dna methylation and cancer shen-chih chang, ph.d epi 243 may 14, 2009

DNA Methylation DNA Methylation

and Cancerand CancerShen-Chih Chang, Ph.DShen-Chih Chang, Ph.D

Epi 243Epi 243May 14, 2009May 14, 2009

Presentation OutlinePresentation Outline

Epigenetics and DNA methylationEpigenetics and DNA methylation DNA methylation and CancerDNA methylation and Cancer Techniques of measuring DNA methylationTechniques of measuring DNA methylation

Methylation-Specific PCR (MSP)Methylation-Specific PCR (MSP) Selected results on lung and head and neck cancerSelected results on lung and head and neck cancer

MethyLight Taqman real-time Methylation AssaMethyLight Taqman real-time Methylation Assayy Selected results on bladder cancerSelected results on bladder cancer Selected results on liver cancerSelected results on liver cancer

EpigeneticsEpigenetics The study of heritable changes of DNA, not involving cThe study of heritable changes of DNA, not involving c

hanges in DNA sequence, that regulate gene expressiohanges in DNA sequence, that regulate gene expression. n. Classic genetics alone cannot explain the diversity of phenotyClassic genetics alone cannot explain the diversity of phenoty

pes within a population.pes within a population. Identical twins or cloned animals have different phenotypes aIdentical twins or cloned animals have different phenotypes a

nd susceptibilities to diseases.nd susceptibilities to diseases.

Epigenetics provides additional instructions on how, wEpigenetics provides additional instructions on how, where, and when the genetic information should be usehere, and when the genetic information should be used. d.

Epigenetics controls gene expression in two main ways:Epigenetics controls gene expression in two main ways: Chemically alteration of DNA: DNA methylationChemically alteration of DNA: DNA methylation Modification of histones: chromatin structure modulationModification of histones: chromatin structure modulation

Epigenetic MechanismsEpigenetic Mechanisms

Qiu J, 2006

Mapping chromosomal regions with differential DNA methylation in Monozygous twins by using comparative genomic hybridization for methylated DNA

Fraga M. F. et.al. PNAS 2005;102:10604-10609

©2005 by National Academy of Sciences

DNA MethylationDNA Methylation

Chemical modification of DNAChemical modification of DNA Addition of a methyl group to the number 5 Addition of a methyl group to the number 5

carbon of the cytosine, to convert cytosine to carbon of the cytosine, to convert cytosine to 5-methylcytosine.5-methylcytosine.

In humans, DNA methylation occurs in a cytIn humans, DNA methylation occurs in a cytosine which is immediately followed by a guosine which is immediately followed by a guanine (dinucleotide CpGs).anine (dinucleotide CpGs).

CpG Sites and CpG CpG Sites and CpG islandsislands

CpG sites are not randomly distributed in the CpG sites are not randomly distributed in the genome - the frequency of CpG sites in human genome - the frequency of CpG sites in human genomes is 1%, which is less than the expectegenomes is 1%, which is less than the expected (~4-6%). d (~4-6%).

Around 60-90% of CpGs are methylated in maAround 60-90% of CpGs are methylated in mammals. DNA methylation frequently occurs in mmals. DNA methylation frequently occurs in repeated sequences, and may help to suppress repeated sequences, and may help to suppress junk DNA and prevent chromosomal instabilitjunk DNA and prevent chromosomal instability. y.

Unmethylated CpGs are grouped in clusters caUnmethylated CpGs are grouped in clusters called “CpG islands” which tend to be located lled “CpG islands” which tend to be located in the promoter regions of many genes.in the promoter regions of many genes.

Function of DNA Function of DNA MethylationMethylation

In humans, DNA is methylated by three enzymes, DNA In humans, DNA is methylated by three enzymes, DNA methyltransferase 1, 3a, and 3b (DNMT1, DNMT3a, DNmethyltransferase 1, 3a, and 3b (DNMT1, DNMT3a, DNMT3b).MT3b).

DNMT3a and 3b are the DNMT3a and 3b are the de novode novo methyltransferases th methyltransferases that set up DNA methylation patterns early in developmeat set up DNA methylation patterns early in development. nt.

DNMT1 is the maintenance methyltransferase that is rDNMT1 is the maintenance methyltransferase that is responsible for copying DNA methylation patterns to thesponsible for copying DNA methylation patterns to the daughter strands during DNA replication. e daughter strands during DNA replication.

DNA methylation is important in:DNA methylation is important in: Transcriptional gene silencingTranscriptional gene silencing Maintain genome stabilityMaintain genome stability Embryonic developmentEmbryonic development Genomic imprintingGenomic imprinting X chromosome inactivationX chromosome inactivation

DNA Methylation and DNA Methylation and CancerCancer

Hypomethylation – decreased methylation Hypomethylation – decreased methylation levelslevels A lower level of DNA methylation in tumors as A lower level of DNA methylation in tumors as

compared to their normal-tissue counterparts was compared to their normal-tissue counterparts was one of the first epigenetic alterations to be found one of the first epigenetic alterations to be found in human cancer. (Feinberg AP, et al., 1983). in human cancer. (Feinberg AP, et al., 1983).

