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Insulin-like Growth Factor Binding Protein 5
(IGFBP5) as a putative biomarker for breast
cancer risk
20th February 2015
Olivia Fletcher
Group leader, Genetic Epidemiology
Making the discoveries that defeat cancer
Structure of my talk
• Background to the 2q35 breast cancer risk locus
• Identifying target(s)
• Tissue specificity of IGFBP5 expression
• Developing IGFBP5 assays in (i) FFPE tumour microarrays
•
(ii) Plasma
• Some preliminary results
• Statistical issues
• What next?
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rs13387042 (Chr2:217,905,832) is associated with a 20% increased risk
of ER+ breast cancer, P=1.3 x 10-13
Replicated in subsequent GWAS and by the Breast Cancer Association
Consortium
Stacey, S et al; Nature Genetics 2007
Milne, R et al; J Natl Cancer Inst 2009
2q35 risk locus – a gene dessert
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Ghoussaini, M et al; Nature Communications 2014
Identifying target(s)
Expression Quantitative Trait Locus (eQTL) analysis
Physical interactions between regulatory elements and the genes they
influence (chromosome conformation capture methods)
Both methods
IGFBP5 (protein coding gene)
DIRC3 (long non-coding RNA)
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IGFBP5 expression
IGFBP5 is expressed and secreted by breast epithelial cells.
eQTL and Chromosome Conformation Capture are reflecting local
expression.
IGFBP5 is also expressed by the liver (endocrine)
Liver expression responsible for (circulating) plasma IGFBP5 levels
Your average epidemiological study has –
• buffy coat,
• plasma/serum,
• possibly urine
• but not breast epithelial cells (at least in controls)
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The plan!
Measure IGFBP5 in (pre-diagnostic) plasma samples from incident cases
and matched controls
Measure IGFBP5 in tumour material from the cases
Qus
(i) Are levels of IGFBP5 in tumours correlated with levels in plasma (in
cases)?
(ii) Are plasma levels of IGFBP5 associated with subsequent risk of breast
cancer?
(iii) Are plasma levels of IGFBP5 associated with established risk factors for
breast cancer?
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Breakthrough Generations Study
Population based cohort study
Over 100,000 female volunteers age 16 or over
UK- based
Recruited between 2003 and 2013
Questionnaire data
Blood samples – entry and after 6 years (subset)
~ 5,000 prevalent cases
~ 1,250 incident cases (excluding DCIS)
PIs Swerdlow/Ashworth
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Measuring IGFBP5 in tumours
Immunohistochemistry (IHC) staining of tissue microarray sections from
one ER-positive and one ER-negative tumour
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Two commercially available IGFBP5
assays……..
A
DELFIA standard curve (R&D systems ELISA kit)
B
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80000
OD = 450nM
Europium counts
100000
ELISA standard curve (Raybiotech ELISA kit)
60000
40000
1
0.10
20000
1
10
100
IGFBP5 (ng/ml)
1000
0.01
0.1
1
10
IGFBP5 (ng/ml)
100
1000
Our IGFBP5 standard curve
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Pilot data (1)
Assayed 224 cases and 223 controls frequency matched on
Age,
Year of entry
Ethnicity
Number of days the blood sample was in the post
Assayed samples in triplicate, and took the mean of the three readings.
Re-assayed
5 samples with IGFBP5>900ng/ml
1 sample with undetectable levels
21 samples with CV>20%
14 random samples
Intra-assay CV=6.2%, Inter-assay CV=12.8%
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Pilot data (2)
IGFBP5 ng/ml
Comparing cases and controls (1)
Mean plasma IGFBP5 in
Controls – 243.0 ng/ml (206.3-279.8)
Cases – 262.9 ng/ml (226.1 – 299.7)
P=0.08
But excluding the two outlier cases
Controls – 249.1 ng/ml (223.0-275.1)
Cases – 256.9 ng/ml (230.8 – 282.9)
P=0.34
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Comparing cases with controls (2)
Controls N (%)
Cases N (%)
OR (95% Ci)
ORadj (95% Ci)
Q1 (63.3 – 174.2)
55 (24.8)
47 (21.2)
1.00
1.00
Q2 (174.3 – 239.3)
56 (25.2)
66 (29.7)
1.38 (0.81 – 2.34)
1.38 (0.77 – 2.48)
Q3 (239.4 – 307.7)
55 (24.8)
49 (21.1)
1.04 (0.60 – 1.80)
1.11 (0.58 – 2.12)
Q4 (307.8 – 986.4)
56 (25.2)
60 (27.0)
1.25 (0.74 – 2.14)
1.35 (0.69 – 2.66)
Ptrend=0.67
Padj=0.55
*three outliers (women whose plasma IGFBP5 levels were >5 standard deviations from the mean), were excluded
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Association with age at FFTP
Number of women*
Mean IGFBP5 (95% CI)
<20
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222.3 (171.7 – 273.0)
20-24
132
248.8 (230.6 – 267.0)
25-29
163
249.5 (233.1 – 265.8)
30-34
53
259.8 (231.2 – 288.5)
≥35
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248.6 (196.4 – 300.8)
P value
Age at FFTP
(years)
Ptrend=0.40 (Padj=0.50)
* three outliers (women whose plasma IGFBP5 levels were >5 standard deviations from the mean), one woman for whom
IGFBP5 levels were undetectable and 12 women for whom the duration of their only pregnancy/pregnancies was less than
26 weeks are excluded from these analyses.
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Association with breast feeding
Number of women**
Mean IGFBP5 (95% CI)
None
70
247.6 (222.4 – 272.8)
<6
138
254.3 (236.5 – 272.1)
6 -11
60
250.6 (223.6 – 277.6)
12 - 24
82
249.9 (226.5 – 273.4)
≥24
29
233.7 (194.2 – 273.3)
P value
Duration of breast feeding
(months)
**data on breastfeeding was missing for six women
Ptrend=0.64 (Padj=0.58)
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Association with parity
Number of women
Mean IGFBP5 (95% CI)
Non-parous
51
259.8 (214.0 – 305.7)
Parous
381
249.4 (239.0 – 259.8)
1 child
40
291.1 (258.5 – 323.8)
2 children
213
250.3 (236.2 – 264.5)
3 children
97
235.5 (214.6 – 256.5)
≥4 children
31
233.0 (195.8 – 270.1)
P value
Parity
P=0.50 (Padj=0.85)
Ptrend=0.01 (Padj=0.02)
Statistical issues
Outliers - to re-assay or not
- remove from quantitative analyses?
- include in analyses of quartiles?
Power – 1,250 cases and 1,250 controls, 70% power at 5% significance
for OR=1.35 (comparing top and bottom quartiles)
• Applied for funding to assay an additional 1,026 cases and 1,026
controls
• Temporal variation – assay plasma samples taken from the same
woman five years apart (subset of 62 women)
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Acknowledgments
ICR - Gene Function
Laura Broome
Nichola Johnson
Richard Elliott
Chris Lord
Alan Ashworth
Bioscale Colin Merrifield
Breakthrough Generations Study Tony Swerdlow
Montse Garcia-Closas
University of Bristol
Nick Orr
Jeff Holly
Minouk Schoemaker
Claire Perks
Michael Jones
Caroline Jarrret
Katarzyna Tomczyk