Triple Negative Breast Cancer - Assignment PDF

Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Assignment: Triple Negative Breast Cancer And The Molecular Markers Literature Review By...

Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Assignment: Triple Negative Breast Cancer And The Molecular Markers Literature Review By Roxana Olteanu [email protected] TU751/3 Research Methods and Applications Sean Kennedy and Kathleen Brosnan BIOL3009 Page 1 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Abstract This area is going to be completed last. It will contain a summary of the overall points within the literature review. Keep note that this assignment is a draft only and I am currently working on putting the information into the Word document. There is a lot of information that is in the wrong area and I wish to rearrange the data, figures and some paragraphs before sending in the official assignment. There are areas of this assignment with highlights. This means that I am due to rephrase these or infiltrate them in another paragraph with a similar layout/wording. There are other paragraphs in light blue which are direct quotations from a paper which are to be rephrased and referenced appropriately. There are other areas of this draft which may not have information or may be highlighted in another colour. This is because I may wish to change the information that I am looking for due to lack of information available, or due to a better proposition for this assignment. This is also a word count added to see how this area would impact the rest of the review layout. 1. Introduction Breast cancer is one of the most common and most talked about forms of cancer. According to the Irish Cancer Society (ICS), “more than 250 people in Ireland get diagnosed with Triple Negative Breast Cancer (TNBC) each year.”i By definition TNBC is the type of cancer that lacks the production of estrogen from the Estrogen Receptor (ER), progesterone from the Progesterone Receptor (PR) and doesn’t make the protein Human Epidermal Growth Factor 2 (HER2) and hence, the triple negative test would indicate the lack of these. (Mongre et al., 2020) Concerning each protein and its receptor, the Hormone Receptor positive (HR+) groups indicate which gene is needed for growth, for example, if the tumour cell is ER+, it denotes that the cancer is receiving signals from the estrogen receptor and so forth.ii However, with TNBC, the diagnosis comes from a lack of ER and PR and a minor/insignificant amount of protein from HER2. Throughout this literature review, we investigated the genes Phosphoinositide 3-kinase (PI3K), Breast Cancer gene (BRCA), and Tumour protein p53 (TP53), which are three significant gene mutations that play a key role in TNBC. We surveyed these gene mutations in order to answer the question: do these mutations have an impact on treatment for the patient if they were included in testing? To understand TNBC, we must first look at the different subtypes which include HR breast cancer, i.e. ER+, PR+, and TRPS+, and the ER-, PR- and TRPS-. A study by (Ai et al., 2021) describes how a research group, published by USCAP, have explored breast cancer patients and found that the “trichorhinophalangeal syndrome type 1 (TRPS1) acts as a specific gene for breast carcinoma across 31 solid tumour types.” The study further elaborates how TPRS1 is a gene which is very dominant in TNBC expression such that it’s much more common than the typical cancer gene that is tested - GATA3 with a difference of 86% vs 21%. It’s been suggested that TRPS1 is a “highly sensitive” and “specific marker” for breast carcinoma, and hence a great tool used for diagnosis. It shows very little to no expression in urothelial carcinoma and other tumour types, compared to the GATA3 gene, which is widespread in other forms of cancer such as renal cell carcinoma. (Ai et al., 2021) To better grasp how these tumours grow, we observed the histopathology of lymphocytes within the breast tissue. Figure 1 represents the difference in a relatively mild mutation through a Haematoxylin and Eosin (H&E) stain, compared to Page 2 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer that of a moderate and intense level of TNBC. The image under the mild title shows very little morphology in relation to the total nuclei (dark purple stains), compared to the intense morphological mutations on the far left. A more detailed explanation can be found in section 1. Histopathology of this paper. Figure 1: “Representative H&E images TNBC tumours that were scored for mild (0–10%) moderate (20–40%) or intense (>50%) levels