Global hypomethylation of DNA sequences that Global hypomethylation of DNA sequences that are normally heavily methylated may result inare normally heavily methylated may result in

Chromosomal instability Chromosomal instability Increased transcription from transposable elements Increased transcription from transposable elements An elevated mutation rate due to mitotic recombinationAn elevated mutation rate due to mitotic recombination

Promoter hypomethylation of proto-oncogenes will Promoter hypomethylation of proto-oncogenes will activate the repressed gene expressionactivate the repressed gene expression

DNA Methylation and DNA Methylation and CancerCancer

Hypermethylation – increased methylation lHypermethylation – increased methylation levelsevels Promoter hypermethylation can suppress gene Promoter hypermethylation can suppress gene

expression in two ways:expression in two ways: Methylated DNA may itself impede the binding of traMethylated DNA may itself impede the binding of tra

nscriptional proteins to the gene nscriptional proteins to the gene Methylated DNA may be bound by proteins which can Methylated DNA may be bound by proteins which can

modify histones to form compact, inactive chromatin.modify histones to form compact, inactive chromatin. Promoter hypermethylation of tumor-suppressoPromoter hypermethylation of tumor-suppresso

r genes is a major event in the origin of many car genes is a major event in the origin of many cancers.ncers.

The profiles of hypermethylation of the CpG islaThe profiles of hypermethylation of the CpG islands are specific to the cancer type.nds are specific to the cancer type.

Baylin et al. 2001; Jones et al. 2002

Laird PW, 1997

Das PM 2004

Factors associated with Factors associated with DNA MethylationDNA Methylation

AgingAging Nutrient intakeNutrient intake Genetic Genetic

polymorphismspolymorphisms Metal exposureMetal exposure Tobacco SmokingTobacco Smoking Alcohol DrinkingAlcohol Drinking

Application of DNA Methylation Application of DNA Methylation MeasurementMeasurement

Early diagnosis –Early diagnosis – Detection of CpG-island hypermethylation in Detection of CpG-island hypermethylation in

biological fluids (serum/plasma)biological fluids (serum/plasma) Prognosis –Prognosis –

Hypemethylation of specific genesHypemethylation of specific genes Whole DNA methylation profilesWhole DNA methylation profiles

Prediction –Prediction – CpG island hypermethylation as a marker of CpG island hypermethylation as a marker of

response to chemotherapyresponse to chemotherapy Prevention –Prevention –

Developing DNMTs inhibitors as Developing DNMTs inhibitors as chemopreventive drugs to reactive silenced chemopreventive drugs to reactive silenced genes genes

Techniques of Measuring Techniques of Measuring Gene-Specific Gene-Specific

HypermethylationHypermethylation Methylation Specific PCR (MSP)Methylation Specific PCR (MSP) MethyLight Taqman Real-Time MethyLight Taqman Real-Time

Methylation AssayMethylation Assay

Methylation Specific PCR Methylation Specific PCR (MSP)(MSP)

•DNA ModificationDNA Modification• C UC U• CCMM C C

•Two set of primersTwo set of primers•MethylatedMethylated•UnmethylatedUnmethylated

•Positive control Positive control (Universal (Universal Methylated DNA)Methylated DNA)

•Negative control Negative control (H(H22O)O)

Results from the MSPResults from the MSP•p1p1

66

•MGMTMGMT •GSTP1GSTP1

Selected Results on Lung Selected Results on Lung and and

Head and Neck CancerHead and Neck CancerShu-Chun Chuang, Ph.D

Aim:Aim:To evaluate the associations between lunTo evaluate the associations between lung and head and neck cancer and promoteg and head and neck cancer and promoter-region methylation of selected genes, inr-region methylation of selected genes, including P16INK4a, MGMT , and GSTP1 gecluding P16INK4a, MGMT , and GSTP1 genes in buccal cell DNA in a population-banes in buccal cell DNA in a population-based case-control study in Los Angeles coused case-control study in Los Angeles county.nty.

Materials and MethodsMaterials and Methods Study design: population-based case-control studyStudy design: population-based case-control study

Subject selection criteria: Cases were newly diagnosed Subject selection criteria: Cases were newly diagnosed and pathologically confirmed. Controls were matched and pathologically confirmed. Controls were matched to cases on age, gender, and neighborhood.to cases on age, gender, and neighborhood.

