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`Molecular analysis of circulating cell-free
`
`DNA in breast cancer patients
`
`Thesis submitted for the degree of Doctor of Philosophy at the
`
`University of Leicester
`
`by
`
`Kevin Anthony Blighe, BSc Honours (Republic of Ireland),
`
`University of Leicester,
`
`Department of Cancer Studies and Molecular Medicine
`
`2012
`
`
`
`Personalis EX2155
`
`

`

`Molecular analysis of circulating cell-free DNA in breast cancer patients
`
`
`
`
`
`Kevin Anthony Blighe
`
`___________________________________________________________________________________
`
`Abstract
`
`Identifying disease-related variants is a primary aim of human genetics. In breast
`cancer, loss of heterozygosity at specific loci was previously demonstrated in paired
`tumour and circulating plasma cell-free DNA (cfDNA) samples. However, alterations
`unique to cfDNA were also found in all cases, suggesting disease progression. These
`results prompted the characterisation of the circulating breast cancer genome in more
`detail in this thesis, to test the hypothesis that cfDNA acts as a surrogate tumour
`marker. This was achieved using Affymetrix SNP 6.0 technology and bioinformatics to
`map SNP and copy number variation (CNV), comparing cfDNA with matched normal
`lymphocyte and tumour DNA in 65 breast cancer patients and 8 healthy female
`controls.
`
`Results in this thesis show that comparison of cfDNA SNP genotypes can distinguish
`between primary breast cancer patients and healthy controls (p<0.0001), and between
`pre-surgical breast cancer patients and those who already had surgery/treatment
`(p=0.0016). A significant difference (p=0.0006) was also found between cfDNA
`samples taken an average of 3 years apart in women on follow-up, again suggesting
`progression. In addition, CNV amplification was observed in matched tumour and
`cfDNA at numerous loci on different chromosome arms. Many of these tumour-specific
`CNVs contributed significantly to disease through logistic regression analysis and
`remained detectable in cfDNA up to 12 years after diagnosis despite no other evidence
`of disease. This finding strongly infers breast cancer dormancy in the majority of
`patients on follow-up.
`
`In addition, candidate CNVs were validated by real-time qPCR and additional
`bioinformatics revealed key SNP and CNV signatures of breast cancer patients. If
`validated in other patient series, the results could alter the diagnostic and prognostic
`landscape of breast cancer.
`
`Future studies will focus on developing high throughput approaches to target common
`SNPs/CNVs with a view to developing a targeted custom DNA chip for screening and
`monitoring.
`
`
`
`Page I
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`Molecular analysis of circulating cell-free DNA in breast cancer patients
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`
`
`
`
`Kevin Anthony Blighe
`
`___________________________________________________________________________________
`
`
`Acknowledgements
`
`This work is dedicated to all of those who have been affected directly or indirectly by
`
`breast cancer, including relatives, friends, and acquaintances. I sincerely hope that my
`
`work has been beneficial to this sensitive area of research and that it will go toward
`
`something of greater potential that can help all afflicted with breast cancer. Also, to my
`
`grandmother: I wish that you were still alive today - my aim has always been to make
`
`you proud of me.
`
` I
`
` also must give thanks to my family - including numerous aunts, uncles, and cousins -
`
`and my three sisters-in-law Amanda, Neasa, and Sabrina - for having an on-going
`
`interest in my research. Also, lest I forget: Lindsay, Angie, Linda, and Janine, for the
`
`smooth running of the laboratory; Dr. Jacqui Shaw for being a great supervisor, friend,
`
`and colleague, but also for being a great role model in research; Dr. Karen Page, with
`
`whom I worked every day, for also being a great colleague and friend; Youssef, Ali Al-
`
`Harth, and their families, Eftychios Papadogeorgakis and Eleni, Drs. J Howard Pringle,
`
`Shona Elshaw, and Chetana Ruangpratheep for being great friends and for putting up
`
`with my antics and personal life brought into the office!
