
`ResearchInstitute GROW, University Maastricht, PO Box 616, 6200
`MD Maastricht, The Netherlands. Tel.: +31 43 3874622; Fax: +31
`
`43 3876613; E-mail: m.vanengeland @path.unimaas.nl
`
`sion model in which genetic alterations as APC and
`TP53 mutations and allelic loss of 5q and 18q play an
`important role [2,16,17,24,43]. Previously, we intro-
`duced the concept that chromosomal instability does
`not merely constitute genetic noise but occurs in non-
`random patterns. Accumulation of losses in 8p21-pter,
`15q11-q21, 17p12-13 and 18q12-21 and gains in 8q23-
`gter, 13q14-31, and 20q13 are strongly associated with
`advanced lesions and can be used as indicator of pro-
`gression towards malignancy [22,32]. Recently, it has
`become clear that
`initiation and progression of can-
`cer also involves epigenetic alterations such as DNA
`methylation and that genetic and epigenetic alterations
`interact
`in driving the development of cancer [34].
`
`1570-5870/06/$ 17.00 © 2006—IOS Press and the authors. All rights reserved
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`248
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`S. Derkset al. / Promoter methylation precedes chromosomalalterations in colorectal cancer development
`
`Colorectal cancer developmentis associated with epi-
`genetic silencing of the DNA repair genes hMLH] [10]
`and O°MGMT[9], the WNTsignal transduction regu-
`lator APC [13], the Ras signalling molecule RASSFIA
`[44], the transcription factors GATA-4 and GATA-5 [1]
`and the cell cycle regulators CHFR [8,40], p16/N&#4
`and pl448F [21,33]. Although extensive knowledge
`exists on epigenetic and genetic changes in colorectal
`cancer, little is known about the exact relationship be-
`tween these two [7,19]. In this cross-sectional study
`we address epigenetic and genetic (at the level of the
`single gene as well as at the level of whole chromo-
`somes) alterations in colorectal cancers and its pre-
`cursor lesions. Using a multi-gene approach we in-
`vestigate the timing of promoter methylation and de-
`fine how these epigenetic events are related to genetic
`events in colorectal cancer development.
`
`2. Materials and methods
`
`2.1. Patient material
`
`This study was performed on a subset (n = 139) of
`colorectal adenoma and carcinomatissues which has
`been analyzed for structural chromosomal abnormali-
`ties by comparative genomic hybridization (CGH)pre-
`viously [22]. Part of this subset has also been ana-
`lyzed for mutation status of APC (n = 96) and KRAS
`(n = 78) [18,22]. We extended this series by adding
`20 colorectal adenoma and carcinoma cases, bringing
`the overall total to 159 tissues. This series consists of
`47 colorectal adenomas without signs of malignancy
`at time of resection (nonprogressed adenomas (nA)),
`41 malignant polyps (colorectal adenomascontaining a
`focus of carcinoma) and 38 additionalsolitary colorec-
`tal carcinomas (Cs). Of the 41 malignant polyps, the
`adenomapart, referred to as progressed adenomas(pA)
`(n = 41), and the carcinoma part (Cmp) (n = 33) were
`microdissected and analyzed separately. If present we
`added morphologically normal mucosa within the re-
`section specimen (n = 18) of patients with solitary
`carcinomas(Cs) to these series. For each tissue sample,
`DNAwasextracted from fifteen 10-j:m paraffin sec-
`tions, dissecting the most tumor-rich areas, allowing a
`maximumof 20% nontumor cell contamination.
`Overall, the tissues were obtained from 95 patients,
`46 males and 49 females (mean age of 67 years: range
`40-89). Twenty-four patients exhibited multiple tu-
`mors; 4 patients presented with multiple adenomas,
`|
`patient presented with multiple carcinomas and 19 pa-
`tients exhibited | or more adenomas adjacent toa car-
`cinoma. The histological characteristics are listed in
`Table 1.
