1

Kim et al Cancer Biomarkers 2012 Web-appendix:

This document is the web-appendix for the following manuscript.

Quantitative DNA methylation and recurrence of breast cancer: A study of 30 candidate genes.

Dae Cheol Kim, Mangesh A. Thorat, Mi Ri Lee, Se Heon Cho, Nataša Vasiljević, Dorota Scibior-

Bentkowska, Keqiang Wu, Amar S. Ahmad, Stephen Duffy, Jack M. Cuzick, Attila T. Lorincz.

Cancer Biomarkers; 11(2011/2012):75-88

DOI: 10.3233/CBM-2012-0266

2

Laboratory Methods

DNA extraction and bisulfite conversion:

A simple macrodissection of tissue slices was performed before DNA extraction to enrich for areas of

cancer, the method employed is quick (approximately 10 min per case) and readily mastered by the

average laboratory technician. Five consecutive sections per specimen (10 µm thickness) were

obtained by cryo-sectioning the cancer tissues and staining the first and fifth sections by H&E for

histopathology review to confirm the areas of cancer and to guide the dissections of the three

central sections. Genomic DNA was extracted from the three slices of tissue material using QIAamp

DNA Mini Kit (Qiagen Inc., Hilden, Germany) and quantified by UV absorption (Nanodrop, Thermo

Scientific, Wilmington, Delaware, USA), a majority of sections yielding a combined >1 µg of gDNA

per specimen. 120 – 300 ng of DNA was used in the bisulfite conversion reactions where

unmethylated cytosines were converted to uracil with EpiTect Bisulfite kit (Qiagen) according to the

manufacturer’s instructions. Briefly, DNA was mixed with water, DNA protect buffer and bisulfite mix

and the conversion was run in a thermocycler (Biometra, Goettingen, Germany) at the

recommended cycle conditions. Converted DNA was purified and eluted in 2 steps into a total 40 µl

Buffer EB and further diluted into 20 µl aliquots of 100 cell-equivalents/µl. (the cell calculations

assumed 6 pg DNA per diploid cell).

Primer design:

Primer sets with one biotin-labelled primer were used to amplify the bisulfite converted DNA

samples. Thirty genes were identified from the literature (Web-table W1) as candidate genes for this

study as previously described [w1]. New primers for each of the 30 genes (Web-table W2) were

designed using PyroMark Assay Design software version 2.0.1.15 (Qiagen), with an aim to keep

amplicons short with lengths between 90 to 140 base pairs (bp) to facilitate later studies on FFPE

3

specimens. Maximum permissible size of the amplicons was 210 bp. All primers were located in

promoter or first exon CpG islands identified by MethPrimer [w2] depending on where the design of

the assay allowed for optimal primers. CG dyads were not allowed in any forward, reverse or

sequencing primer positions to prevent any amplification bias. Mean size of all of the amplicons was

117 bp. For genes, previously investigated by other methods, primers were positioned to investigate

the same CGs or ones in close vicinity. To provide the internal control for total bisulfite conversion, a

non-CG cytosine in the region for pyrosequencing was included where possible. Three to six CG

positions were investigated in each gene.

PCR and Pyrosequencing:

PCRs were performed using bisulfite converted DNA equivalent of 200 to 400 cells employing the

PyroMark PCR kit (Qiagen). Briefly, 12.5 µl master mix, 2.5 µl Coral red, 5pmol of each primer, 7 µl of

water and 2 µl sample were mixed for each reaction and run at thermal cycling conditions: 95°C for

15min and then 45 cycles: 30 sec at 94°C; 30 sec at the optimized primer-specific annealing

temperature (Web-table W2); 30 sec at 72°C and a final extension for 10 min at 72°C. The correct

amplified DNA was confirmed by electrophoresis in a 2% low melting point agarose gel (Sigma-

Aldrich, Steinheim, Germany) in TBE buffer or by the QiaExel capillary electrophoresis instrument