of infiltrating lymphocytes relative to total nuclei.” (Lehmann et al., 2016) It’s important to note that when it comes to cancer treatment, TNBC is known to be a more aggressive cancer. This is because in a typical breast carcinoma, the tumour tissue is usually positive for either ER or PR and so the treatment can involve the use of these proteins to prevent any further growth. This is not possible in patients with TNBC and hence, other treatments must be used, such as chemotherapy, radiation, and in some cases surgery. (American Cancer Society, 22 C.E.) Due to TNBC being the type of cancer which is lacking in three different receptors and protein interactions, the testing and diagnosis of this type of cancer is critical and is discussed under different headings throughout this paper. This study also investigates how Illumina - next-generation sequencing, plays an immense role in identifying and diagnosing TNBC, and how the treatment drug Tamoxifen functions on a molecular level/pathway. Before we get to the treatment, however, we must first discuss how TNBC can become so aggressive by looking at the receptors and pathways of this cancer. 2. Receptors and Pathways 2.1 Histopathology A dysfunction as early as the cell cycle is where the cancer cells develop, where cells undergo mitosis. The cell cycle has many phases, but we turn to the G1 phase to study the inactive negative regulator (tumour suppressor) TP53. This area will be discussed in greater detail in section 2.5 TP53. When discussing TNBC, one cannot discuss a single “type” as this is a form of cancer, and not a carcinoma itself. Due to the many different forms and metastasis tied to TNBC, we investigated the tissue which houses the tumour cells; the female breast. In Figure 2 below, the image describes the anatomical view of the breast and the corresponding areas which are capable of cell mutations, in which tumour cells Page 3 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer can grow. An example of a TNBC tumour cell is Ductal Carcinoma (in situ) which is a form of cancer of the lactic ducts. The term “in situ” refers to a defined location, so in the case of this tumour, it has not yet metastasised.iii Figure 3: DCIS is a form of TNBC where cell mutations occur within the walls of the lactic ducts of the breast. The image shows a comparison of a healthy duct to that of a duct with DCIS. iv DCIS is an example of a tumour type that women are advised to search for when performing a regular breast check. This involves holding/pulling the breast to a side with one hand and palpating with two/three fingers in different areas i.e. behind the nipple, under the breast, at the top of the breast (around the fatty tissue), and at the lymph nodes located at the neck and arm-pit. Further investigation of these tumours suggests that there are different pathways in which TNBC can occur. The three genes PI3K, BRCA, and TP53, all have their own signalling pathways where they can mutate and proliferate into malignant growths. Figure 4: The six sub-types of Tripple Negative Breast Cancer.(Dass et al., 2021) Due to TNBC being such a complex and violent type of cancer, diagnosis is simplified by separating common trends into subtypes, as shown above in Figure 4; Basal-Like-1 (BL-1), Basal-Like-2 (BL-2), Luminal Androgen Receptor (LAR), Mesenchymal (M), Mesenchymal Stem-Like (MSL), and Immunomodulatory (IM). These six classifications come from a study conducted by Lehmann et al. in which the classes were split based on “GE cluster analysis” from a larger number of TNBC patient samples. “Based on gene ontologies and differential GE, the analysis revealed seven Page 4 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer tumour clusters, with six stable clusters each displaying a distinctive gene signature … and one additional unstable (UNS) tumour cluster that expresses genes that are found across the other six clusters.” (Mahmoud et al., 2022) An example of the main histopathology of the breast can be seen in Figure 4, where the images in the top row illustrate some of the sub-types of tumours. The bottom row of Figure 4 describes the markers and pathways that lead to the growth of these tumours. Figure 4: A visual example of TNBC subtypes, histology and biomarkers/pathways. (Mahmoud et al., 2022) We interpreted these tumour growths on a histological level by studying Figure 5 below. This expands our view into the breast tissue by looking at the different metamorphoses of the transformed stages TNBC – from low-grade to high- grade. Each biopsy seen below shows the pathways in which certain mutations can