`
`
`
`Also, special thanks to: Karen Kulbicki for always making me smile; Nathalie Zahra
`
`for sticking by me through highs and lows; the Maria Tilton ‘Hope’ Foundation, Cancer
`
`Research UK, and Breast Cancer Campaign; Joan in Finance for administrative
`
`assistance; Jim Strupish for keeping the corridors alive with the sound of whistle; the
`
`Leicester Market for providing the fruit that fuelled the passion for my research;
`
`Theresa Visser, Amy Moore, and Ush Taylor for administrative help on Floor 3;
`
`Malcolm Rae and Sally Munton for purchasing help; Jon Naylor for our refreshing
`
`conversations on computers; all of my former lecturers and friends at the Institute of
`
`Technology in Carlow, Ireland; and a ‘go rabh míle maith agat’ to all of my other Irish
`
`friends back home. I would also like to acknowledge Professors R Charles Coombes
`
`and Justin Stebbing at Imperial College London for being personal role models, and
`
`Barb, Sharon, Anne-Marie, Chris, Rebecca (Becksteropilopidous), and Colette for their
`
`friendship and support.
`
`
`
`Page II
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`Molecular analysis of circulating cell-free DNA in breast cancer patients
`
`
`
`Kevin Anthony Blighe
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`___________________________________________________________________________________
`
`
`
`
`
`Publications relating to this thesis
`
`Page K, Hava N, Ward B, Brown J, Guttery DS, Ruangpratheep C, Blighe K, Sharma
`
`A, Walker RA, Coombes RC, and Shaw JA (April, 2011), Detection of HER2
`
`amplification in circulating free DNA in patients with breast cancer, British
`
`Journal of Cancer 104(8): 1342-1348
`
`
`
`Payne R, Hava N, Page K, Blighe K, Ward B, Slade M, Brown J, Guttery D, Zaidi
`
`SAA, Stebbing J, Jacob J, Tat T, Yagüe E, Shaw JA, and Coombes RC (January,
`
`2012), The presence of disseminated tumour cells in the bone marrow is
`
`inversely related to circulating-free DNA in plasma in breast cancer dormancy,
`
`British Journal of Cancer 106(2): 375-382
`
`
`
`Shaw JA, Page K, Blighe K, Hava N, Guttery D, Ward B, Brown J, Ruangpratheep C,
`
`Stebbing J, Payne R, Palmieri C, Cleator S, Walker RA, and Coombes RC
`
`(February, 2012), Genomic analysis of circulating cell free DNA infers breast
`
`cancer dormancy, Genome Research 22(2): 220-231
`
`
`
`Stebbing J, Filipovic A, Lit LC, Blighe K, Grothey A, Sommer A, Beckmann G,
`
`Yasuhiro M, Chow LWC, Coombes RC, Sasano H, Shaw JA, and Giamas G,
`
`LMTK3 is implicated in intrinsic endocrine resistance via multiple signaling
`
`pathways (manuscript in preparation)
`
`
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`Page III
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`Molecular analysis of circulating cell-free DNA in breast cancer patients
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`
`
`Kevin Anthony Blighe
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`___________________________________________________________________________________
`
`
`
`
`
`Abbreviations
`
`Acronym
`A
`aCGH
`ACS
`ADHD
`ANOVA
`ASD
`BCAS3
`BCSC
`BD
`BIDD
`
`BLAST
`bp
`BRLMM
`cDNA
`cfDNA
`CEPH
`
`CHB
`CIN
`CQC
`CO2
`CN
`CNA
`CN-LOH
`CNV
`COSMIC
`cov
`CRUK
`Cт
`CTC
`DBC1
`DCIS
`DGV
`DIRC3
`DLC1
`DTC
`DMEM
`e
`ECM
`ECMC
`EDTA
`EGFR
`EM
`EMT
`ER
`
`Definition
`Eigenvector
`Array comparative genomic hybridisation
`American Cancer Society
`Attention deficit hyperactivity disorder
`Analysis of variance
`Autism spectrum disorder
`Breast carcinoma amplified sequence 3
`Breast Cancer Surveillance Consortium
`Benign breast disease
`Biomarkers and Imaging Discovery and
`Development
`Basic Local Alignment Search Tool
`Base-pair
`Bayesian robust linear model with mahalanobis
`Complementary DNA
`Cell-free DNA
`Utah, USA residents with ancestry from northern
`and western Europe
`Han Chinese in Beijing, China
`Chromosomal instability
`Contrast QC
`Carbon dioxide
`Copy number
`Copy number alteration
`Copy neutral-LOH
`Copy number variation
`Catalogue of Somatic Mutations in Cancer
`Covariance
`Cancer Research UK
`Cycle threshold
`Circulating tumour cell
`Deleted in bladder cancer 1
`Ductal carcinoma in-situ
`Database of genomic variants
`Disrupted in renal carcinoma
`Deleted in liver cancer 1
`Disseminated tumour cell
`Dulbecco's Modified Eagle Medium
`Eigenvalue
`Extracellular matrix
`Experimental Cancer Medicine Centre
`Ethylenediaminetetraacetic acid
`Epidermal growth factor receptor
`Expectation maximisation
`Epithelial-to-mesenchymal transition
`Oestrogen receptor
`
`
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`___________________________________________________________________________________
`
`ERBB2
`ESR1
`FAM
`FDA
`FDR
`FGF
`FGFR
`FISH
`FRS2
`g
`GTC
`H&E
`hESC
`HMM
`HR
`HRT
`htSNP
`IARC
`IGF1
`IGFBP3
`IGFR
`IHC
`HSP
`JPT
`Kb
`KDa
`L
`LCIS
`LD
`LH
`LHRH
`LOH
`Lowess
`LR
`M
`MAPD
`
`MAPK
`Mb
`mGy
`ml
`MM
`MRD
`MRI
`MTA1
`NaCl
`NAHR
`NAIP
`NCBI
`NCI
`NeoCEnT
`
`HER-2/neu
