Chromosomal radiosensitivity and instability in triple negative and/or young breast cancer and Fanconi Anaemia patients in South Africa
Date
2018
Authors
Francies, Flavia Zita
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Abstract
Introduction: Breast cancer is the leading cancer in women in South Africa (SA). Triple
negative breast cancer (TNBC) is clinically characterised by the lack of expression of
estrogen, progesterone and HER2/NEU receptors. These breast cancers occur frequently in
young African women and are associated with aggressive disease progression, poor prognosis
and BRCA1 mutations. TN patients with operable tumours may undergo surgery under
general anaesthetics. Treatment of TNBC poses a clinical challenge as these tumours are
unresponsive to hormonal or HER2 targeted therapy. Defects in BRCA1 and other DNA repair
genes contribute to chromosomal instability and radiosensitivity and cause irregularities in the
cell cycle checkpoints in the S/G2 phase.
Studies have shown the overlap of breast cancer susceptibility genes and Fanconi Anaemia
(FA) genes. FA is an autosomal recessive disorder defined by cellular hypersensitivity to
DNA cross-linking agents such as mitomycin C (MMC) and defects in DNA repair genes. FA
patients are known to be radiosensitive and have defects with DNA repair. These patients are
at high risk to develop leukaemia and solid tumours that may require radiotherapy. Diagnosis
of FA patients often includes detecting chromosomal aberrations induced by a cross-linking
agent. Molecular tests are also conducted to identify mutations in FA genes. It has previously
been shown that FA patients undergoing radiotherapy display increased clinical
radiosensitivity. Evidence suggests that FA patients are chromosomally radiosensitive to
ionising radiation (IR).
Chromosomal radiosensitivity can be evaluated using the cytokinesis-block micronucleus
(CBMN) assay in different phases of the cell cycle. Micronuclei (MNi) serve as biomarkers
for radiation-induced DNA damage repair and defects in DNA repair mechanisms can be
reflected in chromosomal radiosensitivity. A number of factors could influence the MNi yield
such as storage time and temperature, and cytotoxic agents such as anaesthetics. As
radiotherapy is considered a principle treatment in the management of TNBC, it is important
to investigate in vitro chromosomal radiosensitivity of South African TN breast cancer
patients. Chromosomal instability and radiosensitivity of FA patients has previously not been
investigated in SA. The overall aim of this study was to investigate chromosomal instability
and radiosensitivity of lymphocytes in South African breast cancer patients, FA patients and
parents compared to healthy individuals using the G0 and S/G2 CBMN assay. The effect of
age, ethnicity and mutations in breast cancer susceptibility genes was also investigated.
Furthermore, storage time and effect of anaesthetics on MNi yield was investigated.
Methods: For the G0 MN assay, heparinised blood in culture medium was irradiated at 0Gy
(Baseline), 2 and 4 Gy followed by the immediate stimulation of lymphocytes using
phytohaemagglutinin (PHA). Cytochalasin B was added 23 hours later to inhibit cell division.
The S/G2 MN assay is a modified version of the G0 MN assay. In this assay, the cultures are
first stimulated with PHA and irradiated 72 hours post stimulation. Eight hours post
irradiation cells were fixed. The Mitomycin C (MMC) MN assay is similar to the G0 MN
assay except the DNA damage is induced using MMC.
Results: Chromosomal instability is significantly elevated in TNBC, young and older breast
cancer patients. Radiation-induced MN values in the G0 MN assay are significantly enhanced
in a total unselected group of breast cancer patients compared to healthy individuals.
However, when subdividing the breast cancer patients in a TNBC group, the enhanced
radiation-induced MNi are not observed. We cannot demonstrate a correlation between the
age of the patients and chromosomal radiosensitivity but an effect of ethnicity is noted in our
breast cancer population. In the S/G2 MN assay, TNBC patients continued to exhibit a
decreased chromosomal radiosensitivity. We also demonstrated that increased storage time
can influence MNi yields in patients and controls; anaesthetics influenced spontaneous MNi
yields.
The FA patients in our study demonstrate higher MNi when compared to parents and controls
indicating chromosomal instability and chromosomal radiosensitivity in the G0 as well as in
the S/G2 phase of the cell cycle. This is not seen in the FA heterozygotes. With the MMC
assay, the detection of significantly higher MN is noted in as well the FA patients as well as
the FA carriers.
Conclusions: Chromosomal instability and radiosensitivity of breast cancer and FA patients
are notably higher when compared to healthy individuals. The association of BRCA
mutations in TN and young patients highlight the importance of radiosensitivity information
in the understudied SA population. FA carriers can be at risk for breast cancer with mutations
associated with breast cancer susceptibility genes. As a functional assay, the MMC MN assay
will be useful in the identification of FA carriers who may be at risk of breast cancer. Data on
radiosensitivity of patients with defects in DNA repair genes could provide important
information for radiotherapy management of cancer.
Description
A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand,
Johannesburg, and the Faculty of Medicine and Health Sciences, Ghent University,
Belgium in fulfilment of the requirements for the joint degree of
Doctor of Philosophy / Doctor in Health Sciences
Johannesburg, 2018.