Eligibility:Eligibility: Current resident of Los Angeles CountyCurrent resident of Los Angeles County 18-65 during the observation period, 1999-200418-65 during the observation period, 1999-2004 either speak English or Spanish or have translators either speak English or Spanish or have translators

availableavailable have no other primary cancers (cases)have no other primary cancers (cases) have no history of lung or head and neck cancers have no history of lung or head and neck cancers

(controls)(controls)

Biological samples: buccal cell samples were collected Biological samples: buccal cell samples were collected during the interviewduring the interview

Response RateResponse Rate

Eligible Interviewed (%) Buccal Biospecimen (%)

Control 1540 1040 (67.5) 928 (89.2)

Lung 1577 611 (38.7) 544 (89.0)

Oral 584 303 (51.9) 195 (64.4)

Pharynx 238 100 (42.0) 77 (77.0)

Larynx 226 90 (39.8) 79 (87.8)

Esophagus 316 108 (34.2) 97 (89.8)

Cancer Cases Reason of non-participation

Lung H & N (1) The patients died before we contacted them. 25% 10% (2) Incorrect addresses. 14% 18% (3) The patients were too ill to get interviewed. 5% 4% (4) The patients were not willing to participate. 16% 21% (5) Physicians refused our requests. 1% 1%

Main Effect of p16 Main Effect of p16 HypermethylationHypermethylation

P16P16HypermethylaHypermethyla

tiontion

ControControlsls

N (%)N (%)

LungLung Head and NeckHead and Neck

N (%)N (%) Crude ORCrude OR(95% CI)(95% CI)

Adjusted Adjusted OROR11

(95% CI)(95% CI)


Adjusted Adjusted OROR22

(95% CI)(95% CI)

NoNo 769 769 (8(84)4)

433 433 ((8811))

1.001.00 1.001.00 293 293 (8(84)4)

1.001.00 1.001.00

YesYes 146 146 (1(16)6)

100 100 ((1199))

1.22 (0.92-1.22 (0.92-1.61)1.61)

1.31 (0.94-1.31 (0.94-1.83)1.83)

57 57 (1(16)6)

1.03 (0.73-1.03 (0.73-1.43)1.43)

1.03 (0.72-1.03 (0.72-1.48)1.48)

1. Adjusted for age, sex, race, and pack-years of smoking.

2. Adjusted for age, sex, race, pack-years of smoking, and drink-years of alcohol consumption

Main Effect of GSTP1 Main Effect of GSTP1 HypermethylationHypermethylation

GSTP1GSTP1HypermethylatHypermethylat

ionion

ControControlsls

N (%)N (%)



Adjusted ORAdjusted OR11

(95% CI)(95% CI)N (%)N (%) Crude ORCrude OR

(95% CI)(95% CI)Adjusted ORAdjusted OR22

(95% CI)(95% CI)

NoNo 675 675 (88)(88)

354 354 (85)(85)

1.001.00 1.001.00 254 254 (86)(86)

1.001.00 1.001.00

YesYes 96 (12)96 (12) 61 (15)61 (15) 1.21 (0.86-1.21 (0.86-1.71)1.71)

1.17 (0.78-1.17 (0.78-1.75)1.75)

40 (14)40 (14) 1.11 (0.75-1.11 (0.75-1.65)1.65)

1.04 (0.67-1.04 (0.67-1.62)1.62)

1.Adjusted for age, sex, race, and pack-years of smoking.2.Adjusted for age, sex, race, pack-years of smoking, and drink-years of alcohol consumption

Main Effect of MGMT Main Effect of MGMT HypermethylationHypermethylation

MGMTMGMTHypermethylatHypermethylat

ionion

ControControlsls

N (%)N (%)



Adjusted ORAdjusted OR11

(95% CI)(95% CI)N (%)N (%) Crude ORCrude OR

(95% CI)(95% CI)Adjusted ORAdjusted OR22

(95% CI)(95% CI)

NoNo 721 721 (82)(82)

380 380 (77)(77)

1.001.00 1.001.00 250 250 (76)(76)

1.001.00 1.001.00

YesYes 157 157 (18)(18)

112 112 (23)(23)

1.35 (1.03-1.35 (1.03-1.78)1.78)

1.19 (0.86-1.19 (0.86-1.65)1.65)

78 (24)78 (24) 1.43 (1.05-1.43 (1.05-1.95)1.95)

1.34 (0.96-1.34 (0.96-1.88)1.88)

1.Adjusted for age, sex, race, and pack-years of smoking.2.Adjusted for age, sex, race, pack-years of smoking, and drink-years of alcohol consumption

MGMTMGMTHypermethylatHypermethylat

ionion

ControControlsls

N (%)N (%)

PharynxPharynx


Adjusted ORAdjusted OR(95% CI)(95% CI)

NoNo 721 721 (82)(82)

38 38 (68)(68)

1.001.00 1.001.00

YesYes 157 157 (18)(18)

18 18 (32)(32)

2.18 (1.21-2.18 (1.21-3.91)3.91)

2.00 (1.09-2.00 (1.09-3.68)3.68)

Adjusted for age, sex, race, pack-years of smoking, and drink-years of alcohol consumption

Stratified by Site

MethyLight Taqman Methylation MethyLight Taqman Methylation AssayAssay

Real-Time PCRReal-Time PCR Real-time PCR is a PCR-based method

using fluorescent molecules to directly measure the reaction while amplification is taking place.