`
`Histological characterics of 159 adenomas and carcinomastissues
`
`
`Table |
`
`Adenoma
`
`nonprogressed
`adenoma(nA)
`
`progressed
`adenoma (pA)
`
`tubular
`
`tubulovillous
`
`villous
`
`serrated
`
`mild
`
`moderate
`
`severe
`
`n
`
`47
`
`41
`
`38
`
`42
`
`5
`
`3
`
`13
`
`50
`
`25
`
`Tissue
`
`Histologic
`
`type
`
`Degree of
`dysplasia
`
`Differentiation
`
`grade
`
`TNM
`
`Carcinoma
`
`carcinoma
`part of malignant
`poly (Cmp)
`solitary carcinoma
`
`n
`
`35
`
`38
`
`Well
`
`Moderate
`
`Poor
`
`I
`
`II
`
`Il
`
`13
`
`52
`
`6
`
`23
`
`30
`
`17
`
`
`
`IV 1
`
`2.2. Promoter methylation analysis
`
`DNAmethylation in the CpG islands of the AMLH1,
`O°MGMT,APC,p14", p16!'NK#4 | RASSFIA, GATA-
`4, GATA-5 and CHFR gene promoters was deter-
`mined by chemical modification of genomic DNA with
`sodium bisulfite and subsequent methylation-specific
`PCR (MSP) as described in detail elsewhere [11,20,
`42]. Briefly,
`1 zg of DNA was denatured by NaOH
`and modified by sodium bisulfite. DNA samples were
`then purified using Wizard DNA purification resin
`(Promega, Madison, USA) again treated with NaOH,
`precipitated with ethanol, and resuspended in H20O.
`To facilitate MSP analysis on DNA retrieved from
`formalin-fixed, paraffin embedded tissue, DNA was
`first amplified with flanking PCR primers that amplify
`bisulfite-modified DNA but do not preferentially am-
`plify methylated or unmethylated DNA. The resulting
`fragment was used as a template for the MSP reac-
`tion. Primer sequences have been described before [1,
`5,42]. All PCRs were performed with controls for un-
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`S. Derks et al. / Promoter methylation precedes chromosomalalterations in colorectal cancer development
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`249
`
`methylated alleles (DNA from normal lymphocytes),
`methylated alleles [normal lymphocyte DNAtreated in
`vitro with Sssl methyltransferase (New England Bio-
`labs)], and a control without DNA.Ten jul of each MSP
`reaction were directly loaded onto nondenaturing 6%
`polyacrylamide gels, stained with ethidium bromide,
`and visualized under UV illumination. The methyla-
`tion status was assessable in 96% of the total number
`of analyses. To asses reproducibility, 234 MSP reac-
`tions have been performed in duplicate starting from
`DNAamplification with flanking PCR primers, the re-
`producibility was 90%. To exclude false priming, se-
`quencing of the methylated amplicon of APC wasper-
`formed, revealing extensive methylation of all ampli-
`cons, including the primerbindingregion.
`
`2.3. P53 immunohistochemistry
`
`P53 immunohistochemistry (n = 146) was per-
`formed using the mouse monoclonal antibody DO7
`(DAKO, Glostrup, Denmark).
`Immunoperoxidase
`staining for p53 in formalin-fixed, paraffin-embedded
`tissue sections was performed by a horseradish per-
`oxidase labeled streptavidin-biotin method. Four jzm
`sections were mounted on 0.1% poly-L-lysine coated
`glass slides, deparaffinized, and rehydrated through
`graded alcohols to water. Endogenous peroxidase ac-
`tivity was blocked by incubation with 0.3% H2O2
`in methanol. Sections were immersed in 10 mM
`sodium citrate buffer, pH 6.0, and subjected to heat-
`induced antigen retrieval with microwave. To block
`non-specific protein binding, sections were pre-incu-
`bated with normal rabbit serum (1:50, DAKO) for 10
`min at room temperature. Mouse monoclonalantibody
`against p53 (1:500 DO7, DAKO)wasapplied,andtis-
`sue sections were incubated overnight at 4°C. Then
`sections were rinsed with PBS, and treated with bi-
`otinylated rabbit anti-mouse IgG (1:500, DAKO) for
`30 min at room temperature, rinsed with PBS, and
`then incubated with streptavidine-biotin-HRP complex
`(1:200, DAKO) for | hour at room temperature. Af-
`ter washing with PBS the complex wasvisualized with
`diaminobenzidine and HO, for 3 min. Sections were
`ihen counter stained with hematoxylin, dehydrated in
`graded alcohols, cleared in xylene and cover slipped.