occur. We take a specific look at the gene mutations that have occurred due to TP53 and PI3K concerning Figure 5. PI3K seems to have a more prevalent appearance in later stages of the low-grade mutations as seen in image three of the top row, and image two of the bottom row. However, once the high-grade mutations have occurred, we can see that the signalling pathways most distinguished are those of TP53 and PI3K. This suggests that these pathways are strong indicators when testing for TNBC. This also indicates that a form of treatment can be deduced and calculated based on these pathways. Page 5 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Figure 5: “Low-grade and high-grade histologic types of triple-negative breast carcinomas (TNBCs). Representative micrographs of low-grade and high-grade variants of TNBC. Several histologic types of low-grade TNBC, including salivary gland–like tumours of the breast and solid papillary carcinoma with reverse polarity, are underpinned by specific/pathognomonic genetic alterations.” (Geyer et al., 2017) 2.2 The Pathway Overview The TNBC genes PI3K, BRCA, and TP53 are the molecular biomarkers that if non-functioning/malfunctioning, can cause growths which can metastasise if left untreated. TNBC tumour growth can be identified by looking at the immunological studies of the cell pathways. We start at the beginning of this malignant evolution, which occurs during the cell cycle; however, we investigate deeper into the cell's genetic information to identify these specific biomarkers. Page 6 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Figure 6: “Classification and therapeutic options for TNBC. ADC: antibody-drug conjugates; AR: androgen receptor; LAR: luminal androgen receptor; M: mesenchymal; MSL: mesenchymal-stem-like; PARP: poly-adenosine diphosphate ribose polymerase; PI3K: phosphoinositol-3 kinase; TKI: tyrosine kinase inhibitor; and TNBC: triple-negative breast cancer”(Y. Li et al., 2022) Figure 6 shows the different sub-types in which chromosome alterations can lead to TNBC through the lens of an immunological response or a lack thereof. In the third quadrant of the diagram, we see the M or MSL sub-types where the differentiation steps occur; here, anti-angiogenesis (the treatment whereby the tumour is prevented from developing its blood supplyv ) is one of the treatments, as is radiation for the Immune Inflammation response in the fourth quadrant. 2.3 BRCA1/BRCA2 The BRCA1 and BRCA2 genes are two of many genes that take up the responsibility for DNA repair. Like TP53, they are also tumour suppressor genes. A strong biomarker for TNBC is the BRCA genes BRCA1 and BRCA2 which can be found on chromosomes 13 and 17, as mentioned previously. The location of the BRCA1 and BRCA 2 genes acknowledges where exactly on the human chromosomes mutations can occur. Figure 7: “Schematic of the location of BRCA1 on Chromosome 17, and the location of BRCA 2 on Chromosome 13. vi Page 7 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer According to an article by (Varol et al., 2018) “BRCA1 is located on the chromosome 17q21 and has 24 exons. BRCA1 codes for a protein consisting of 1863 amino acids (5.6 kb). BRCA2 is located on chromosome 13q12 and consists of 27 exons, with exon 11 being the largest one (4.9 kb). BRCA2 codes for a protein consisting of 3418 amino acids (10.2 kb).” The schematics shown in Figure 8 show how the “zoomed in” pathway of the BRCA1 and BRCA2 genes from a tumour. On the right-hand side, the BRCA1 and BRCA2 genes show a similar representation to Figure 7, and also demonstrate to have genomic integrity, meaning that the “entire genome is intact during cell division”. vii Following the continuation of this pathway, we see that TP53 plays a role in preventing tumour growth that leads to either apoptosis (programmed cell death) or cell-cell restart ( cell cycle continuation). Figure 8: (A) “BRCA1, BRCA2 and TP53 in DNA damage repair pathway resulting in cellular and genetic instability with potential points for targeted therapy. PIK3CA cellular pathway effects on cell cycle, invasiveness and survival with potential points for targeted therapy. (B) Number and per cent of mutations for genes of interest. The most frequently mutated somatic genes were PIK3CA (12.9%), BRCA2 (11.7%), BRCA1 (10.2%), TP53 (6.0%)”(Barakeh et al., 2021) Page 8 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer A more detailed explanation of the specific pathway can be seen on the left-hand side of Figure 8 in section (A). To study the relative frequency of these genetic mutations, the specific biomarkers we