(Qiagen). A standard pyrosequencing sample preparation protocol was applied [w3]. 3 µl

streptavidin beads (GE Healthcare, UK), 37 µl PyroMark binding buffer (Qiagen), 20 µl PCR product

and 20 µl water were mixed and incubated for 10 min on a shaking table at 1300 rpm. Using the

Biotage Q96 Vaccum Workstation, amplicons were separated, denatured, washed and added to 45µl

annealing buffer containing 0.33 µM of pyrosequencing primer. Primer annealing was performed by

incubating the samples at 80°C for 2 min and allowed to cool to room temperature prior to

pyrosequencing. PyroGold reagents were used for the pyrosequencing reaction and the signal was

analyzed using the PSQ 96MA system (Biotage, Uppsala, Sweden). Target CGs were evaluated by

4

instrument software (PSQ96MA 2.1) which converts the pyrograms to numerical values for peak

heights and calculates proportion of methylation at each base as a C/T ratio. All runs contained

standard curves, which comprised a range of control methylated DNA (0%, 25%, 50%, 75%, and

100%) to allow standardized direct comparisons between different experiments. For the standard

curves a total of 300 ng of unmethylated (Qiagen) and hypermethylated DNA (Millipore, Billerica,

MA, USA) were mixed to obtain the different ratios of DNA methylation and then bisulfite converted

as described above.

A further selection of preferred genes from the initial 30 candidate genes was performed after the

first 30 samples were processed. Genes (n = 20) correlating to (p <0.1) any of Age, Nodal status,

Histological grade, ER, PgR, HER2 were selected as preferred. The remaining 10 genes (Web-table

W2) had very low methylation frequency and levels and therefore were unlikely to succeed as

biomarkers. These genes were therefore not investigated further in this study; we report findings on

20 selected preferred genes.

We have previously established reproducibility of the PCR-PSQ method [w1]; therefore, all samples

were assayed once except for the samples which did not yield pass results on first assay. Such

samples were assayed once more and data were recorded as missing if the samples were

unsuccessful in the second instance as well. Failed samples constitute a very low proportion of the

samples investigated.

5

Web-table W1: Candidate genes and the studies that formed the basis for their selection.

No. Gene References

1 APC [w4-21]

2 RARB [w8, 13, 14, 17, 18, 20-25]

3 RASSF1A [w6-9, 11, 14, 16-25]

4 GSTP1 [w8, 9, 12, 16, 18, 20-22]

5 MDR1 [w21, 26-28]

6 TIG1 [w21, 29, 30]

7 EDNRB [w27, 31, 32]

8 CDH13 [w23, 24, 33, 34]

9 HIN1 [w14, 18, 20, 23-25, 30]

10 DPYS [w35]

11 NKX2-5 [w35, 36]

12 EGFR5 [w35]

13 PTGS2 [w21, 37-39]

14 BCL2 [w21, 40]

15 PDLIM4 [w24, 39, 41]

16 CCND2 [w9, 19, 20]

17 P16 [w15-17]

18 CDH1 [w5, 12, 17, 22]

19 SFN [w20, 42]

20 SERPINB5 [w43, 44]

21 CNR1 [w45, 46]

22 MCAM [w47]

23 ESR1 [w11, 12, 16, 17, 42]

24 HSPB1 [w48]

25 TWIST1 [w14, 25, 49]

26 HLA-A [w50]

27 SLIT2 [w51, 52]

28 THRB [w53]

29 MAL [w54]

30 DAPK1 [w7, 14, 17, 20]

6

Web-table W2 - Primers used for pyrosequencing

Primers used for pyrosequencing, size and position of the amplicons with number of investigated CGs in each gene. Genes in BLUE Italics were not included

in this study after interim decision as described in methods.

