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METHODS
Subjects and samples
Subjects were selected from participants in the Melbourne Collaborative Cohort Study, a
prospective cohort study of 41,514 volunteers (24,469 women) aged between 27 and 76 years at
baseline (99.3% of whom were aged 40-69)35. At baseline attendance, participants completed
questionnaires that measured demographic characteristics and lifestyle factors including diet.
Height and weight were directly measured and a blood sample was collected and stored. For a
large proportion of individuals (75%) only dried blood spots (i.e. DBS) on Guthrie cards were
available while for others buffy coat or lymphocyte samples were available.
Nested case-control study
To estimate the association between aberrant DNA methylation from samples collected at
baseline and breast cancer risk we conducted a nested case-control study and used the Infinium
HM450 produced by Illumina to quantify DNA methylation in more than 480,000 CpG sites
covering up to 96% of CpG islands as well as a large number of non-island CpG sites 22,23.
The study sample included women in the nested case-control study that we designed to
estimate the association between mammographic density and breast cancer risk 36. In the
mammographic density study, cases were women with a first diagnosis of ductal carcinoma in
situ (DCIS; 120 cases) or invasive adenocarcinoma of the breast (International Classification of
Diseases for Oncology, C50.0-C50.9; 680 cases) occurring between baseline interview up until
31 December 2007 and ascertained by record linkage to the population-based Victorian Cancer
Registry (VCR), and to the Australian Cancer Database. For each breast cancer case, we
randomly selected 4 women as controls from among those who had not been diagnosed with
breast cancer at the age of diagnosis of the case (reference age). The controls were also matched
on year of birth, year of baseline attendance and country of origin (classified as Australia/New
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Zealand/UK/others, Italy or Greece). To be eligible for the methylation case-control study
women in the mammographic density case-control study had to have at least one mammogram
already retrieved at the time the study sample was assembled (first trimester 2010) and available
blood collected at baseline interview. The sample included 498 invasive breast cancer cases and
one control per case randomly selected from the four controls matching, when possible, on type
of biospecimen (DBS, buffy coat, or lymphocyte).
Tumour characteristics
Tumour characteristics were extracted from the VCR database; these include tumour grade,
size, nodal status, oestrogen receptor status (ER), progesterone receptor status (PR) and
epidermal growth factor 2 status (Her2). Additional assessment of ER, PR, HER2, EGRF1 and
CK56 status using immunohistochemistry techniques was performed by a single pathologist
(CM) 37. For the analyses presented in this paper, we used our immunohistochemistry measures
of ER, PR and HER2 and given the good agreement with the results from the original pathology
report 37, when archival tumour tissue was not available, ER, PR and HER2 status was assigned
according to the histopathology reports held at the VCR.
Biospecimens, DNA extraction and DNA methylation measures
In 485 matching sets both case and control had the same biospecimen available (373 DBS,
103 lymphocytes, 9 buffy coats); of the remaining 13 sets, in 5 the biospecimens were buffy coat
and lymphocytes and in 8 DBS and lymphocytes. This resulted in 754 samples from DBS (76%),
219 from lymphocytes (22%) and 23 from buffy coats (2%).
DNA extraction from lymphocytes and buffy coats were performed using Qiagen mini spin
columns (Hilden, Germany) while dried blood spot DNA was extracted using a method
developed in-house 38: twenty 3·2 mm diameter archived blood spot punches were added to 200
ul phosphate buffered saline and homogenised using the TissueLyser (Qiagen). The resulting
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supernatant was transferred to a clean 1·5 ml tube and DNA extracted using Qiagen mini spin
columns according to the manufacturer’s protocol. The quality and quantity of DNA was
assessed using the Quant-iT™ Picogreen® dsDNA assay measured on the Qubit® Fluorometer
(Life Technologies, Grand Island, NY).
Bisulfite conversion (EZ DNA Methylation-Gold kit, Zymo Research, Irvine, CA), quality
control analyses and the Infinium HM450 DNA methylation assays were performed at the
Australian Genome Research Facility as per the manufacturers’ instructions. The bisulfite
conversion control dashboard on the Illumina arrays was primarily used to check success of the
conversion. Also, a bioanalyser run was performed on a subset of samples in each batch to further
check the success of the conversion.
Samples were distributed into 96-well plates and processed by Infinium HM450 in chips of
12 arrays (8 chips per plate) with case-control pairs arranged consecutively on the same chip.