Data are collected throughout the PCR process rather than the end of the process.

It measures the point in time when amplification of a target is first detected during cycling rather than by the amount of target accumulated at the end of PCR.

Can be used to achieve both qualitative and quantitative measurements.

DenaturationPrimer Annealing

Elongation

5’ 3’

5’3’5’ 3’

5’3’

5’ 3’

5’3’5’

5’

Taq

Taq

Repeat

Traditional Standard Traditional Standard PCRPCR

In theory, product accumulation is proportional to 2n, where n is the number of amplification cycle repeats

However, in reality...However, in reality...

• A linear increase follows exponential

•

Eventually plateaus

Cycle #

Theoretical

Real Life

Log

Targ

et

DN

A

Limitation in standard PCRLimitation in standard PCR

Amplification is exponential, but the Amplification is exponential, but the exponential increase is limited:exponential increase is limited:

Geometric

linear

plateau

Standard PCR as Standard PCR as endpointendpoint

Standard PCR as Standard PCR as endpointendpoint

Identical reactions will have very different final amounts of fluorescence at endpoint

•The point at which the fluorescence rises appreciably above threshold is called CT

•Identical reactions will have identical CT values

Real-Time PCRReal-Time PCR

CT

Threshold

How to measure DNA How to measure DNA concentration?concentration?

Setup for MethyLight MSPSetup for MethyLight MSP Modified DNA as templatesModified DNA as templates Methylation sequence specific forward Methylation sequence specific forward

and backward primersand backward primers Taqman Probes: 5’-FAM---------TEMRA-3’Taqman Probes: 5’-FAM---------TEMRA-3’ Taqman Universal PCR Master MixTaqman Universal PCR Master Mix Negative control contains PCR reagents Negative control contains PCR reagents

but without DNA – ddHbut without DNA – ddH22OO Replicate wells -- using two or more Replicate wells -- using two or more

replicate reactions per sample to ensure replicate reactions per sample to ensure statistical significance.statistical significance.

A calibrator -- The sample used as the basis A calibrator -- The sample used as the basis for comparative results. for comparative results. A universal methylated positive control was A universal methylated positive control was

used in this study as a calibrator. used in this study as a calibrator. An endogenous control gene -- A gene An endogenous control gene -- A gene

present at a consistent expression level in present at a consistent expression level in all experimental samples. An endogenous all experimental samples. An endogenous gene is used as an internal control of the gene is used as an internal control of the difference amount of input DNA.difference amount of input DNA. ACTB gene without CpG dinucleotides was used ACTB gene without CpG dinucleotides was used

as endogenous control gene in this study.as endogenous control gene in this study.

Setup for MethyLight MSPSetup for MethyLight MSP

Calibrator

Negative control

Endogenous control gene; others are all target genes

Setup for MethyLight MSPSetup for MethyLight MSP

Analyzing Relative Analyzing Relative Quantification DataQuantification Data

∆∆∆∆CCTT Method Method

∆∆CCT T (sample) = C(sample) = CT T (marker)- C(marker)- CT T (ACTB)(ACTB)

∆∆CCT T (calibrator) = C(calibrator) = CT T (marker)- C(marker)- CT T (ACTB)(ACTB)

∆∆∆∆CCTT = ∆C = ∆CT T (sample) - ∆C(sample) - ∆CT T (calibrator) (calibrator) Relative quantification of methylated 5’-Relative quantification of methylated 5’-

cytosine = Ecytosine = E(-∆∆CT)(-∆∆CT) E: efficiency of amplificationE: efficiency of amplification Assumption: E = 2 for both marker and Assumption: E = 2 for both marker and

endogenous geneendogenous gene

Analyzing Relative Quantification Analyzing Relative Quantification DataData

-- Amplification Plot (linear plot of reporter signal vs cycle nu-- Amplification Plot (linear plot of reporter signal vs cycle number) --mber) --

negative control ACTB gene


-- Amplification plot of positive control---- Amplification plot of positive control--

linear plot of reporter signal vs cycle numberlinear plot of reporter signal vs cycle number logarithmic plot of baseline-corrected reporter signal vs. cycle number


-- Amplification plot of p16 gene hypermethylation---- Amplification plot of p16 gene hypermethylation--

logarithmic plot of baseline-corrected reporter signal vs. cycle number

After adjusting baseline and threshold, software automatically calculates relative quantity (RQ) of the sample compared to the calibrator

Selected Results on Bladder Selected Results on Bladder CancerCancer

Aim:Aim:

To evaluate the associations To evaluate the associations between between promoter hypermethylation status of promoter hypermethylation status of genes involved in bladder tumorigenesis genes involved in bladder tumorigenesis (including P16INK4a, P14ARF, APC, (including P16INK4a, P14ARF, APC, CDH1, RASSF1A, MGMT, and GSTP1) in CDH1, RASSF1A, MGMT, and GSTP1) in WBC, NBC, CIS, and, cancer tissues from WBC, NBC, CIS, and, cancer tissues from 73 bladder cancer patients.73 bladder cancer patients.

