Kim et al Cancer Biomarkers 2012...
Transcript of Kim et al Cancer Biomarkers 2012...
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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
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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
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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
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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.
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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]
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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
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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
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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
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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
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