Chromosomal radiosensitivity and instability in triple negative and/or young breast cancer and Fanconi Anaemia patients in South Africa

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.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.LG201

Aberrant splicing events and epigenetics in viral oncogenomics : current therapeutic strategies

Cancer is a global burden and is the second leading cause of mortality. It is largely a non-communicable disease attributable to the accumulation of damaged DNA and deleterious mutations in vital genes caused by exposure to carcinogens. Besides, viruses with oncogenic potential are also known to cause cancer through infections. Approximately, 12–20% of all cancers have a viral aetiology. Oncovirus infections are potentially modifiable risk factors, and targeting infections can be useful in prevention measures. In 2018, the global cancer cases attributable to infections were estimated to be 2.2 million. The International Agency for Research on Cancer (IARC) has identified seven different cancer-causing viruses namely the Human papillomaviruses (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Epstein–Barr virus (EBV), Human T cell leukaemia virus 1 (HTLV-1), Kaposi’s Sarcoma Herpesvirus (KSHV) and Human immunodeficiency virus 1 (HIV-1). HIV-1 contributes to cancer development through immunosuppression by permitting the co-infection of other oncogenic viruses. With the exception of KSHV, the IARC classified these viruses as group one human carcinogens and further categorised these based on the viral genome as DNA viruses or RNA viruses. HPV, HBV and HCV are major contributors to cancers associated with viral infections, and the number of cases varies based on geographic locations. In 2018, Eastern Asia had the highest number of infection-related cancer, with 37.9 cases per 100,000 person-years, closely followed by Sub-Saharan Africa (SSA) with 33.1 cases per 100,000 person-years. A number of these infection-related cancers can be prevented with effective infection control through available vaccines, awareness and understanding of the risk factors.The Medical Research Council of South Africahttp://www.mdpi.com/journal/cellspm2021Medical Virolog

Chromosomal radiosensitivity of human immunodeficiency virus positive/negative cervical cancer patients in South Africa

Cervical cancer is the second most common cancer amongst South African women and is the leading cause of cancer-associated mortality in this region. Several international studies on radiation-induced DNA damage in lymphocytes of cervical cancer patients have remained inconclusive. Despite the high incidence of cervical cancer in South Africa, and the extensive use of radiotherapy to treat it, the chromosomal radiosensitivity of South African cervical cancer patients has not been studied to date. Since a high number of these patients are human immunodeficiency virus (HIV)-positive, the effect of HIV infection on chromosomal radiosensitivity was also investigated. Blood samples from 35 cervical cancer patients (20 HIV-negative and 15 HIV-positive) and 20 healthy controls were exposed to X-rays at doses of 6 MV of 2 and 4 Gy in vitro. Chromosomal radiosensitivity was assessed using the micronucleus (MN) assay. MN scores were obtained using the Metafer 4 platform, an automated microscopic system. Three scoring methods of the MNScore module of Metafer were applied and compared. Cervical cancer patients had higher MN values than healthy controls, with HIV-positive patients having the highest MN values. Differences between groups were significant when using a scoring method that corrects for false positive and false negative MN. The present study suggested increased chromosomal radiosensitivity in HIV-positive South African cervical cancer patients

Artificial intelligence (AI) and big data in cancer and precision oncology

Artificial intelligence (AI) and machine learning have significantly influenced many facets of the healthcare sector. Advancement in technology has paved the way for analysis of big datasets in a cost- and time-effective manner. Clinical oncology and research are reaping the benefits of AI. The burden of cancer is a global phenomenon. Efforts to reduce mortality rates requires early diagnosis for effective therapeutic interventions. However, metastatic and recurrent cancers evolve and acquire drug resistance. It is imperative to detect novel biomarkers that induce drug resistance and identify therapeutic targets to enhance treatment regimes. The introduction of the next generation sequencing (NGS) platforms address these demands, has revolutionised the future of precision oncology. NGS offers several clinical applications that are important for risk predictor, early detection of disease, diagnosis by sequencing and medical imaging, accurate prognosis, biomarker identification and identification of therapeutic targets for novel drug discovery. NGS generates large datasets that demand specialised bioinformatics resources to analyse the data that is relevant and clinically significant. Through these applications of AI, cancer diagnostics and prognostic prediction are enhanced with NGS and medical imaging that delivers high resolution images. Regardless of the improvements in technology, AI has some challenges and limitations, and the clinical application of NGS remains to be validated. By continuing to enhance the progression of innovation and technology, the future of AI and precision oncology show great promise.The South African Medical Research Council (SAMRC)http://www.elsevier.com/locate/csbjam2020Obstetrics and Gynaecolog