`rhe area percentage ofpositive nuclei was scored with
`a point counting approach using a video overlay mea-
`suring system (Qprodit Leica, Cambridge, UK). An
`area percentage of 20 was usedas threshold for posi-
`tivity [31].
`
`2.4. KRAS mutation analysis
`
`KRAS mutation status of 78 colorectal adenoma and
`carcinomatissues have been analyzed previously [22].
`Fifty-two additional
`tissues were analyzed by PCR
`using an oligonucleotide 20-mer panel of codons 12
`and 13 (TIB Molbiol, Advanced Biotechnology Cen-
`ter, Genova, Italy) as previously described [18]. Ex-
`tracted DNA from peripheral blood lymphocytes from
`healthy donors was used as wild type KRAS codon
`12 GGT-gly and codon 13 GGC-gly controls, and ex-
`tracted DNA from 6 different colon cancer cell lines
`was used as control for known KRAS mutations.
`
`2.5. Array-CGH analysis
`
`One hundred-thirty-nine colorectal adenoma and
`carcinomatissues have been analyzed by conventional
`CGHpreviously [22]. The 20 additionally collected
`neoplasms were analyzed by array CGH analysis using
`5 K BAC arrays [6,36]. In short, we used a full-
`genomearray printed in the house containing approxi-
`mately 5000 clones with an average resolution of 1 Mb
`(http://www.vumce.nl/microarrays/index.html). After
`amplification of BAC clone DNAbyligation-mediated
`polymerase chain reaction (PCR) according to Snijders
`et al. (2001) all clones were printed in triplicate. Print-
`ing of clones was performed on codelinkTM slides
`(Amersham BioSciences, Roosendaal, NL) at a con-
`centration of | g/l, in 150 mM sodium phosphate,
`pH 8.5, using a SpotArray72 printer (Perkin Elmer
`Life Sciences, Zaventum, BE). After printing slides
`were processed according to the manufacturer’s pro-
`tocol. Labeling and hybridization of tumor and refer-
`ence DNAs were performed as described in detail by
`Snijderset al. (2001) with some modifications, namely,
`hybridizations took place in a hybridization station
`(HybArray12TM — Perkin Elmer Life Sciences, Za-
`ventum, BE) and slides were scanned with Agilent
`DNA Microarray scanner (Agilent technologies, Palo
`Alto, USA), omitting the DAPI staining step. Segmen-
`tation and quantification of the spots was done us-
`ing Imagene 5.6 software (Biodiscovery Ltd, Marina
`del Rey, California). Local background median inten-
`sity was subtracted from the signal median intensity
`for both test and reference channels and aratio tu-
`
`mor/reference was calculated. The ratios were normal-
`ized against the mode ofall ratios of the autosomes.
`As the clones were spotted in triplicate,
`the median
`value of the corresponding three intensities was taken
`into account for each clone in the array. Clones from
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`250
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`S. Derks et al. / Promoter methylation precedes chromosomalalterations in colorectal cancer development
`
`which the intensities of the three spots had a standard
`deviation >0.2 were excluded. Furthermore, clones
`with more than 20% missing values in all carcino-
`mas were excluded for further analysis. All the analy-
`ses were done excluding chromosome X,as in every
`hybridization a sex-mismatched reference DNA was
`used for quality control of the experiment. Clone po-
`sitions were considered according to freeze May2004.