are investigating, BRCA1, BRCA2, TP53 and PI3K, show to have the highest frequency of genetic mutation with TNBC specifically, with BRCA1 at a rate of 11.7%, TP53 at 6%, BRCA2 at 10.2%, and the highest frequency of PI3K at a rate of 12.9%. In Figure 9 below, we see the pathway in a less complex status than in Figure 8, however; this allows us to better understand the difference between a wild-type (healthy) sample, compared to a mutant sample. On the left-hand side, the drug Berberine is present. This is a naturally occurring chemical in certain plants which helps with immunological responses, nevertheless, this study does not concern this drug, as we focus on the damage compared to the natural pathways. Figure 9: Side-by-side comparison of the pathways a Wildtype BRCA1 gene takes vs the mutant genes of BRCA1/BRCA2 including the genomic errors and checkpoint failures which lead to breast tumour development.(Jabbarzadeh Kaboli et al., 2014) Significant data shows that the N-terminus of Chromosome 17 has been found to have an ability to “bind to zinc” and also has a “nuclear localisation signal in the centre region”. Because a nuclear localisation region is present, TP53 is also implicated via a mechanism of nuclear transfer – predominantly linked to DNA damage which can be seen in Figure 9 revealing the pathway of both Wild Type BRCA1 and the Mutant BRCA1/BRCA2 above. (Jabbarzadeh Kaboli et al., 2014) 2.4 PI3K It has been discovered that PI3K (specifically PI3KCA) is a strong molecular biomarker for tumour growth and has been labelled to be “oncogenic”. A study by (Ascione et al., 2022) explains the three classes associated with PI3K; class I, II, and III. The study states that “the PIK3CA gene encodes for the catalytic alpha subunit (p110α) of class I PI3K and is commonly mutated in human cancers, constantly stimulating tumour growth and survival.” This suggests that the protein is a good target for therapeutics, and hence, “About 80% of PIK3CA somatic mutations are hot-spot mutations, the most common being single nucleotide changes resulting in aminoacidic substitutions: E542K and E545K in the exon Page 9 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer 9, H1047R and H1047L in the exon 20.” Figure 10 below shows the relevant hotspot mutations of PI3K for TNBC. Examining part (B) of this pi-chart, we can see the variant frequency of the mutated gene with a strong focus on Exon 9 and Exon 20 of the chromosome; similar to BRCA1/BRCA2 in Figure 7. Figure 10: Somatic mutations of the PI3K gene with a focus on Exon 7, Exon 9, and Exon 20. (A) A pi-chart showing the percentage of mutations versus no mutations detected in TNBC. (B) Exon 7, Exon 9, Exon 20, and other mutations in a visual chart with the specific respective mutations. (C) “Lollipop plot representing the distribution of PIK3CA mutations positioned by their amino acid coordinates across the protein domain. Data were retrieved from The Cancer Genome Atlas (TCGA) breast cancer (n = 825) genomic dataset through cBioportal and reanalysed by RStudio software (version 3.6.3). Available at: https://www.cbioportal.org (accessed on 10 January 2021).” (Criscitiello et al., 2021) To further understand the PI3K mutations, an additional investigation taken directly from the cancer research database known as Depmap Portal shows the dependent cell lines of the gene effects under CRISPR; an area of study in genomic engineering which involves targeting specific “stretches of genetic code to edit DNA at precise locations”.viii Figure 11 (i) states that the PI3K has a strong selectivity, which makes it an important molecular biomarker. (ii) represents the expression of PI3K mutations. (i) (ii) Page 10 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Figure 11: Gene expression for PI3K: (i) The Left-hand side shows the Dependent Cell Lines visualised under the lens of CRISPR where the Blue map represents the strong selectivity regions, and the Purple region is depicting strong selectivity under the lens of RNAi gene effect. (ii) Right- hand side shows the Expression and Copy Number (CN) under a logarithmic representation.ix Due to PI3K being such a strong biomarker, we use Depmap Portal to confirm our theory that if this mutation is used for diagnosis, it could have a big impact on the treatment of the patient. Figure 12 (i) explores the graph of the expresses the relationship between the different expressions and the hot mutations. (i) (ii) Figure 12: (i) CRISPR and RNAi predictability with reference to relative importance, correlation, and feature type graph. (ii) A strong focus on the hot mutations of