Genomics and splicing events of type II endometrial cancers in the black population : racial disparity, socioeconomic and geographical differences

Endometrial cancer, also known as uterine cancer, is the most common gynaecological malignancy with burgeoning incidence and mortality rates globally. Racial disparity, socioeconomic and geographical differences are important determinants of endometrial cancer incidence and mortality. Endometrial cancer is mainly categorised as type I and type II. Although less prevalent, type II is the most aggressive form of the disease and typically diagnosed at a late stage, contributing to higher mortality. Black women are at higher risk of developing aggressive, type II disease. Type I tumours are related to higher levels of circulating estrogen with lower-grade tumours that have a good prognosis and frequently related to PTEN mutations. In comparison, type II tumours are estrogen-independent, typically have poor prognosis and associated with the p53, HER2, PPP2R1A, FBXW7 and PIK3R1 mutations. The risk of developing type II malignancy is higher in women with Lynch syndrome as a result of mutations in the MMR gene family. Genetic modifications contribute to aberrant alternative splicing events that are related to tumour development, progression and resistance to therapy. Alternative splicing events are rapidly emerging as potential biomarkers and therapeutic targets. Type II endometrial cancer lacks targeted therapy and biomarkers for novel therapeutic strategies. Recent advances have illustrated a number of molecular targets that are currently explored for the treatment of advanced, late-stage endometrial cancer. The aim of this review is to outline 1) the epidemiology of type II endometrial cancer in black women, 2) discuss the correlated risk factors that contribute to the development of type II endometrial cancer and 3) the associated molecular mechanisms and genetic factors underlying the disease, and 4) aberrant splicing events and biomarkers with therapeutic potential as novel drug targets.The Medical Research Council of South Africa (SAMRC)http://www.ajcr.ushj2021Medical Oncolog

MicroRNA and alternative mRNA splicing events in cancer drug response/resistance : potent therapeutic targets

Cancer is a multifaceted disease that involves several molecular mechanisms including changes in gene expression. Two important processes altered in cancer that lead to changes in gene expression include altered microRNA (miRNA) expression and aberrant splicing events. MiRNAs are short non-coding RNAs that play a central role in regulating RNA silencing and gene expression. Alternative splicing increases the diversity of the proteome by producing several different spliced mRNAs from a single gene for translation. MiRNA expression and alternative splicing events are rigorously regulated processes. Dysregulation of miRNA and splicing events promote carcinogenesis and drug resistance in cancers including breast, cervical, prostate, colorectal, ovarian and leukemia. Alternative splicing may change the target mRNA 30UTR binding site. This alteration can affect the produced protein and may ultimately affect the drug affinity of target proteins, eventually leading to drug resistance. Drug resistance can be caused by intrinsic and extrinsic factors. The interplay between miRNA and alternative splicing is largely due to splicing resulting in altered 30UTR targeted binding of miRNAs. This can result in the altered targeting of these isoforms and altered drug targets and drug resistance. Furthermore, the increasing prevalence of cancer drug resistance poses a substantial challenge in the management of the disease. Henceforth, molecular alterations have become highly attractive drug targets to reverse the aberrant effects of miRNAs and splicing events that promote malignancy and drug resistance. While the miRNA–mRNA splicing interplay in cancer drug resistance remains largely to be elucidated, this review focuses on miRNA and alternative mRNA splicing (AS) events in breast, cervical, prostate, colorectal and ovarian cancer, as well as leukemia, and the role these events play in drug resistance. MiRNA induced cancer drug resistance; alternative mRNA splicing (AS) in cancer drug resistance; the interplay between AS and miRNA in chemoresistance will be discussed. Despite this great potential, the interplay between aberrant splicing events and miRNA is understudied but holds great potential in deciphering miRNA-mediated drug resistance.This research was funded by the South African Medical Research Council (SAMRC).The South African Medical Research Council (SAMRC)https://www.mdpi.com/journal/biomedicinesam2022Maxillo-Facial and Oral SurgeryMedical OncologySurger

Aberrant Splicing Events and Epigenetics in Viral Oncogenomics: Current Therapeutic Strategies