`After using a smoothing algorithm [25], DNA copy
`numberratios obtained by array CGH were recoded
`as gains andlossesat the resolution of whole chromo-
`some arms compatible with the data obtained by chro-
`mosome CGH.
`
`2.6. Data analysis
`
`Differences in frequencies of gene methylation be-
`tween the different stages of disease progression and
`associations between promoter methylation and muta-
`tions were evaluated by the Pearson’s 7 or Fisher ex-
`act test where appropriate. The total number of methy-
`lated genes, referred to as methylation index (MI), is
`defined as the number of genes methylated divided by
`the numberof genes analyzed. The same accounts for
`the total number of gains and losses, referred to as
`chromosomal events, and the numberoflosses (8p21-
`pter, 15ql1-q21, 17p12-13 and 18q12-21) and gains
`(8q23-qter, 13q14-31, and 20q13) associated with ad-
`vanced lesions, referred to as cancer associated events
`[22]. The Mann—Whitney U and Kruskal Wallis non-
`parametric test were used for comparing meansof con-
`tinuous variables. The McNemartest for paired cases
`was usedto test the methylation differences between a
`subset of solitary carcinomas (Cs) and paired normal
`tissue.
`Simple linear regression analysis was performed
`to investigate the correlation between the number of
`methylated genes and the number of chromosomal ab-
`normalities. For all analyses SPSS software version
`11.0 was used. All reported P values are two-sided,
`and a P value < 0.05 was consideredstatistically sig-
`nificant.
`
`3. Results
`
`3.1. Promoter methylationin relationto
`adenoma-carcinomaprogression
`
`In order to investigate the timing of promoter methy-
`lation in colorectal cancer development, we studied
`
`the frequency of promoter methylation of genes which
`function in regulating diverse cell functions in the
`colorectal adenoma and carcinoma tissues. Our data
`demonstrate that nonprogressed adenomas (nA), pro-
`gressed adenomas (pA), carcinoma parts of malignant
`polyps (Cmp) and solitary carcinoma (Cs) showed sim-
`ilar frequencies of promoter methylation for the major-
`ity of genes (Table 2). No difference in mean methy-
`lation index (MI) (total number of methylated genes
`divided by the number of genes analyzed) between
`the different categories of neoplasm’s was observed.
`However, p/44** methylation was found in 71.1% and
`73.5% of the nonprogressed adenomas (nA) and pro-
`gressed adenomas (pA) respectively and decreases to
`53.3% and 37.1% of the carcinomaparts of malignant
`polyps (Cmp) andsolitary carcinoma (Cs) respectively
`(P value: 0.006).
`Since we observed that promoter methylation of the
`studied genesis already present in nonprogressed ade-
`nomas (nA), we were interested in the presence ofpro-
`moter methylation in matching normal mucosa. For 18
`solitary carcinomas (Cs) morphological normal tissue
`from the resection specimen wasavailable (Table 3a).
`We performed a nonparametric test for matched pairs
`and compared the methylation profile of 18 solitary
`carcinomas to their corresponding normal mucosa. In
`158 of the 162 (9 genes x 18 pairs) possible combi-
`nations the gene methylation status was assessable in
`the carcinoma as well as in the normaltissue. In 48.7%
`of the pairs (77/158) no difference in gene methyla-
`tion was observed within pairs (N = C) (Table 3b). In
`66.2% (51/77) of these pair both components were un-
`methylated while in 33.8% (26/77) the carcinomasas
`well as the corresponding normal tissue showed pro-
`moter methylation. In 43% (68/158) ofall pairs gene
`methylation was present in the carcinomas while ab-
`sent in the corresponding normal tissue (C > N), and
`only in 8.2% (13/158) of the pairs promoter methyla-
`tion was only observedin the normal tissue (N > C).