Yu-Ching Kelly Yang, Ph.D

Materials and MethodsMaterials and Methods Hospital-based case-only studyHospital-based case-only study Memorial Sloan-Kettering Cancer CenterMemorial Sloan-Kettering Cancer Center Recruitment period: Oct 1993 to June 1997Recruitment period: Oct 1993 to June 1997 Cases selection criteria:Cases selection criteria:

Newly diagnosed and pathologically confirmed blNewly diagnosed and pathologically confirmed bladder cancer casesadder cancer cases

In stable medical conditionIn stable medical condition Have lived in the US for at least one yearHave lived in the US for at least one year

Fresh bladder tissues were obtained from radFresh bladder tissues were obtained from radical cystectomy ical cystectomy

Study PopulationStudy PopulationPatients with tissue blocks

N = 152

Only have cancerous tissuesN = 33

Did not conform to pathological criteria

N = 46

Patients with cancerous tissue andnon-cancerous tissue (NBC)

N = 73

Proportion of methylation detected in seven tumor-related genes in white blood cells (WBC), in situ (CIS), non-cancerous (NBC), and cancerous tissue specimens of the bladder cancer patients. WBC CIS NBC Cancer

Gene analyzedMethylation

detected

analyzedMethylation

detected

analyzedMethylation

detected

analyzedMethylation

detected

P16 0/11 0.00 0/6 0.00 0/73 0.00 21/73 0.29

APC 0/11 0.00 3/6 0.5 21/73 0.29 42/73 0.58

MGMT 0/11 0.00 0/6 0.00 0/73 0.00 3/73 0.04

RASSF1A 1/11 0.09 2/6 0.33 10/73 0.14 28/73 0.38

CDH1 2/11 0.18 4/6 0.67 22/73 0.30 45/73 0.62

GSTP1 0/11 0.00 0/6 0.00 1/73 0.01 2/73 0.03

ARF 0/11 0.00 0/6 0.00 0/73 0.00 0/73 0.00

WBC:white blood cell; CIS: carcinoma in situ; NBC: epithelium showing no remarkable histological change

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

WBC NBC CIS Cancer

Av

era

ge

nu

mb

er

of

me

thy

late

d g

en

e

The mean and standard deviation of average number of The mean and standard deviation of average number of methylated genes in white blood cells (WBC), carcinoma in situ methylated genes in white blood cells (WBC), carcinoma in situ (CIS), epithelium showing no remarkable histological change (CIS), epithelium showing no remarkable histological change

(NBC) and cancer tissues of bladder cancer patients (NBC) and cancer tissues of bladder cancer patients

age

(>=65 vs. <65 years)

Gender

(male vs. female)

Cigarette smoking

(ever vs. never)

Alcohol drinking

(ever vs. never) Gene

Y (%) N (%) P-value Y (%) N (%) P-value Y (%) N (%) P-value Y (%) N (%) P-value

Promoter hypermethylation

P16

normal 31 66 21 81 37 71 12 67 43 74 3 38 33 73 11 58

hypermethylation 16 34 5 19 0.186 15 29 6 33 0.721 15 26 5 63 0.048 12 27 8 42 0.227

APC

normal 28 60 19 73 35 67 10 56 39 67 5 63 28 62 14 74


MGMT

normal 45 96 25 96 49 94 18 100 56 97 8 100 43 96 19 100

hypermethylation 2 4 1 4 0.933 3 6 0 0.980 2 3 0 0.983 2 4 0 0.983

RASSF1A

normal 34 72 19 73 36 69 15 83 44 76 5 63 34 76 14 74


CDH1

normal 26 55 17 65 30 58 11 61 35 60 3 38 27 60 10 53


No. of methylated genes

none 19 40 13 50 21 40 9 50 27 47 1 13 21 47 6 32

at least one 28 60 13 50 0.431 31 60 9 50 0.479 31 53 7 88 0.101 24 53 13 68 0.268

The association between hypermethylation in promoter-region of The association between hypermethylation in promoter-region of tumor-related genes and environmental exposures, including tumor-related genes and environmental exposures, including

cigarette smoking and alcohol drinkingcigarette smoking and alcohol drinking

Stage

(>=3a vs. <3a)

Lymph node metastatsis

(yes vs. no)

Vascular invasion

(yes vs. no)

TP53 mutation

(mutated vs. normal) Characteristics

Y (%) N (%) P-value Y (%) N (%) P-value Y (%) N (%) P-value Y (%) N (%) P-value

Promoter hypermethylation

P16

normal 34 69 15 71 22 71 27 69 23 62 25 78 23 66 24 75


APC

normal 30 61 15 71 22 71 23 59 26 70 19 59 27 77 17 53


MGMT

normal 46 94 21 100 30 97 37 95 35 95 31 97 34 97 30 94

hypermethylation 3 6 0 0.979 1 3 2 5 0.699 2 5 1 3 0.647 1 3 2 6 0.513

RASSF1A

normal 34 69 17 81 24 77 27 69 27 73 24 75 23 66 28 88


CDH1

normal 25 51 16 76 18 58 23 59 17 46 23 72 22 63 18 56



none 18 37 12 57 14 45 16 41 10 27 19 59 15 43 14 44

at least one 31 63 9 43 0.118 17 55 23 59 0.728 27 73 13 41 0.008 20 57 18 56 0.941