`Oestrogen receptor 1-α
`Family with sequence similarity
`Food and Drug Administration
`False discovery rate
`Fibroblast growth factor
`Fibroblast growth factor receptor
`Fluorescent in situ hybridisation
`Fibroblast growth factor receptor substrate 2
`Gram
`Genotyping Console
`Haematoxylin and Eosin
`Human embryonic stem cell
`Hidden Markov model
`High risk
`Hormone replacement therapy
`Haplotype-tagging SNP
`International Agency for Research on Cancer
`Insulin-like growth factor 1
`IGF-binding protein-3
`Insulin-like growth factor receptor
`Immunohistochemistry
`Heat-shock protein
`Japanese in Tokyo, Japan
`Kilobase
`KiloDalton
`Litre
`Lobular carcinoma in-situ
`Linkage disequilibrium
`Luteinising hormone
`Luteinising hormone releasing hormone
`Loss of heterozygosity
`Locally-weighted linear regression
`Low risk
`Molar
`Median of the absolute values of all pairwise
`differences
`Mitogen activated protein kinase
`Megabase
`MilliGray
`Millilitre
`Mismatch
`Minimal residual disease
`Magnetic resonance imaging
`Metastasis associated 1
`Sodium chloride
`Non-allelic homologous recombination
`Neuronal apoptosis inhibitory protein
`National Centre for Biotechnology Information
`National Cancer Institute
`Neo-adjuvant chemo- or endocrine therapy
`
`
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`Kevin Anthony Blighe
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`___________________________________________________________________________________
`
`ng
`NGS
`NHS
`NHSBSP
`nm
`NTN1
`OMIM
`PAM
`PCA
`P/C/IAA
`pH
`PI3K
`pM
`PM
`PR
`PRAME
`QC
`qPCR
`REC
`ROC
`RPIA
`rpm
`RPMI
`RQ
`RS
`SAP
`
`SDS
`SERM
`SNP
`TERT
`UCSC
`µl
`var
`VNTR
`WGA
`YRI
`σ
`φ29
`°C
`
`X
`
`
`Nanogram
`Next-generation sequencing
`National Health Service
`NHS Breast Screening Programme
`Nanometre
`Netrin-1
`Online Mendelian Inheritance in Man
`Partitioning around means
`Principal component analysis
`Phenol/chloroform/isoamylalcohol
`Power of the [hydrogen ion]
`Phosphoinositide kinase-3
`Picomolar
`Perfect match
`Progesterone receptor
`Preferentially expressed antigen in melanoma
`Quality control
`Quantitative PCR
`Research Ethics Committee
`Receiver operating characteristic
`Ribose 5-phosphate isomerase A
`Revolutions per minute
`Roswell Park Memorial Institute
`Relative quantitation
`Recurrence score
`Application of SNP arrays (S) to monitor the post-
`adjuvant (A) period (P)
`Sodium dodecyl sulphate
`Selective estrogen receptor modulator
`Single nucleotide polymorphism
`Telomerase reverse transcriptase
`University of California Santa Cruz
`Microlitre
`variance
`Variable number of tandem repeats
`Whole genome amplification
`Yoruba in Ibadan, Nigeria
`Standard deviation
`Phi29
`Degrees Celsius
`Mean
`
`
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`Molecular analysis of circulating cell-free DNA in breast cancer patients
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`
`
`Kevin Anthony Blighe
`
`___________________________________________________________________________________
`
`
`
`
`
`Table of contents
`
`ABSTRACT .................................................................................................................. I
`
`ACKNOWLEDGEMENTS ....................................................................................... II
`
`PUBLICATIONS RELATING TO THIS THESIS ............................................... III
`
`ABBREVIATIONS ................................................................................................... IV
`
`TABLE OF CONTENTS ........................................................................................ VII
`
`INDEX OF FIGURES ............................................................................................. XV
`
`INDEX OF TABLES ............................................................................................ XXII
`
`1
`
`INTRODUCTION ............................................................................................... 1
`
`1.1
`
`1.2
`
`THE BREAST ....................................................................................................... 2
`
`BREAST CANCER: HISTORY AND CURRENT RESEARCH FOCUS ............................ 3
`
`1.2.1
`
`Incidence ..................................................................................................... 3
`
`1.2.1.1
`
`Future trends ........................................................................................ 5
`
`1.2.2
`
`Types of breast cancer ................................................................................ 6
`
`1.2.3 Morphological and molecular factors ......................................................... 7
`
`1.2.4 Risk factors ................................................................................................. 8
`
`1.2.5 Detection and screening ............................................................................ 10
`
`1.2.5.1
`
`Breast mammography ........................................................................ 10
`
`1.2.5.2