`The McNemartest for paired cases showedthat pro-
`moter methylation of APC, p/6!\**4, GATA-4, and
`GATA-S5 occurred significantly more frequent
`in the
`carcinomas when compared to corresponding normal
`mucosa (?-values 0.02, 0.02, 1.1
`x 107> and 0.008
`respectively) (Table 3b). Although promoter methyla-
`tion of AMLH/, O°MGMTand CHFR waspresentin
`the normal tissues, this was predominantly when the
`carcinomas was also methylated. For example, pro-
`moter methylation of hMLH/ was observedin 50.0%
`of the adenomas and 72.2% ofthe paired carcinomas
`(this high frequency could be explained bythe fact
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`S. Derkset al. / Promoter methylation precedes chromosomalalterations in colorectal cancer development
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`251
`
`Timing of promoter methylation, genetic and chromosomalalterations
`
`
`Table 2
`
`
`nA
`pA
`Cmp
`47
`41
`33
`
`Numberof cases
`
`44.7
`66.7
`19.1
`
`61.7
`vale
`28.9
`Fie
`
`95.7
`
`48.6
`
`0.57
`
`31-7
`72.5
`24.4
`
`se)
`73.5
`36.6
`TES
`
`87.8
`
`56.7
`
`0.56
`
`36.4
`54.5
`24.2
`
`333
`53.3
`36.4
`75.8
`
`84.4
`
`50.0
`
`0.49
`
`Cs
`38
`
`55:3
`60.5
`29.7
`
`44.7
`37.1
`31.6
`86.5
`
`82.9
`
`55.9
`
`0.53
`
`P value
`
`NS
`NS
`NS
`
`NS
`0.006
`NS
`NS
`
`NS
`
`NS
`
`NS
`
`Promoter methylation (%)
`hMLH1
`0°-MGMT
`RASSFla
`
`APC
`plane
`pIGiNK4A
`GATA-4
`
`GATA-5
`
`CHFR
`
`Mean methylation index
`Genetic alterations
`p53 immunopositivity (n = 146)
`APC mutation (n = 96)
`KRAS mutation (n = 130)
`Chromosomalalterations
`number of chromosomalevents
`number of CAE
`
`14.3
`50 (17/34)
`34.3
`
`6.2
`0.9
`
`34.2
`63.2 (24/38)
`40.0
`
`12.4
`3.0
`
`a2
`58.3 (7/12)
`28.6
`
`ee
`3.3
`
`59:5
`66.7 (8/12)
`28.1
`
`10.6
`3.0
`
`1.1 x 1075
`NS
`NS
`
`4.1 x 107°
`BEBO = 10
`
`Note: Results of epigenetic and genetic analyses of nonprogressed adenomas(nA), progressed adenomas(pA), carcinoma parts of a malignant
`polyps (Cmp) and solitary carcinomas (Cs). Listed are the frequencies of promoter methylation of 9 genes, methylation index (total number
`of methylated genes divided by the number of genes analyzed), p53 immunopositivity, APC and KRAS mutation, number of chromosomal
`abnormalities (chromosomal events) and numberof cancer associated events (losses at 8p21-pter, 15q11-q21, 17p12-13, 18q12-21, and gain at
`8q23-qter, 13q14-31 and 20q13) per group. Data on p53 immunopositivity, APC and KRAS mutations were available for a subset of cases; MSP
`and CGH have been done onall cases.
`
`that in 6 of the 18 pairs (33.3%) the carcinomas part
`showed microsatellite instability, data not shown. In 5
`of these 6 pairs, the carcinoma as well as the paired
`normal tissue showed AMLH/ methylation). In 8 of the
`9 pairs in which normal tissue showed hMLH1/pro-
`moter methylation, carcinomatissue was methylated as
`well. In 6 cases a difference in methylation was present
`of which in 5 cases hMLHI was methylated in the
`carcinomas while unmethylated in the paired normal
`mucosa. Comparable patterns were observed for pro-
`moter methylation of O°7MGMT, RASSFIA and CHFR.
`More difference between paired carcinomas and nor-
`mal tissues were observed for p/4“*methylation. In
`4 of the 9 cases in which a difference within pairs
`was observed, normal tissue displayed gene methyla-
`tion while p/44*" was not methylated in the corre-
`sponding carcinomas.