The association between hypermethylation in promoter-region of The association between hypermethylation in promoter-region of six tumor-related gene and clinicopathological factors six tumor-related gene and clinicopathological factors

Overall survival Disease-specific survival Progression-free survival

No. D HRa 95%CI D HRa 95%CI D HRa 95%CI

P16 hypermethylation

Yes 49 38 1.00 ref 30 1.00 ref 13 1.00 ref

No 21 17 1.18 ( 0.66- 2.12) 13 1.07 ( 0.55- 2.07) 5 0.97 ( 0.34- 2.77)

P-chisq 0.580 0.847 0.953

APC hypermethylation

Yes 45 35 1.00 ref 26 1.00 ref 12 1.00 ref

No 25 20 0.84 ( 0.47- 1.49) 17 0.93 ( 0.49- 1.78) 6 0.73 ( 0.26- 2.02)

P-chisq 0.547 0.836 0.540

MGMT hypermethylation

Yes 67 52 1.00 ref 40 1.00 ref 17 1.00 ref

No 3 3 1.34 ( 0.41- 4.42) 3 1.64 ( 0.49- 5.46) 1 1.18 ( 0.15- 9.20)

P-chisq 0.627 0.421 0.871

RASSF1A hypermethylation

Yes 51 40 1.00 ref 31 1.00 ref 12 1.00 ref

No 19 15 1.10 ( 0.59- 2.04) 12 1.19 ( 0.59- 2.39) 6 1.37 ( 0.49- 3.84)

P-chisq 0.774 0.630 0.552

CDH1 hypermethylation

Yes 41 33 1.00 ref 26 1.00 ref 8 1.00 ref

No 29 22 0.89 ( 0.50- 1.59) 17 0.82 ( 0.43- 1.58) 10 1.41 ( 0.52- 3.85)

P-chisq 0.693 0.552 0.503


none 30 23 1.00 ref 18 1.00 ref 6 1.00 ref

at least one 40 32 1.19 ( 0.67- 2.11) 25 1.12 ( 0.59- 2.13) 12 1.41 ( 0.50- 3.99)

P-chisq 0.545 0.731 0.512

HRa: hazard ratio adjusted for age, gender, and stage

The association between hypermethylation in six tumor-related The association between hypermethylation in six tumor-related genes and survival time genes and survival time

Selected Results on Liver Selected Results on Liver CancerCancer

Aim:Aim: To evaluate the associations between HCC To evaluate the associations between HCC

and promoter-region methylation of and promoter-region methylation of selected genes, including APC, CDH1, selected genes, including APC, CDH1, P16INK4a, and MGMT genes in peripheral P16INK4a, and MGMT genes in peripheral blood DNA in a Chinese population. Also, to blood DNA in a Chinese population. Also, to examine the associations of examine the associations of hypermethylation with age, gender, tobacco hypermethylation with age, gender, tobacco smoking, and alcohol consumption. smoking, and alcohol consumption.

Shen-Chih Chang, Ph.D

Materials and MethodsMaterials and Methods Population-based case-control study Population-based case-control study Taixing, ChinaTaixing, China Recruitment period: Jan 1, 2000 to Jun 30, 2000Recruitment period: Jan 1, 2000 to Jun 30, 2000 Cases selection criteria:Cases selection criteria:

Newly diagnosed liver cancer casesNewly diagnosed liver cancer cases Age 25-70Age 25-70 Have no history of any previous diagnosis of cancerHave no history of any previous diagnosis of cancer Have lived in Taixing for at least 10 yearsHave lived in Taixing for at least 10 years

A group of healthy population controls were A group of healthy population controls were frequency-matched (on age and gender) to cases frequency-matched (on age and gender) to cases with a control-to-case ratio of 2:3 from the general with a control-to-case ratio of 2:3 from the general population in Taixing (one common control group population in Taixing (one common control group to three case groups)to three case groups)

Epidemiology data collectionEpidemiology data collection Face-to-face interviewFace-to-face interview Blood samples collected during interviewBlood samples collected during interview

Study PopulationStudy Population

358 incident liver 358 incident liver cancer cases were cancer cases were diagnoseddiagnosed 204 (57%) 204 (57%)

recruited recruited 199 blood 199 blood

samplessamples 194 DNA 194 DNA

extractedextracted

464 potential 464 potential controls were controls were identified identified 415 (90%) 415 (90%)

recruited recruited 410 blood 410 blood

samplessamples 393 DNA 393 DNA

extractedextracted

Case (%)

Control (%)

Crude OR (95% CI)