`
`Therapy .............................................................................................. 12
`
`1.3 MOLECULAR PROFILING AND PERSONALISED MEDICINE ................................... 13
`
`1.3.1 Breast gene expression subtypes ............................................................... 16
`
`1.3.2 Commercial profiling ................................................................................ 18
`
`1.3.3
`
`Profiling with SNPs and CNV .................................................................. 20
`
`1.3.4 Next-generation sequencing ..................................................................... 21
`
`1.3.5
`
`Potential of DNA arrays ........................................................................... 24
`
`1.3.5.1 Affymetrix microarrays ..................................................................... 25
`
`1.3.5.2
`
`The use of Affymetrix SNP 6.0 .......................................................... 26
`
`1.3.5.2.1 Genotyping calling algorithms ...................................................... 27
`
`
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`Kevin Anthony Blighe
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`___________________________________________________________________________________
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`1.3.5.2.2 Capabilities .................................................................................... 28
`
`1.4
`
`CIRCULATING CELL-FREE DNA ....................................................................... 28
`
`1.4.1
`
`Origins of cfDNA ..................................................................................... 29
`
`1.4.1.1
`
`Tumourigenic origin .......................................................................... 30
`
`1.4.1.2
`
`Other sources of cfDNA .................................................................... 31
`
`1.4.2
`
`Biological importance of cfDNA .............................................................. 33
`
`1.4.2.1
`
`Cell-free DNA as a diagnostic and prognostic marker ...................... 33
`
`1.4.2.2
`
`The search for markers of minimal disease ....................................... 34
`
`1.5
`
`1.6
`
`1.7
`
`1.8
`
`BACKGROUND TO THESIS ................................................................................. 35
`
`HYPOTHESIS TO BE TESTED .............................................................................. 35
`
`AIMS ................................................................................................................ 36
`
`OBJECTIVES ..................................................................................................... 36
`
`2 MATERIALS AND METHODS ...................................................................... 37
`
`2.1
`
`2.2
`
`2.3
`
`2.4
`
`2.5
`
`PROJECT FUNDING AND SPONSORSHIP DETAILS ................................................ 38
`
`ASSOCIATED CLINICAL STUDIES AND TRIALS ................................................... 38
`
`SAMPLE INFORMATION..................................................................................... 40
`
`SAMPLE PROCESSING ....................................................................................... 43
`
`DNA EXTRACTION AND WHOLE GENOME AMPLIFICATION ............................... 43
`
`2.6 MICROARRAY PROCESSING .............................................................................. 44
`
`2.6.1
`
`The scanning process ................................................................................ 45
`
`2.7
`
`BIOINFORMATIC DATA ANALYSIS ..................................................................... 45
`
`2.7.1
`
`Normalisation ............................................................................................ 46
`
`2.7.1.1
`
`Referencing and derivation of log2 values ......................................... 47
`
`2.7.1.2
`
`Variations to normalisation ............................................................... 48
`
`2.7.2
`
`2.7.3
`
`Initial QC .................................................................................................. 49
`
`Genotyping and generation of CN segments ............................................ 49
`
`2.7.3.1
`
`Single nucleotide polymorphism concordance checks ...................... 49
`
`2.7.3.2
`
`Genomic segmentation ...................................................................... 50
`
`2.7.4
`
`Principal component analysis and clustering ............................................ 51
`
`2.7.4.1
`
`Genetic signature of breast cancer ..................................................... 51
`
`2.7.5
`
`2.7.6
`
`Loss of heterozygosity .............................................................................. 52
`
`Copy number variation frequency analysis .............................................. 53
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`___________________________________________________________________________________