`
`3.2. Promoter methylation in relation to genetic
`alterations
`
`In order to studythe relationship between promoter
`methylation and genetic alterations we analyzed mu-
`
`tations of three key genes involved in developmentof
`colorectal cancer, i.e. 7P53, APC and KRAS.
`Disruption of the p53 pathway, amongst others, can
`occurby loss of function of TP53 itself and by p/ 44%"
`methylation [46]. The frequency of p/44"" methyla-
`tion significantly decreased in tumor progression (Ta-
`ble 2). In contrast, aberrant p53 status, indicated by p53
`immunopositivity, increased from 14.3% in the non-
`progressed adenomas (nA) through 34.2% in the pro-
`gressed adenomas(pA) to 55.2% and 59.5% in the car-
`cinoma parts of malignant polyps (Cmp) andsolitary
`carcinomas (Cs) respectively (P value: 0.001). Case
`by case, p/44%" methylation showsan inverse relation
`with p53 immunopositivity, approaching statistical sig-
`nificance (P value: 0.07).
`For APC and KRAS, a similar pattern was found.
`APC was mutated in 50% and 63.2% of the nonpro-
`gressed and progressed adenomas (nA and pA), re-
`spectively, compared to 58.3% and 66.7% ofthe car-
`cinomasparts of malignant polyps (Cmp) and solitary
`carcinomas (Cs) [22]. KRAS mutation was observed in
`34.3% and 40.0% of the nonprogressed and progressed
`adenomas(nA and pA), respectively, and in 28.6% and
`
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`252
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`S. Derks et al. / Promoter methylation precedes chromosomalalterations in colorectal cancer development
`
`Promoter methylation frequencies in 18 paired carcinoma and normaltissues
`
`Table 3a
`
`
`Patient AMLHI MGMT RASSFIA pl4*™ pi6o™4~4pC_ GATA-4_ GATA-5—CHFRTissue
`
`
`
`
`
`Res methylated gene
`
`[_Junmethylated gene
`
`hI no amplification
`
`Promoter methylation frequencies in 18 paired carcinoma and normaltissues
`
`Table 3b
`
`N (%)
`
`C(%)
`
`hMLH1
`50
`
`72.2
`
`MGMT
`38.9
`
`61.1
`
`RASSFIA_—
`16.7
`
`p14ARF
`op JgINK4A
`APC
`GATA-4
`GATA-5
`CHFR
`
`33.3
`0
`16.7
`16.7
`16.7
`27.8
`
`44.4
`
`38.9
`
`38.9
`
`61.1
`
`94.4
`
`72.2
`
`55.6
`
`N=C(%)
`
`66.7(12/18)
`
`44.4(8/18)
`
`38.9(7/18)
`
`50.0(9/18)
`
`61.1 (11/18)
`
`44.4(8/18)
`
`22.2(4/18)
`
`46.7(7/15)
`
`64.7 (12/17)
`
`C>N(%) 50 (9/18)=77.8 (14/18)27.8(5/18) 38.9(7/18) 44.4(8/18) 27.8 (5/18) 38.9(7/18) 53.3 (8/15) 29.4 (5/17)
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`N>C(%)
`P value
`
`5.6(1/18)
`NS
`
`16.7(3/18)
`NS
`
`16.7(3/18)
`NS
`
`22.2 (4/18)
`NS
`
`0 (0/18)
`0.02
`
`5.6 (1/18)
`0.02
`
`0 (0/18)
`4.1 x 10-6
`
`0 (0/15)
`0.008
`
`5.6 (1/18)
`NS
`
`Note: Results of promoter methylation in 18 paired carcinoma (C) and normal tissue (N). N = C: no difference in gene methylation between
`carcinoma and normal tissue. C > N: gene methylated in carcinoma and unmethylated in normal tissue. N < C: gene methylated in normal
`tissue and unmethylated in carcinoma.