PI3KCA at 25.8% relative importance, compared to 23.9% in the RNAi model.x 2.5 TP53 TP53 is a tumour suppressor gene which is meant to regulate cells and prevent unnecessary proliferation, however, in the case of this investigation, a mutation occurs where this gene is either unresponsive or damaged. xi In healthy cells, damaged DNA is recognised by TP53 and apoptosis occurs. This protein is an important factor in the diagnosis of TNBC; this gene is found to be “altered in almost all cases of TNBC.” However, in a non-functional TP53, the protein can no longer bind to the damaged DNA and will allow it to pass to the next phase of replication, the S-phase.xii Page 11 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer 2.6 Gene Prevalence Figure x: “Somatic mutations affecting cancer genes in TNBCs from The Cancer Genome Atlas (TCGA). Bars indicate the frequency of somatic mutations affecting the 50 cancer genes most frequently mutated in TNBCs”(Pareja et al., 2016) 2.7 Find the statistics table for each 2.8 Include a heat map if possible Figure x: “Heatmap shows the expression of immuno-regulatory genes across 587 TNBC tumours ranked by increasing correlations to the IM TNBC centroid.”(Lehmann et al., 2016) 3. Testing – Mutations and Detections Mutations and their Detection 3 History of how cancer and ultimately TNBC was first discovered 4 Reference first study and evolution of TNBC testing “The study showed that with an adequate patient sample size, TNBC GE analysis can determine distinct subtypes and reveal molecular targets, providing predictive biomarkers to help stratify patients for tailored treatments and, consequently, help improve patient response to therapy with the design of appropriate clinical trials.” (Mahmoud et al., 2022) 5 Next-generation sequencing and Illumina (in detail) 6 Discuss modern testing, detection of mutations, correlation on how and why these specific genes are targeted biomarkers 7 Tamoxifen Page 12 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Figure x: Tamoxifen and its relationship with cancer and receptors.xiii 8 In-depth details on Illumina and next-generation sequencing Figure x: Next-generation sequencing workflowxiv - Include heat maps here, images of specific genes 9 Explain the meaning of overexpression, and include some percentages 10 Include potential causes (external where applicable) 4. Statistics and Epidemiology 1.1 Statistics and epidemiology 11 Introduce and discuss statistics of patients for: - Testing Page 13 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Figure x: The enriched ligases in CRISPR and RNAi with a focus on the highest functioning target for the PI3K gene. Row 3 on the left-hand side shows the expression found in breast cancer in high amounts under CRISPR, and on the right-hand side, the purple lines, we investigate the 1st, 2nd and 4th lines which show the highest reading of RNAi in invasive breast cancer.xv - Diagnosis (add phases and metastasizing) - Misdiagnosis (why and how this could be eliminated) - Treatment - Success rate - Deaths - Also to include – correlation between three mutations (Dep-map portal) Page 14 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Figure x: Depmap representation of the corresponding genes which have been found as biomarkers for TNBC, specifically in relation to testing for invasive breast carcinoma.xvi 12 Discuss some clinical trials from the past and present 13 Include graphs and another heat map/diagram of global statistics (population graph) A study dating between 2005 and 2015 shows a generalised data collection of the different types of cancer recorded in Ireland, with the leading percentage of it being breast cancer in women. Although this data isn’t specific to TNBC, the collection shows the average case per year, the percentage of the patients who tested positive, the incidence and the death rate per year. More specific data will be discussed later on in this review. 14 Figure x: Key facts and most common cancers in Ireland with a data sheet of the composition of cancers. xvii Page 15 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer Figure x: “Estimated major-market sales of key therapies for breast cancer, by drug class. The figure shows the 2019–2029 forecast for the seven major markets: the USA, France, Germany, Italy, Spain, United Kingdom and Japan.” (Wilcock & Webster, 2021) 1.2 Treatments and Further Research – Targeting and Elimination 5. Treatment 15 Discuss specific tests performed in Ireland in big detail Figure x: “(A) ER and PI3K/AKT/mTOR signalling and endocrine therapy in ER+ early breast cancer. Schematic interaction of the two signalling pathways and dominant effects of adjuvant endocrine therapy on the cellular output and its overall impact within 10 years after surgery.”