REPORT FOR PATHOLOGICAL SOCIETY: BSc INTERCALATED DEGREE
Title: Plasma markers in familial breast cancer
Name & Address:
Sarah Pennington, Department of Cancer Sciences and Molecular Medicine, Clinical
Sciences Building, University of Leicester, Leicester Royal Infirmary, Leicester, LE2
7LX
Background:
Breast cancer is the most common cancer in women, with one in eight women
affected during their lifetime. Women with a family history of breast cancer are at
increased risk, particularly those who inherit predisposing gene mutations, such as in
the BRCA genes. However, known dominant genes only account for around 10% of
inherited breast cancers, and polygenic factors rather than a single dominant gene
are responsible for most inherited breast cancers.
Current screening for early breast cancer detection, using mammography and/or
MRI is insufficiently sensitive and specific for malignancy and is uncomfortable for
patients. It is hoped that a blood test can be developed using circulating free DNA as
a biomarker, representing a non-invasive “liquid biopsy” of the tumour. This could
potentially also provide prognostic data and enable individualised monitoring. It is
not possible to give women at increased familial risk but with low risk of a gene
mutation individualised counselling, and the identification of biomarkers in the blood
could enable better stratification of individual risk.
In this preliminary study, 44 women aged 35-39 at moderate risk of breast cancer
undergoing early mammography as part of the FH-02 trial consented to giving blood
samples for analysis. 2 women were later excluded due to positive BRCA mutation
testing in a relative. Participants had their breast cancer risk scores at 1, 5 and 10
years calculated using the BOADICEA web application (version 2), the results of their
mammograms recorded and their blood samples analysed. The plasma was
separated and DNA isolated. The DNA was quantified using real-time qPCR and was
subsequently analysed for amplifications in the CDKN2A, FGFR1, CCND1, MYC and
ERBB2 genes. Amplifications in the FGFR1, CCND1, MYC and ERBB2 genes have been
identified in over 10% of breast tumours (Stephens et al, 2012), and CDKN2A was
chosen based on previous research within the team. Samples were analysed using
GAPDH/RPPH1 as endogenous control genes.
Original Aims (copied from original application):
1. Carry out family history of breast cancer computer modelling
2. Assist in developing an algorithm of breast cancer risk
3. Assist in recruitment of patients to novel biomarker identification in familial
breast cancer
4. Perform related laboratory techniques for development of plasma nucleic acid
signatures
Results:
BOADICEA calculations confirmed that the cohort fitted within the moderate risk
category but showed little variation between individuals, demonstrating that
individualised counselling could not be developed based on the BOADICEA risk
scores alone. None of the individuals were found to have breast cancer on their
initial mammogram at the time of blood sampling, and none were diagnosed within
the follow-up period (8-12 months).
Quantification values were shown to be within the expected range for healthy
individuals, but values for the whole cohort fell within a small range (Figure 1A). This
therefore also prevented the stratification of individuals based on quantification. No
correlation was found between BOADICEA breast cancer risk scores at 10 years and
DNA quantity (Figure 1B).
A
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0.06
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10 year risk
DNA quantity (ng)
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Participant no.
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DNA quantity (ng)
Figure 1: (A) Bar chart showing DNA quantitation values for the cohort. (B) Dot plot
demonstrating DNA quantity plotted against 10 year risk score for each individual.
The highest numbers of amplifications were seen in the FGFR1 (26.2%) and CDKN2A
(26.2%) genes in the cohort. Six participants (highlighted in Figure 2) accounted for
the majority of amplifications seen, suggesting that these individuals may form a
separate group clinically, possibly due to early tumour development.
10
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RQ
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RQ = 2.5
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Gene
Figure 2: Dot plot demonstrating relative quantitation values for the FGFR1, CCND1,
ERBB2, CDKN2A and MYC genes within the cohort (RQ values >2.5 represent
amplification).
No relationship was identified between gene amplifications and benign breast
disease, BOADICEA risk scores above the 75th percentile or those with family
members with negative BRCA mutation testing in a family member. This may suggest
that cfDNA testing could be used as an independent biomarker.
The ERBB2 gene showed unexpected results for the cohort, with all participants
showing relative quantitation (RQ) values below 1. Some samples were reanalysed
without the preamplification step before qPCR and showed higher RQ values,
suggesting that the preamplification step affected ERBB2 analysis.
Buffy coat samples were analysed to compare lymphocyte DNA to that isolated from
the plasma. RQ values were found to differ between the buffy coat and plasma
samples for individuals, suggesting that the circulating free DNA was not simply
germline DNA.
Conclusions:
A group of individuals has been identified within the cohort that shows
amplifications in the genes of interest in plasma DNA. This group may be showing
signs of early tumour development that cannot yet be detected clinically. Long-term
follow-up over many years would identify whether this group differs from the rest of
the cohort in terms of breast cancer risk. Larger-scale studies with long-term followup would identify whether analysis of these genes in the plasma could be used to
develop a signature for breast cancer risk prediction. It will also be important to
investigate how these plasma signatures change over time for individuals, alongside
their clinical status.
The differences in gene analysis results between the lymphocyte and plasma DNA
suggests that the plasma DNA harboured gene aberrations different from their
germline status, and could therefore represent another source, such as an
underlying tumour. There is therefore potential for circulating free DNA to be
identified as a biomarker for breast cancer detection in future studies.
How closely have the original aims been met:
Breast cancer risk modelling was successfully carried out for all of the individuals in
the cohort. Laboratory techniques were carried out for the development of plasma
DNA signatures as planned. The patients were recruited prior to the commencement
of the BSc project by the Clinical Genetics department at the Leicester Royal
Infirmary. The results from this preliminary study cannot yet be used to develop any
new algorithms for predicting breast cancer risk, but will hopefully contribute to
future developments in this area.
Reference:
Stephens, P.J. et al., 2012. The landscape of cancer genes and mutational processes
in breast cancer. Nature. 486, 400.