Data processing
Methylation data were normalised to the internal built-in controls as provided by the
standard Illumina software and subset-quantile within array normalization (SWAN) for type I and
II probe bias correction 39. We excluded the 416 CpGs sites on the Y chromosome and the 65
corresponding to single nucleotide polymorphisms. We assigned the methylation measures at
CpG sites with a detection p-value higher than 0·01 as missing. Samples for which more than 5%
of the CpG measures were missing were considered as “failed” and excluded from further
analyses as were the CpG sites whose measures were missing for more than 20% of the samples.
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Online Resource Table 1
Characteristics of the study sample
Age at blood collection
Cases
Controls
N=420
N=420
Mean: 56
Mean=56
SD=8
SD=8
Range=(38 to 70)
Range=(38 to 70)
Mean=56
Biospecimen
Country of Origin
Guthrie Card
325
320
Lymphocytes
84
92
Buffy Coats
11
8
355
356
65
64
Australia/NZ/UK
Italy/Greece
Reference age (*)
Mean: 64
SD: 8
Range: 44 to 83
Time from blood
collection to reference age
Grade
<5years
131
5-9years
148
10 years or more
141
Well differentiated
Moderately differentiated
179
Highly differentiated
120
Missing
Size
Nodal Status
ER
PR
HER2
Subtype
87
34
Less than 2 cm
286
2 cm or more
123
Missing
11
Negative
398
Positive
86
Missing
36
Negative
98
Positive
297
Missing
25
Negative
173
Positive
217
Missing
30
Negative
281
Positive
109
Missing
30
Luminal A
221
Luminal B
87
HER2+
20
Triple Negative
54
Missing
38
(*) Age at diagnosis for the cases and age of the case in the matching set for the controls
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Online Resource Table 2
Proportion of the total variance explained by the first 10
principal components and their correlation with global methylation overall and by CpG
genomic feature
PC1
PC2
PC3
PC4
PC5
PC6
PC7
PC8
PC9
PC10
Proportion
0·274
0·062
0·023
0·017
0·015
0·009
0·008
0·006
0·005
0·004
Cumulative Proportion
0·274
0·336
0·359
0·376
0·391
0·400
0·408
0·414
0·419
0·423
All
0·56
0·67
0·31
0·15
-0·30
-0·03
-0·09
0·02
-0·02
-0·02
Island/Shore
0·79
0·15
0·30
0·33
-0·36
0·07
-0·08
-0·04
-0·05
0·00
Shelf/None
0·22
0·89
0·30
-0·04
-0·20
-0·10
-0·06
0·04
0·00
-0·02
Functional Promoter
0·38
-0·25
0·19
0·43
-0·65
-0·01
-0·26
-0·14
-0·05
0·07
Non Functional Promoter
0·54
0·73
0·32
0·10
-0·23
-0·03
-0·05
0·03
-0·02
-0·03
Repetitive Elements
0·33
0·88
0·21
0·02
-0·22
-0·06
-0·07
0·01
0·00
-0·04
Non Repetitive Elements
0·62
0·58
0·34
0·20
-0·32
-0·02
-0·09
0·02
-0·02
-0·01
Total variance explained
Correlations
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Online Resource Table 3
Relative risk of breast cancer for 1 standard deviation of the first,
second, fifth and eight principal components
Overall
PC1
PC2
PC5
By time since blood collection
RR (95% CI)
p
0·74 (0·56-0·99)
0·04
0·66 (0·51-0·86)
0·83 (0·62-1·09)
0·002
0·18
RR (95% CI)
p
<5 years
0·44 (0·25-0·79)
0·006
5-9 years
0·99 (0·61-1·61)
0·98
10 years or more
0·82 (0·49-1·37)
0·44
test for linear trend
0·21
<5 years
0·45 (0·27-0·74)
0·002
5-9 years
0·66 (0·43-1·02)
0·06
10 years or more
0·83 (0·51-1·34)
0·44
test for linear trend
0·1
<5 years
1·2 (0·73-1·95)
0·48
5-9 years
0·73 (0·45-1·2)
0·22
0·61 (0·35-1·09)
0·1
test for linear trend
0·05
<5 years
1·83 (1·11-3·01)
0·02
5-9 years
1·10 (0·76-1·60)
0·61
10 years or more
1·04 (0·66-1·64)
0·86
test for linear trend
0·02
10 years or more
PC8
1·27 (1·00-1·62)
0·05
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Online Resource Figure 1
Difference in epigenome-wide methylation between cases and
controls by time between blood collection and diagnosis (number of sets in each time frame in
red)
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Online Resource Figure 2
Score plots of principle components analysis showing the first
component (PC1, x-axis) versus the second component (PC2, y-axis). Incident breast cancer
cases are shown in red with controls in black
12