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`2.7.7
`
`Genomic aberration detection ................................................................... 53
`
`2.8
`
`2.9
`
`CULTURING CELL-LINES ................................................................................... 54
`
`DESIGN OF PRIMERS AND PROBES ..................................................................... 55
`
`2.10 REAL-TIME QUANTITATIVE PCR ...................................................................... 56
`
`2.11
`
`STATISTICAL ANALYSIS ................................................................................... 57
`
`3 MOLECULAR CHARACTERISATION OF CIRCULATING CELL-FREE
`
`DNA IN BREAST CANCER PATIENTS USING SNP 6.0 MICROARRAYS ... 58
`
`3.1
`
`3.2
`
`3.2.1
`
`3.2.2
`
`3.2.3
`
`OVERVIEW ....................................................................................................... 59
`
`INTRODUCTION ................................................................................................ 59
`
`Genome-wide characterisation ................................................................. 60
`
`Sequential analysis .................................................................................... 60
`
`Aims .......................................................................................................... 61
`
`3.3
`
`RESULTS .......................................................................................................... 62
`
`3.3.1
`
`Reproducibility and sensitivity of qPCR for measuring ERBB2 copy
`
`number in breast cancer cell-lines .......................................................................... 62
`
`3.3.2
`
`Affymetrix SNP 6.0 QC analysis of whole genome amplification cfDNA,
`
`tumour, and lymphocyte DNA samples .................................................................. 69
`
`3.3.3
`
`Single nucleotide polymorphisms and the comparison of genomic profiles
`
`74
`
`3.3.3.1 HapMap comparisons ........................................................................ 74
`
`3.3.3.2 Matched lymphocyte comparisons .................................................... 74
`
`3.3.3.3 Matched tumour comparisons ........................................................... 77
`
`3.3.4 Copy number variation analysis ............................................................... 78
`
`3.3.4.1
`
`Status ................................................................................................. 78
`
`3.3.4.2 Number of CNVs called .................................................................... 78
`
`3.3.4.3 Amplification and deletion ................................................................ 82
`
`3.3.4.3.1 Distribution ................................................................................... 82
`
`3.3.4.3.2 Thresholding amplifications by copy number ............................... 83
`
`3.3.4.3.3 Logistic regression to reveal significant copy number variant
`
`regions
`
`97
`
`3.3.5
`
`Principal component analysis and hierarchical clustering to interrogate
`
`both SNP and CN data ............................................................................................ 99
`
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`Molecular analysis of circulating cell-free DNA in breast cancer patients
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`Kevin Anthony Blighe
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`___________________________________________________________________________________
`
`3.3.5.1
`
`Principal component analysis ............................................................ 99
`
`3.3.5.2
`
`Hierarchical clustering ..................................................................... 108
`
`3.3.6
`
`Loss of heterozygosity analysis .............................................................. 110
`
`3.4
`
`DISCUSSION ................................................................................................... 113
`
`3.4.1
`
`3.4.2
`
`3.4.3
`
`ERBB2 assay validation experiments using breast cell-lines .................. 114
`
`Formalin-fixed Vs fresh frozen tissue .................................................... 114
`
`Single nucleotide polymorphism concordance segregated cfDNA of
`
`healthy controls from breast cancer patients ......................................................... 115
`
`3.4.4
`
`Principal component analysis reveals ethnicity and may predict breast
`
`cancer 116
`
`3.4.5
`
`Copy number variation ........................................................................... 117
`
`3.4.5.1 Many novel CNVs ........................................................................... 118
`
`3.4.5.2
`
`Amplifications and deletions ........................................................... 119
`
`3.4.5.3
`
`Commonly-amplified intervals in plasma cfDNA .......................... 120
`
`3.4.5.4
`
`Receptor status ................................................................................. 121