`
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`S. Derks et al. / Promoter methylation precedes chromosomalalterations in colorectal cancer development
`
`253
`
` 44
`
`Sa
`
`38
`
`N=
`
`47
`nA
`
`Ca N=
`
`2o
`
`>
`o
`3
`Ee
`2
`E
`£
`oO
`es°a
`ooO
`Ea
`
`B
`
`47
`nA
`
`41
`33
`38
`pA Cmp Cs
`
`A
`
`1.0
`
`08
`
`5
`z
`i=
`S 06
`8
`
`<:
`
`® 04
`E
`
`0.2
`
`Cc numberofcancer
`
`associatedevents
`
`41
`pA
`
`33
`38
`Cmp Cs
`
`pA Cmp Cs
`
`nA
`
`Fig. 1. Promoter methylation and chromosomalalterations in colorectal cancer development. nA = nonprogressed adenoma; pA = progressed
`adenoma; Cmp = carcinomapart of a malignant polyp; Cs = solitary carcinoma. (A) Promoter methylation has been analyzed for 9 DNA repair-
`and tumor suppressor genes. The methylation index (total number of methylated genes divided by the number of genes analyzed) stays stable
`during tumor development, while (B) the total number of chromosomalalterations (chromosomalevents), analyzed on genomiclevel, and (C) the
`numberof cancer associated events (losses at 8p21-pter, 15q11-q21, 17p12-13, 18q12-21, and gain at 8q23-qter, 13q14-31 and 20q13) increase.
`*P value: 4.1 x 10—©, +P value: 3.6 x 1079,
`
`28.1% of the carcinomas parts of malignant polyps
`(Cmp) and solitary carcinomas (Cs) (Table 2). While
`neither the frequencies of APC mutation nor APC pro-
`moter methylation differ between the different stages
`of tumor development, case by case analysis indicated
`an inverse relation (P value: 0.06). A similar pattern
`and inverse relation was observed for KRAS mutation
`and promoter methylation of RASSF/JA and hMLH1 (P
`values: 0.01 and 0.001 respectively).
`
`3.3. Promoter methylation in relation to
`chromosomal alterations
`
`The timing and interrelationship of promoter methy-
`lation and chromosomalalterations in tumor progres-
`sion were analyzed by studying promoter methylation
`in cases without chromosomal abnormalities and by
`relating gene methylation status to the mean number
`of chromosomal and cancerassociated events. Simple
`linear regression analyses revealed no correlation be-
`tween the MI and the numberof chromosomal abnor-
`
`malities or number of cancer associated events. As de-
`scribed previously, the number of chromosomal abnor-
`malities and especially the number of cancer associ-
`ated events are associated with progressed lesions (P
`values: 4.1 x 107° and 3.6 x 107!respectively) (Ta-
`ble 2) [22]. Figure | showsthat while the mean number
`of chromosomal events and cancer associated events
`increases during tumor progression the mean MI re-
`mains stable. In 13 cases (11 nonprogressed adenomas
`(nA) and 2 solitary carcinomas (Cs)) no chromosomal
`
`alterations were observed. The 12 adenomas without
`
`chromosomal abnormalities did not differ in MI from
`adenomas with chromosomal abnormalities. Interest-
`ingly, the 2 solitary carcinomas (Cs) without chromo-
`somal abnormalities (both cases exhibit microsatellite
`instability; data not shown) were characterized by a MI
`of 0.96, while the MI of carcinomas harboring chro-
`mosomalalterations was 0.56 (P value: 0.006). No as-
`sociation between promoter methylation of a specific
`tested gene and the numberof chromosomal abnormal-
`ities was observed.