Adjusted OR1

(95% CI)Adjusted OR2

(95% CI)APC

- 181 (95.6) 332 (94.1) 1.00 1.00 1.00+ 8 (4.2) 21 (6.0) 0.70 (0.30, 1.61) 0.52 (0.19, 1.41) 0.70 (0.24, 2.08)

p-value0.3998 0.1981 0.5233

CDH1

- 100 (52.9) 173 (49.4) 1.00 1.00 1.00+ 89 (47.1) 177 (50.6) 0.87 (0.61, 1.24) 0.93 (0.61, 1.42) 1.05 (0.65, 1.68)

p-value0.4406 0.7287 0.8548

MGMT

- 190 (100.0) 351 (100.0)

+ 0 (0.0) 0 (0.0)

p-value

P16

- 190 (100.0) 348 (99.4)

+ 0 (0.0) 2 (0.6)

p-value


None 99 (52.4) 171 (48.9) 1.00 1.00 1.001 83 (43.9) 160 (45.7) 0.90 (0.62, 1.29) 0.98 (0.63, 1.51) 1.04 (0.64, 1.69)2 7 (3.7) 19 (5.4) 0.64 (0.26, 1.57) 0.50 (0.17, 1.47) 0.78 (0.24, 2.59)

Associations between promoter hypermethylation of APC, Associations between promoter hypermethylation of APC, CDH1, MGMT, and P16 gene and HCCCDH1, MGMT, and P16 gene and HCC

1: Adjusted on age, gender, BMI, education, smoking pack-years, alcohol drinking, HBsAg, and plasma AFB1-albumin adducts2: Further adjusted on plasma levels of folate, vitamin B12, and homocysteine

≦12.76 nM >12.76 nM

Case/Control

Crude OR (95% CI)

Adjusted OR*

(95% CI)Case/

ControlCrude OR (95% CI)

Adjusted OR* (95% CI)

APC

- 93/169 1.00 1.00 84/159 1.00 1.00

+ 2/10 0.36 (0.08, 1.69)

0.20 (0.04, 1.06) 6/11 1.03 (0.37, 2.89)

1.01 (0.26, 3.86)

p-value 0.1974 0.0583 0.9513 0.9920

CDH1

- 48/82 1.00 1.00 49/89 1.00 1.00

+ 47/96 0.84 (0.51, 1.38)

0.98 (0.54, 1.77) 41/79 0.94 (0.56, 1.58)

0.82 (0.43, 1.59)

p-value 0.4825 0.9412 0.8218 0.5653

APC+CDH1 (continuous)

0.78 (0.50, 1.22)

0.81 (0.48, 1.34) 0.96 (0.63, 1.47)

0.87 (0.50, 1.51)

p-value 0.2727 0.4084 0.8646 0.6294

Associations between promoter hypermethylation of APC and Associations between promoter hypermethylation of APC and CDH1 gene and HCC, stratified on plasma folate levelsCDH1 gene and HCC, stratified on plasma folate levels

*Adjusted on age, gender, BMI, education, smoking pack-years, alcohol drinking, HBsAg, and plasma AFB1-albumin adducts

≦228.88 pM >228.88 pM

Case/Control

Crude OR (95% CI)

Adjusted OR*

(95% CI)Case/



APC

- 33/160 1.00 1.00 145/166 1.00 1.00

+ 1/15 0.32 (0.04, 2.53) 0.24 (0.03, 2.04) 7/6 1.34 (0.44, 4.07)

1.62 (0.39, 6.69)

p-value 0.2824 0.1922 0.6096 0.5060

CDH1

- 19/79 1.00 1.00 80/92 1.00 1.00

+ 15/94 0.66 (0.32, 1.39) 0.78 (0.33, 1.82) 72/79 1.05 (0.68, 1.62)

1.05 (0.59, 1.85)

p-value 0.2773 0.5628 0.8334 0.8726


0.64 (0.34, 1.21) 0.68 (0.34, 1.36) 1.07 (0.73, 1.58)

1.10 (0.67, 1.83)

p-value 0.1728 0.2781 0.7185 0.7060

Associations between promoter hypermethylation of APC and Associations between promoter hypermethylation of APC and CDH1 gene and HCC, stratified on plasma vitamin B12 levelsCDH1 gene and HCC, stratified on plasma vitamin B12 levels


≦9.50 µM >9.50 µM

Case/Control

Crude OR (95% CI)

Adjusted OR*

(95% CI)Case/



APC

- 79/168 1.00 1.00 100/161 1.00 1.00

+ 4/10 0.85 (0.26, 2.80) 0.95 (0.24, 3.81) 4/11 0.59 (0.18, 1.89)

0.26 (0.05, 1.23)

p-value 0.7899 0.9468 0.3706 0.0890

CDH1

- 44/92 1.00 1.00 55/79 1.00 1.00

+ 39/84 0.97 (0.58, 1.64) 1.00 (0.52, 1.92) 49/92 0.77 (0.47, 1.25)