`
`3.4.5.5
`
`Profiles of relapsed patients ............................................................. 121
`
`3.4.5.6
`
`Surgery and therapy received .......................................................... 122
`
`3.4.6
`
`Genes overlapping commonly-amplified CNVs ..................................... 123
`
`3.4.6.1
`
`4q13.2 .............................................................................................. 123
`
`3.4.6.2
`
`5q13.2 .............................................................................................. 123
`
`3.4.6.3
`
`7q11.23 ............................................................................................ 124
`
`3.4.6.4
`
`8p23.1 .............................................................................................. 124
`
`3.4.6.5
`
`10q11.22 and 10q11.23 ................................................................... 125
`
`3.4.6.6
`
`15q25.2 ............................................................................................ 126
`
`3.4.6.7
`
`16p12.3 ............................................................................................ 126
`
`3.4.6.8
`
`Other CNVs with potential genes of interest ................................... 126
`
`3.4.7
`
`Breast cancer dormancy .......................................................................... 128
`
`3.5
`
`3.6
`
`CONCLUSION ................................................................................................. 130
`
`FUTURE STUDIES ............................................................................................ 131
`
`4 MONITORING CIRCULATING CELL-FREE DNA IN RESPONSE TO
`
`THERAPY IN BREAST CANCER PATIENTS USING SNP 6.0 MICROARRAYS
`
`132
`
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`Molecular analysis of circulating cell-free DNA in breast cancer patients
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`Kevin Anthony Blighe
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`___________________________________________________________________________________
`
`4.1
`
`4.2
`
`4.2.1
`
`4.2.2
`
`4.2.3
`
`4.2.4
`
`OVERVIEW ..................................................................................................... 133
`
`INTRODUCTION .............................................................................................. 133
`
`Cancer therapy ........................................................................................ 133
`
`Resistance to therapy .............................................................................. 134
`
`The NeoCEnT clinical trial ..................................................................... 135
`
`Aims ........................................................................................................ 136
`
`4.3
`
`RESULTS ........................................................................................................ 136
`
`4.3.1
`
`Affymetrix SNP 6.0 QC analysis of cfDNA, tumour, and lymphocyte
`
`samples 136
`
`4.3.2
`
`Single nucleotide polymorphisms and the comparison of genomic profiles
`
`139
`
`4.3.3
`
`Copy number variation analysis ............................................................. 141
`
`4.3.3.1
`
`Number of CNVS called ................................................................. 141
`
`4.3.3.2
`
`Amplifications and deletions ........................................................... 143
`
`4.3.3.2.1 Frequency .................................................................................... 143
`
`4.3.3.2.2 Distribution ................................................................................. 144
`
`4.3.3.2.3 Thresholding amplifications by copy number ............................. 145
`
`4.3.3.2.4 Logistic regression to reveal significant copy number variant
`
`regions
`
`149
`
`4.3.3.3
`
`Copy number variation in growth factor receptor genes ................. 150
`
`4.3.3.4
`
`Detection of genomic aberrations .................................................... 151
`
`4.3.4
`
`Principal component analysis to interrogate both SNP and CN data ..... 155
`
`4.4
`
`DISCUSSION ................................................................................................... 158
`
`4.4.1 Microarray detects genomic aberrations ................................................. 158
`
`4.4.2
`
`Single nucleotide polymorphism/copy number variation changes with time
`
`and therapy ............................................................................................................ 159
`
`4.4.2.1
`
`Single nucleotide polymorphism concordance checks give mixed
`
`results
`
`159
`
`4.4.2.2
`
`Plasma cfDNA CNV profile becomes less heterogeneous .............. 161
`
`4.4.2.3
`
`Principal component analysis indicates subtle changes in the plasma
`
`cfDNA profile ................................................................................................... 161
`
`4.4.2.4
`