Global cancer incidence and mortality are on the rise. Although cancer is fundamentally a non-communicable disease, a large number of cancers are known to have a viral aetiology. A high burden of infectious agents (Human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis B virus (HBV)) in certain Sub-Saharan African countries drives the rates of certain cancers. About one-third of all cancers in Africa are attributed to infection. Seven viruses have been identified with carcinogenic characteristics, namely the HPV, HBV, Hepatitis C virus (HCV), Epstein–Barr virus (EBV), Human T cell leukaemia virus 1 (HTLV-1), Kaposi’s Sarcoma Herpesvirus (KSHV), and HIV-1. The cellular splicing machinery is compromised upon infection, and the virus generates splicing variants that promote cell proliferation, suppress signalling pathways, inhibition of tumour suppressors, alter gene expression through epigenetic modification, and mechanisms to evade an immune response, promoting carcinogenesis. A number of these splice variants are specific to virally-induced cancers. Elucidating mechanisms underlying how the virus utilises these splice variants to maintain its latent and lytic phase will provide insights into novel targets for drug discovery. This review will focus on the splicing genomics, epigenetic modifications induced by and current therapeutic strategies against HPV, HBV, HCV, EBV, HTLV-1, KSHV and HIV-1

Breast cancer in low-middle income countries : abnormality in splicing and lack of targeted treatment options

Breast cancer is a common malignancy among women worldwide. Regardless of the economic status of a country, breast cancer poses a burden in prevention, diagnosis and treatment. Developed countries such as the U.S. have high incidence and mortality rates of breast cancer. Although low incidence rates are observed in developing countries, the mortality rate is on the rise implying that low- to middle-income countries lack the resources for preventative screening for early detection and adequate treatment resources. The differences in incidence between countries can be attributed to changes in exposure to environmental risk factors, behaviour and lifestyle factors of the different population groups. Genomic modifications are an important factor that significantly alters the risk profile of breast tumourigenesis. The incidence of early-onset breast cancer is increasing and evidence shows that early onset of breast cancer is far more aggressive than late onset of the disease; possibly due to the difference in genetic alterations or tumour biology. Alternative splicing is a pivotal factor in the progressions of breast cancer. It plays a significant role in tumour prognosis, survival and drug resistance; hence, it offers a valuable option as a therapeutic target. In this review, the differences in breast cancer incidence and mortality rates in developed countries will be compared to low- to middle-income countries. The review will also discuss environmental and lifestyle risk factors, and the underlying molecular mechanisms, genetic variations or mutations and alternative splicing that may contribute to the development and novel drug targets for breast cancer.The South African Medical Research Council (SA-MRC)http://www.ajcr.ushj2021Medical Oncolog

Micronucleus assay for Fanconi Anaemia diagnosis with ionising radiation and mitomycin C

Introduction: Fanconi Anaemia (FA) is a cancer - prone chromosomal instability disorder characterized by congenital and developmental abnormalities, bone marrow failure, defects in DNA repair and cellular hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). Twenty different genetic subtypes of FA have been described and are associated with DNA repair pathways. Mutations in DNA repair genes could be reflected in chromosomal radiosensitivity. Clinical radiosensitivity in FA patients has previously been described. This study aimed to evaluate the in vitro chromosomal radiosensitivity and genomic instability of 14 FA patients and their parents by comparing it to healthy controls using 3 different micronucleus (MN) assays. Materials and Methods: In the cytokinesis block G0 MN assay, heparinised blood in culture medium was irradiated to 2 and 4Gy ionising radiation and immediately stimulated with phytohaemagglutinin (PHA). Cytochalasin B (Cyto B) blocked cytokinesis 23 hours after stimulation. Seventy hours post irradiation, cells were harvested. In the S/G2 MN assay, the cultures were first stimulated with PHA. The cultures were then irradiated 72 hours after stimulation with similar doses as in the G0 MN assay. Cytokinesis was immediately blocked by addition of Cyto B after irradiation. Cells were harvested 8 hours post irradiation. The MMC MN assay was conducted in similar manner as the G0 MN assay; however, the cell damage was induced by the addition of MMC. Slides were stained with acridine orange. Micronuclei were scored using a fluorescent microscope. Results: FA patients demonstrated significantly higher spontaneous and radiation-induced MN frequencies when compared to parents and healthy controls in both the G0 and S/G2 phase. FA patients also exhibited higher MMC-induced chromosomal aberrations. Conclusions: FA patients present with higher chromosomal radiosensitivity and genomic instability when compared to healthy controls. The MMC MN assay is the most sensitive assay to demonstrate differences between FA patients, parents and control