Gene Primer name Sequence 5’ 3’ Size (bp2) Position in the gene No of CpG sites Temp(°)

1 APC

pAPCp1f GGGTTAGGGTTAGGTAGGTTGT

95 -180 to -85 5 52 (B)pAPCp1r B1 - AATTACACAACTACTTCTCTCTCC

pAPCp1s GGTTAGGGTTAGGTAGGTT

2 RARB

RARbp2f GTATAGAGGAATTTAAAGTGTGGGT

90 53 to 143 5 52 (B)RARbp2R B - ACCCAAACAAACCCTACT

RARbp2s GTTTGAGGATTGGGATG

3 RASSF1A

pRASF3f1 AAGGAGGGAAGGAAGGGTAA

89 -100 to -11 6 53 (B)pRASF3r1 B-AAACCTAAATACAAAAACTATAAAACCC

pRASF3s1 GGAAGGAAGGGTAAG

4 GSTP1 pGSTPr1f GGGAGTAAATAGATAGTAGGAAGAG 140 -378 to -238 4 49

7

(B)pGSTPr1r B-CCCTCTCCCCTACCCTATAA

pGSTPr1s AGATAGTAGGAAGAGGA

5 MDR1

MDR1p1F GTTTAGGTTTTTTGTGGTAAAG

168 11292 to

11124 5 52 (b)MDR1p1R B-TTCCTCCTAAAAATTCAACCTATT

MDR1p1s GTTTTTTGTGGTAAAGAGAG

6 TIG1

TIG1p1F GGAAGTTGAAGAAGTGAAG

84 289 to 205 6 53 (B)TIG1p1R B-ACCCTAAACAACCTCAAA

TIG1p1s GGAAGTTGAAGAAGTGAAG

7 EDNRB

EDNRBf AGAGGGTATTAGGAAGGAGTTT

169 56713 to 56881 5 52 (B)EDNRBr B-ACAAAACACTTAAATCAACTACCC

EDNRBs AGGGTATTAGGAAGGAGTTT

8 CDH13

CDH13p2f AGTTTTTTTTTTTTATTTGGGATAGGAG

153 28 to 181 4 53 (B)CDH13p2r B-ACCTCTCCTCAAAACCTAACTC

CDH13p2s TTTTTTTATTTGGGATAGGAGAA

8

9 HIN1

pHIN1p1f GGTTTGTTTGTTTTTAGAGGGTTTTAG

119 -233 to -112 4 53 (B)pHIN1p1r B-ACCCTAACCAACTTCCTACT

pHIN1p1s GTAGGGAAGGGGGTA

10 DPYS

DPYS F GGTTTGGGGTGTTTTTTTGTAAGG

145 -85 to 50 4 56 (B) DPYS R B-TAAACTCCAACCCAACCTTCC

DPYS s AGTTTTGTTTTAGGTTGTAAATT

11 NKX2-5

NKX25F GGTTAGTATGTAGGAGGAGG

116 209 to 325 5 51 (B) NKX25 R B-CCTTCTCAATCAAAAACATCCT

NKX25 s AGTATGTAGGAGGAGGG

12 EGFR5

EGFR5F GTTGGGGAAGTTAGTTGTAGAGG

90 238 to 328 6 50 (B) EGFR5R B-AAACTACTCCCAACTTAAATCTAT

EGFR5s GGAAGTTAGTTGTAGAGGG

13 PTGS2

PTGS2 F AGATTTTTGGAGAGGAAGTTAAGT

185 251 to 436 4 53

(B) PTGS2 R B-CTCCTATCTAATCCCTCCCTCT

9

PTGS2 s GATTAGTTTAGAATTGGTTTT

14 BCL2

BCL2F AGGTGTAGTTGGTTGGATAT

98 977 to 1075 5 52 (B)BCL2R B- ATACCACCTATAATCCACCT

BCL2s GTGTAGTTGGTTGGATATT

15 PDLIM4

(B)PDLIM4 F B-GATAGTTGGGTTTGGGTT

104 -196 to -300 6 52 PDLIM4R CACCCCCACTCAACTCTC

PDLIM4 s CAACTCTCAAAAATCCCC

16 CCND2

