Biological characterisation of HER2 amplified breast cancer. PhD thesis, University of N

336 Pages·2017·6.67 MB·English

by  Christine Middleton

Biological
Characterisation of
HER2 amplified breast
cancer

Fabrício Félix Tabuada Barros
(Eng, MSc Biochemistry)

Thesis Submitted to the University of Nottingham
for the Degree of Doctor Philosophy

March 2013
ABSTRACT

Breast carcinoma is the most frequent type of cancer affecting women. Among
the recently described molecular and phenotypic classes of breast cancer,
human epidermal growth factor receptor 2 (HER2)-positive tumours are
associated with a poor prognosis. HER2 status is currently assessed in routine
breast cancer reporting using immunohistochemistry (IHC) in addition to in
situ hybridisation (ISH) in borderline cases. The ability of HER2 gene status to
predict response to targeted therapy (Trastuzumab) is well documented.
However, prognostic information provided by IHC expression categories and
prognostic value added by using ISH in borderline cases remains
unclear. HER2 plays an important role in cancer progression being targeted to
provide predictive and prognostic information. Moreover, HER2 is related to
cancer resistance against a variety of therapies; however, trastuzumab has
proved successful in treatment of this subgroup. Nevertheless, patients may
acquire resistance to this drug after a period of treatment, which indicates that
other molecular mechanisms might influence success of this therapy.
Dimerisation between members of the HER family may contribute to
resistance against treatments due to different combinations that trigger different
downstream pathways. This is promoted by ligands, which are expressed as
transmembrane precursor protein molecules and have a conserved epidermal
growth factor-like domain. Through resistance to trastuzumab, other drugs are
being developed to interact in different domains of HER2 protein. The study of
interaction between receptors/ligands will characterise specifically their
signalling pathway and understand which strategy to acquire.
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The main aim of this thesis was to assesses the status of HER2 protein (IHC),
HER2 gene (chromogenic ISH) and HER heterodimers (in situ proximity
ligation assay (PLA)), including HER2/EGFR, HER2/HER3 and HER2/HER4,
in two BC series prepared in tissue microarray format; a series of consecutive
primary operable BC cases (n = 1858) including HER2+ trastuzumab naïve
cases (TrN, n = 221), the second series of HER2+ trastuzumab adjuvant treated
cases (TrT, n = 143). Therefore determining the biological characterisation of
these biomarkers by associating against clinicopathological parameters,
survival outcome and understand the trastuzumab therapy value.

There was excellent overall concordance between HercepTest negative (scores
0/1+) and positive (3+) with CISH positive/negative (defined as HER2/Chr17
copy number ratio of ≥ 2; p < 0.001). Kaplan-Meier analysis for breast cancer
specific survival (BCSS) and disease free interval (DFI) revealed statistically
significant differences between HER2 positive and negative cases detected by
HercepTest and CISH (p < 0.001). Interestingly, it was identified that
HercepTest 2+ non-amplified cases were not significantly different with those
amplified 2+ or 3+ cases with respect to their behaviour (BCSS and DFI).

The results revealed an inverse association between the HER
heterodimerisation status and hormone receptor status (p < 0.001), and a
significantly worse outcome amongst cases revealing high levels of all
heterodimers (p < 0.001). Among ER+ cases, the heterodimers high levels
were significantly associated with worse prognosis (p < 0.001) overall.
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However amongst the two HER2+ populations dimerisation status did not
show an association with patient outcome.

The overall concordance between HercepTest and CISH analysis for HER2
status was excellent. All HercepTest 2+ cases identified were observed to have
poor outcomes similar to those HercepTest 3+ cases regardless of gene
amplification status. In the current clinical environment cases exhibiting IHC
2+ and non-amplified gene HER2 status will not be offered targeted HER2
therapy but do exhibit aggressive clinical behavioural characteristics. Even
though those patients with high levels of the HER2 truncated form, p95HER2,
have shown poor outcome, this biomarker does not reveal any extra findings
comparing with the HER2 expression results. Beside HER2, EGFR is the only
monomer that reveals prognostic value amongst the breast cancer patients.
Tumours exhibiting high levels of HER heterodimerisation have an adverse
prognosis, however in the context of HER2+ breast cancer no association with
clinical outcome was observed regardless of use of trastuzumab treatment.
HER2/HER3 heterodimerisation status assessed in multivariate analyses has
shown that this protein-protein interaction is associated with a poor prognostic
outcome, which needs further investigation and assessment of clinical utility to
use in the future in breast cancer treatment decision. Further quantification
analysis of dimer/ligand complex using PLA of other HER family members
may be useful to identify subset of patients associated with distinct outcome,
response to treatment and relationships with HER signalling related
biomarkers.

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PUBLICATIONS

Peer Reviewed Publication:

Barros FFT, Powe DG, Ellis IO, Green AR, Understanding the HER family in
breast cancer: interaction with ligands, dimerisation and treatments,
Histopathology, Vol:56, 560-572, doi:10.1111/j.1365-2559.2010.03494.x,
(2010)

Storr SJ, Woolston CM, Barros FFT, Green AR, Shehata M, Chan SY, Ellis
IO, Martin SG, Calpain-1 expression is associated with relapse-free survival in
breast cancer patients treated with trastuzumab following adjuvant
chemotherapy, International Journal of Cancer, Vol:129, 1773-1780,
DOI: 10.1002/ijc.25832, (2011)