0.75 (0.41, 1.39)

p-value 0.9115 0.9938 0.2825 0.3625


0.96 (0.61, 1.49) 1.00 (0.58, 1.70) 0.76 (0.50, 1.16)

0.67 (0.39, 1.15)

p-value 0.8393 0.9842 0.2020 0.1427

Associations between promoter hypermethylation of APC and Associations between promoter hypermethylation of APC and CDH1 gene and HCC, stratified on plasma homocysteine levelsCDH1 gene and HCC, stratified on plasma homocysteine levels


Hypermethylation

APC CDH1 No. of methylated genes

No(N, %)

Yes(N, %) P*

No(N, %)

Yes(N, %) P*

0 (N, %)

1 (N, %)

2 (N, %) P*

Age

< 55 124 (37.4)

8 (38.1)

0.95

70 (40.5) 60 (33.9)

0.20

69 (40.4) 54 (33.8) 7 (36.8)

0.46≥ 55 208

(62.7)13 (61.9) 103

(59.5)117

(66.1)102 (59.7) 106 (66.3) 12

(63.2)

Gender

Female 104 (31.3)

6 (28.6)

0.79

54 (31.2) 54 (30.5)

0.89

53 (31.0) 50 (31.3) 5 (26.3)

0.91Male 228

(68.7)15 (71.4) 119

(68.8)123

(69.5)118 (69.0) 110 (68.8)

14 (73.7)

Smoking

Never 173 (52.3)

12 (57.1)

0.66

99 (57.6) 84 (47.5)

0.06

97 (57.1) 76 (47.5)10

(52.6)0.22

Ever 158 (47.7)

9 (42.9) 73 (42.4) 93 (52.5)73 (42.9) 84 (52.5) 9 (47.4)

Alcohol Drinking

Never 168 (51.1)

9 (42.9)

0.47

90 (52.6) 85 (48.0)

0.39

88 (52.1) 80 (50.0) 7 (36.8)

0.45Ever 161

(48.9)12 (57.1) 81 (47.4) 92 (52.0)

81 (47.9) 80 (50.0)12

(63.2)

Associations between promoter hypermethylation of APC and Associations between promoter hypermethylation of APC and CDH1 gene and age, gender, smoking, and alcohol drinking CDH1 gene and age, gender, smoking, and alcohol drinking

habits in the control grouphabits in the control group

*P-value from χ2 tests or Fisher’s exact test

Compare MSP and MethyLightCompare MSP and MethyLightMethylation Specific PCRMethylation Specific PCR MethyLight Methylation assayMethyLight Methylation assay

AdvantageAdvantage

•Inexpensive Inexpensive •Easy to performEasy to perform•Less prone to human errorLess prone to human error•Faster, more efficient than MSPFaster, more efficient than MSP•More specific by adding Taqman More specific by adding Taqman probe probe

DisadvantageDisadvantage

•Prone to human errorProne to human error•Easily get contaminatedEasily get contaminated•Labor-intensiveLabor-intensive

•Higher expenses for equipment Higher expenses for equipment maintenancemaintenance

Other TechniquesOther Techniques

Gene-Specific DNA MethylationGene-Specific DNA Methylation SequencingSequencing MicroarrayMicroarray High-resolution Melting Method (Roche)High-resolution Melting Method (Roche) BeadChip Technology (Illumina)BeadChip Technology (Illumina)

Global DNA MethylationGlobal DNA Methylation Liquid Chromatography-Mass SpectrometLiquid Chromatography-Mass Spectromet

ryry ELISA-based global methylation analysis aELISA-based global methylation analysis a

ssay (Sigma Aldrich)ssay (Sigma Aldrich)

ReferencesReferences

http://www.appliedbiosystems.comhttp://www.appliedbiosystems.com http://pathmicro.med.sc.edu/pcr/realtime-home.htmhttp://pathmicro.med.sc.edu/pcr/realtime-home.htm http://www.biorad.comhttp://www.biorad.com Eads CA., et al. MethyLight: a high-throughput assay to mEads CA., et al. MethyLight: a high-throughput assay to m

easure DNA methylation. Nucleic Acids Res., 28: e32, 200easure DNA methylation. Nucleic Acids Res., 28: e32, 2000.0.

Esteller M. Epigenetics in cancer. New England Journal oEsteller M. Epigenetics in cancer. New England Journal of Medicine, 358: 1148-59, 2008f Medicine, 358: 1148-59, 2008

Qiu J. Epigenetics: unfinished symphony. Nature, 441: 14Qiu J. Epigenetics: unfinished symphony. Nature, 441: 143-145, 2006.3-145, 2006.

Zeschniqk M., et al. Zeschniqk M., et al. A novel real-time PCR assay for quanA novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of titative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus. Nucleic Acids Res., 7: 3125, 200the retinoblastoma locus. Nucleic Acids Res., 7: 3125, 2004.4.

Thank you!Thank you!

dna methylation and cancer shen-chih chang, ph.d epi 243 may 14, 2009

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