(B)CCND2F B-GGGTTATTTTTTAGAAAGTTGTAT

80 -860 to -940 5 52 CCND2R CCCCTACATCTACTAACAA

CCND2s CCCTACATCTACTAACAAAC

17 P16

p16F AGGGGTTGGTTGGTTATTAGA

75 120 to 195 6 54 (B)P16R B-CTACCTACTCTCCCCCTCT

p16s GGTTGGTTGGTTATTAGAG

18 CDH1 (B)p2CDH1p1f B-GGAAGTTAGTTTAGATTTTAGT 103 37 to 140 5 49

10

p2CDH1p1r ACTCCAAAAACCCATAACTA

CDH1p1s CAAAAACCCATAACTAACC

19 SFN

p14-3-3p2f GGAGAAGGTGGAGATTGAGTTTTA

121 326 to 447 3 52 (B)p14-3-3p2r B-ACCCTTCATCTTCAAATAAAAAACC

p14-3-3p2s1 GGTGGAGATTGAGTTTTAG

20 SERPINB5

SRP1F AGGTTTGAGTAGGAGAGGAGTGT

221 -71 to 150 6 56 (B)SRP1R B-CCCACCTTACTTACCTAAAATCACA

SRP1s TTGAGTAGGAGAGGAGTGT

21 CNR1

CNR1 F GTTTAGTTTAGGGGTTGGTTG

157 ?? 5 56 (B) CNR1 R B-CTTCCTTCTCCACTTCTTTTCC

CNR1 s GAGTTTTGTAGGGAGT

22 MCAM

MCAM F GGTTGTAAATTGGTTGTAAAGAAGA

74 207 to 281 6 56 (B)-MCAM R B-AACCTCCTCCCTAAATCC

MCAM S GGTTGTAAAGAAGAGTTGT

11

23 ESR1

ERalfaF GGGTTGTGTTTTTTTTTTAGGTGG

136 504 to 640 5 49 (B)-ERalfaR B-ACAATAAAACCATCCCAAATACTTTAATA

ERalfa seq GGTTTTTGAGTTTTTTGTTTTG

24 HSPB1

Hsp27p4F AGTTGGGGAGTGAGTAGT

112 864 to 976 5 54 Hsp27p4R B-CAACCCCATCCCCAAATAA

Hsp27p4s TGGGGAGTGAGTAGTA

25 TWIST1

(B)-TWIST1 F B-GGGGTAGAGGAGAAGAG

98 221 to 319 5 52 TWIST1 R TCCTCCTACTCTCTCCT

TwIST1 seq TCCTACTCTCTCCTCC

26 HLA-A

HLA-A F GGGTTTTGGTTTTGATTTAGATTT

88 44 to 132 4 54 (B)-HLA-A R B-CAAAAAAACCCCTTACTTCTCC

HLA-A Seq GGTTGTAAAGAAGAGTTGT

27 SLIT2

SLIT2 F GGTTGTTAAGATGTAGGGG

112 -151 to -263 4 52

(B)-SLIT2 R B-AAATCCCCTCTTCTATCTTATAC

12

SLIT2 seq GTTGTTGTGGGGAGGG

28 THRB

THRB F TTAGGGTATTGGTAATTTGGTTAGA

90 79 to 169 4 54 (B)-THRB R B-CCACCCTATAAACAATTAAAACTATC

THRB seq GTATTGGTAATTTGGTTAGAGG

29 MAL

MALp1F GGGTTTGTAGTGGGGGATG

146 -504 to -650 5 54 (B)-MALR B-ACTAAAAACAACCTCCTACTCTCA

MAL seq TGTAGTGGGGGATGGGAT

30 DAPK1

DAPKp2F TAGTTAGGGAGTGAGTGGG

210 -39 to 169 4 51 DAPKp2R ACAAAATCCCCATTAACC

DAPKp2s GGAGGGAATAAAGTTTT

* B = Biotinylated primer at 5’ end 2 bp = base pair

† The position of 0 is start of the exon 1

13

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