`Plasma cfDNA CNVs become less prevalent ................................. 162
`
`4.4.3
`
`Genes overlapping commonly-amplified CNVs ..................................... 163
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`Molecular analysis of circulating cell-free DNA in breast cancer patients
`
`Kevin Anthony Blighe
`
`___________________________________________________________________________________
`
`4.4.4
`
`Affymetrix SNP 6.0 reproducibility ......................................................... 165
`
`4.5
`
`4.6
`
`CONCLUSION ................................................................................................. 165
`
`FUTURE STUDIES ............................................................................................ 166
`
`5
`
`PAINTING A PORTRAIT OF BREAST CANCER THROUGH PRINCIPAL
`
`COMPONENT ANALYSIS .................................................................................... 167
`
`5.1
`
`5.2
`
`OVERVIEW ..................................................................................................... 168
`
`INTRODUCTION .............................................................................................. 168
`
`5.2.1
`
`Principal component analysis as a tool for analysing large bodies of data
`
`168
`
`5.2.1.1
`
`Limitations of PCA .......................................................................... 169
`
`5.2.2 Deriving a genetic ‘signature’ ................................................................. 169
`
`5.2.3 Aims ........................................................................................................ 171
`
`5.3
`
`RESULTS ........................................................................................................ 171
`
`5.3.1 Genes with known roles in breast cancer ................................................ 171
`
`5.3.1.1 Markers overlapping exons ............................................................. 179
`
`5.3.1.2 Markers overlapping introns ............................................................ 180
`
`5.3.1.2.1 Breast cancer patients on follow-up ............................................ 180
`
`5.3.1.2.2 Relapsed breast cancer patients on follow-up ............................. 180
`
`5.3.1.2.3 Unaffected BRCA1 mutation carriers ......................................... 182
`
`5.3.1.2.4 Pre-surgical breast cancer patients on neoadjuvant therapy ....... 183
`
`5.3.2
`
`Towards a breast cancer signature through cfDNA analysis .................. 184
`
`5.4
`
`DISCUSSION ................................................................................................... 189
`
`5.4.1
`
`5.4.2
`
`Exons were infrequently affected by variation ....................................... 190
`
`Principal component analysis revealed numerous existing and novel gene
`
`targets of oncogenic potential ............................................................................... 191
`
`5.4.2.1
`
`Patients on follow-up ....................................................................... 192
`
`5.4.2.1.1 Lymphocyte Vs Tumour DNA.................................................... 192
`
`5.4.2.1.2 T1N0 (T≤20mm) Vs T>20mm/node-positive (first and second
`
`cfDNA) 194
`
`5.4.2.2
`
`Relapsed patients’ final cfDNA samples ......................................... 195
`
`5.4.2.2.1 First Vs Final/second cfDNA ...................................................... 195
`
`5.4.2.2.2 Tumour DNA Vs Final cfDNA ................................................... 196
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`Molecular analysis of circulating cell-free DNA in breast cancer patients
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`Kevin Anthony Blighe
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`___________________________________________________________________________________
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`5.4.2.2.3 Non-relapsed T1N0 (T≤20mm) Vs Relapsed T1N0 (T≤20mm)
`
`final cfDNA .................................................................................................. 197
`
`5.4.2.2.4 Non-relapsed T>20mm/node-positive Vs Relapsed T>20mm/node-
`
`positive final cfDNA ..................................................................................... 198
`
`5.4.2.3
`
`Healthy control Vs Unaffected BRCA1 carriers’ cfDNA ................ 199
`
`5.4.2.4
`
`Pre-surgical patients’ cfDNA .......................................................... 199
`
`5.4.3
`
`5.4.4
`
`5.4.5
`
`ER receptor activity is altered in tumour and cfDNA ............................ 200
`
`SNP is more conducive to variation than CNV ...................................... 202
`
`Principal component analysis creates breast cancer cfDNA genetic
`
`signatures .............................................................................................................. 203
`
`5.4.5.1
`
`Genetic signatures are highly defined and narrow .......................... 204
`
`5.5
`
`5.6
`
`CONCLUSION .................