Aleskandarany MA, Green AR, Benhasouna AA, Barros FFT, Neal KR, Reis-
Filho JS, Ellis IO, Rakha EA, Prognostic value of proliferation assay in the
luminal, HER2 positive and triple negative biological classes of breast cancer,
Breast Cancer Research, Vol:14, doi:10.1186/bcr3084, (2012)

Publications in preparation:
Barros FFT, Al-Fatah T, Moseley P, Nolan CC, Durham A, Rakha EA, Chan
S, Ellis IO, Green AR, Characterisation of HER Heterodimers in Breast Cancer
Using in situ Proximity Ligation Assay, (2012)

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Barros FFT, Al-Fatah T, Moseley P, Nolan CC, Durham A, Rakha EA, Chan
S, Ellis IO, Green AR, Understanding HER family heterodimers and respective
ligands on Breast Cancer Prognosis, (2012)

Oral Presentation:
Barros FFT, Al-Fatah T, Moseley P, Nolan CC, Durham A, Rakha EA, Chan
S, Ellis IO, Green AR. Quantification of HER2/HER3 Dimerisation Status in
Primary Invasive Breast Cancer. 202nd Scientific Meeting of the Pathological
Society of Great Britain & Ireland, 3-5 July 2012, University of Sheffield, UK.

Barros FFT, Al-Fatah T, Moseley P, Nolan CC, Durham A, Rakha EA, Chan
S, Ellis IO, Green AR. In situ Proximity Ligation Assay (PLA) as a Novel
Approach on Breast Cancer Research, Euroscicon, 2012 Histopathology Event:
Advances in research and techniques, London, UK.

Barros FFT, Powe DG, Ellis IO, Green AR. Borderline HER2 Protein Positive
Breast Cancers Have a Similar Patient Outcome Regardless of HER2 Gene
Amplification Status, 1st British Breast Cancer Research Conference,
University of Nottingham, UK.

Conference Poster Presentations:
Barros FFT, Powe DG, Ellis IO, Green AR. Borderline HER2 Protein Positive
Breast Cancers Have Similar Patient Outcome Regardless of HER2 gene
Amplification Status, 198th Scientific Meeting of the Pathological Society of
Great Britain & Ireland 2010, University of St Adrews, UK.
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Barros FFT, Powe DG, Ellis IO, Green AR. Borderline HER2 Protein Positive
Breast Cancers Have Similar Patient Outcome Regardless of HER2 gene
Amplification Status, NCRI – Cancer Conference 2009, The International
Convention Centre, Birmingham, UK.

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Acknowledgement

Taking this PhD project changed my life and I would never be able to complete
without the support and supervision that I had from a colossal list of people.

First of all I would like to thank Professor Ian Ellis and Dr Andrew Green for
their supervision and the intensive support and encouragement they gave me.
Without their continuous guidance this project would never be achieved.
Additionally I would like to thank Chris Nolan and Catherine Morgan that
were a crucial when they sacrificed their work time to help me. I also have to
thank my examiners Dr James Going and Dr Ian Spendlove for their
suggestions in general.

I am grateful to all my friends in Nottingham who were at all times so helpful
in numerous ways even if they did not understand a single idea of my PhD.
Special thanks to Renze Lowrie (the adviser), Nyary Tamas (the supporter),
Zolta Genda (my friend’s boyfriend) and Claire Michel (la mademoiselle).

A very exceptional thank to Shanow Uthman (the friend) and Eva Jordan (the
lazy) for their priceless advices and understanding all that I was felling,
especially because they were living the same challenge of completing a PhD.

Also I have to thank the very special friends that were far (Holland, Spain and
Portugal) from me but that know me better than no one and always made me
forget for a while my responsibilities and just enjoy life. Big thanks to Ana
Roque (the blond), Eugenia Silva (the funniest), Claúdia Jesus (the nicest),
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Gonçalo Silva (the coolest), Celine Xavier (the patient), Anselmo Fernandes
(the clown), Juan Pereira (the friend), Liliana Pereira and kids (the punk) and
Liliana Cunha (the lady).

And finally the most important support, which has been always on my side
during this marathon in every single minute, I have to thank David Richards
(the best) for not quitting on me and for standing all the bad times next to me.
When I felt desperate and alone while I was writing during those long days,
someone by just jumping to my lap and cuddle a bit, use to make me feel
better, Mika (THE Cat).

Finally I could never forget to thank those two that without them my life would
be completely different and I would never complete a PhD or I would never be
the person that I am now. A massive thank to Félix de Barros (THE DAD) and
Maria da Conceição Tabuada Barros (THE MUM).

I dedicate this work to my grandmother, Ana Videira (THE Nan), which
passed away recently and I always looked at her as my second mother.

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ABBREVIATIONS

ADCC Antibody Dependent Cell Cytotoxicity
Ang-1 Angiopoietin-1
AP-1 Activator Protein 1
AREG Amphiregulin
ASCO American Society of Clinical Oncology
ASK1 Apoptosis Signal Regulating Kinase 1
ATP Adenosine Triphosphate
Bcl-2 B Cell Lymphoma 2
BCSS Breast Cancer Specific Survival
BP Basal Phenotype
BRCA1 Breast Cancer 1
BTC Betacellulin
CAF Cyclophosphamide, Adriamycin and Fluorouracil
CAP College of American Pathologists
CDK Cyclin Dependent Kinase
CGH Comparative Genomic Hybridisation
CISH Chromogenic in situ Hybridisation
CK Cytokeratin
CMF Cyclophosphamide containing regime
CTFs Carboxy Terminal Fragments
DC Dendritic Cell
DFI Disease Free Interval
DNA Deoxyribonucleic Acid
ECD Ectodomain
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