(Yang et al., 2016) Page 16 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer 16 Explain how the mutations are targeted Figure x: “Metabolism pathway of tamoxifen and its interaction with estrogen receptors. Copyright © 2009. Nature Publishing Group. Adapted and reprinted with permission; Hoskins JM, Carey LA, McLeod HL. CYP2D6 and tamoxifen: DNA matters in breast cancer. Nat Rev Cancer 2009;9:576–586. 131 Abbreviations: CYP, cytochrome P450; ER, estrogen receptor; FMO, flavin-containing monooxygenases; SULT1A1, sulphotransferase 1A1; UGT, uridine diphosphate glucuronosyltransferase.”(J. Li & Bluth, 2011) 17 Investigate how these mutations impact testing and treatment 18 Go into details on short titles in section 1.3 – testing, diagnosis, etc. 19 Include a study that proves this testing to be the most successful 6. Conclusions a. Conclusion We mentioned that there are six different sub-types of TNBC classification and that there are three mutations in particular in which we chose to study as molecular biomarkers for this investigation. Recap summary to include: - The three gene mutations - Next generation sequencing - Recap on discussed clinical trials and short result - Treatment conclusion - Refer back to the question and answer it again Page 17 of 18 Roxana Olteanu [email protected] TU751 Triple Negative Breast Cancer References i Irish Breast Cancer (2021) Breast-Predict, Triple Negative Breast Cancer - https://www.cancer.ie/sites/default/files/2021- 06/short%20public%208731%20Irish%20Cancer%20Society%20Breast%20Predict%20booklet%20screen.pdf [09/11/23] ii NCI (2023), Hormone Receptor-Positive (HR+) Breast Cancer, Penn Medicine – Abramson Cancer Centre https://www.pennmedicine.org/cancer/types-of-cancer/breast-cancer/types-of-breast-cancer/hormone-positive-breast-cancer [01/11/23] iii Whitlock J, RN, MSN, FN (2023), Dotdash Meredith, How in situ is used during surgery and cancer treatment - https://www.verywellhealth.com/in-situ-explained-3157097 [20/11/23] iv Cancer Counsil Victoria (2023) Ductal Carcinoma in Situ https://www.cancervic.org.au/cancer-information/screening/breasts- health/ductal-carcinoma-in-situ [15/11/23] v Cancer Research UK (2023) Drugs that block cancer blood vesicles growth (anti-angiogenesis ) https://www.cancerresearchuk.org/about-cancer/treatment/targeted-cancer-drugs/types/anti-angiogenics [22/11/23] vi Kochetkova E.O, Degemerzanova N.K, (2013), Breast and Ovarian Cancer Risk Due to BRCA1, BRCA2, CHEK2 Gene Mutations, Kazakhstan Medical Journal, https://treegene.kz/en/brca/ [27/11/23] vii H. Collins, Collins English Dictionary (2023), Genomic Integrity definition https://www.collinsdictionary.com/dictionary/english/genome-integrity [05/12/23] viii Broad Institute Cambridge (2023), Questions And Answers About CRISPR, https://www.broadinstitute.org/what-broad/areas- focus/project-spotlight/questions-and-answers-about-crispr [05/12/23] ix PI3K gene overview (2023), Depmap portal, Board Institute https://depmap.org/portal/gene/PIK3CA?tab=overview [22/11/23] x PI3K gene overview, Predictability (2023), Depmap portal, Board Institute https://depmap.org/portal/gene/PIK3CA?tab=predictability [05/12/2023] xi American Society of Clinical Oncology (2023), Genetic testing for cancer risk https://www.cancer.net/navigating-cancer- care/cancer-basics/genetics/genes-and-cancer [15/11/23] xii Khan Academy (2023), Cancer and the cell cycle, How cancer can be linked to the overactive positive cell cycle regulators (oncogenes) or inactive negative regulators (tumour suppressors) https://www.khanacademy.org/science/ap-biology/cell- communication-and-cell-cycle/regulation-of-cell-cycle/a/cancer [24/11/23] xiii Wise (2023) Tamoxifen vs. Aromatase Inhibitors – How do they work? – Blog - https://owise.uk/tamoxifen-aromatase-inhibitors/ [ 01/11/23] xiv S. Aryal, Generation Sequencing (NGS) – Definition, Types, 5th August 2022 - https://microbenotes.com/next-generation- sequencing-ngs/ [3/11/23] xv PI3K gene overview (2023), Depmap portal, Board Institute https://depmap.org/portal/gene/PIK3CA?tab=overview [22/11/23] xvi TP52 and PI3K gene expression of invasive breast carcinoma, Depmap Portal (2023) https://depmap.org/portal/cell_line/ACH- 001389?tab=overview [22/11/23] xvii National Cancer Registry Ireland (2018), Cancer Factsheet – Overview and most common cancers https://www.ncri.ie/sites/ncri/files/factsheets/Factsheet%20all%20cancers.pdf [15/11/23] Page 18 of 18