`Oneof the cancer associated chromosomal changes,
`deletion of 8p21, includes the GATA-4 gene, which was
`also a frequent target of epigenetic changes in these
`tumors. Promoter methylation as well as loss of het-
`erozygosity could combine leading to loss of func-
`tion of GATA-4. GATA-4 methylation was found in
`respectively 77.3% and 77.5% of the nonprogressed
`adenomas (nA) and progressed adenomas (pA) and in
`75.8% and 86.5% of the carcinoma parts of malignant
`polyps (Cmp) and solitary carcinoma (Cs) respectively.
`In contrast,
`the frequency of loss of 8p21-pter in-
`creases in tumor development from 12.8% and 39.0%
`of the nonprogressed adenomas (nA) and progressed
`adenomas (pA) respectively to 45.5% and 42.1% of
`the carcinomaparts of malignant polyps (Cmp) and
`solitary carcinoma(Cs) respectively (? value: 0.005).
`The frequency in which loss of 8p21-pter is com-
`bined with GATA-4 methylation (GATA-4 M/8p-) in-
`creases during tumor development from 9% in nonpro-
`gressed adenomas(nA) to 25%, 30% and 32% in pro-
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`S. Derks et al. / Promoter methylation precedes chromosomalalterations in colorectal cancer development
`
`gressed adenomas (pA), carcinomaparts of malignant
`polyps (Cmp) andsolitary carcinoma(Cs) respectively.
`The frequency in which only GATA-4 is methylated
`(GATA-4/8p)is stable and occurs in 68.2% of the non-
`progressed adenomas (nA) and 52.2%, 45% and 54%
`of the progressed adenomas (pA), carcinoma parts of
`malignant polyps (Cmp) and solitary carcinoma (Cs)
`respectively. Loss of 8p21-pter without concomitant
`methylation of GATA-4 (GATA-4 U/8p-) is infrequent
`occurring in 4.5% and 15.2% of the nonprogressed
`adenomas (nA) and progressed adenomas(pA)and in
`15% and 8.1% of the carcinoma parts of malignant
`polyps (Cmp) and solitary carcinoma (Cs).
`
`4. Discussion
`
`In this study we attempt to elucidate the timing and
`interrelation of promoter methylation and genetic al-
`terations in colorectal cancer development. Therefore
`we studied genetic and epigenetic events known to be
`associated with colorectal cancer development.
`Considering the timing of epigenetic events in tumor
`progression, our results indicate that promoter methy-
`lation of the studied genes can be regarded as an early
`event in colorectal carcinogenesis. A high frequency of
`promoter methylation of multiple DNA repair- and tu-
`mor suppressor genes is already present in adenomas
`without any histological signs of progression, and ma-
`lignant lesions showedsimilar frequencies of methyla-
`tion. Even in morphologically normal mucosa from pa-
`tients with solitary carcinomas (Cs) promoter methyla-
`tion of hMLH1, MGMT, RASSFIA, p14“*" and CHFR
`was observed, but, with exception of p/44*" methy-
`lation, in lower frequencies compared to the carcino-
`mas. P16'NK44, APC, GATA-4, and GATA-5 methyla-
`tion occurred predominantly in the carcinomas. Pro-
`moter methylation in normal tissues was in most cases
`consistent with the methylation profile of the paired
`carcinoma. However, additional studies involving nor-
`mal colonic mucosa from individuals without cancers
`are required to determine the exact timing of promoter
`methylation of the studied genes.
`Interestingly, hypermethylation of p/44%"was more
`frequently present
`in nonprogressed adenomas (nA)
`and progressed adenomas (pA) when comparedto car-
`cinomaparts of malignant polyps (Cmp) andsolitary
`carcinomas (Cs). This observation can possibly be ex-
`plained bythe conceptthat the transition from an ade-
`noma to a carcinoma can be considered as a transi-
`tion from a heterogeneous cellular population to one
`
`that is more homogeneous[17]. Even though promoter
`methylation is a dynamic process, this indicates that
`p14methylation is not necessarily associated with
`a definitive growth advantage.
`Furthermore, since the tumor suppressor functions
`ofp/44F is dependent uponthe presence of functional
`p53 [14], p/44** methylation is possibly of greater im-
`portance in early stag