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Background: Breast cancer is the most commonly diagnosed cancer in women and is one of the leading causes of cancer related deaths worldwide. Although many risk factors have been identified, the aetiology of 50-80% of breast cancer cases remain unknown. This has resulted in an attempt to identify other possible causes of breast cancer such as viral infections. The aim of this study is to investigate the association of high risk human papillomavirus types 16 and 33 with breast cancer.
Methods:Paraffin-embedded tissues from breast cancer of four patients were analysed for the presence of human papillomavirus using real-time polymerase chain reaction. DNA extraction was achieved using a MasterPureâ„¢ Complete DNA and RNA Purification Kit (Epicentre Biotechnologies).
Results: None of the four breast carcinomas were positive for human papillomavirus types 16 or 33.
Conclusion: These results do not support a role of human papillomavirus in breast cancer however controversial reports from around the world suggest further research is needed before a definitive relationship can be attained.
Key words: Breast cancer; Human papillomavirus; Real-time polymerase chain reaction.
Table of Contents
List of Figures vii
List of Tables viii
1. Introduction 10
1.1 Cancer 10
1.1.1 Genes associated with cancer 12
1.2 Breast Cancer 15
1.3 Viruses and Cancer 18
1.4 Human Papillomavirus 19
1.5 HPV Transmission 22
1.6 HPV and Cancer 23
1.7 HPV and Breast Cancer 24
1.8 Aims and Objectives 27
2. Materials and Methods 28
2.1 Materials 28
2.1.1 DNA Extraction Materials 28
2.1.2 PCR Materials 29
2.2 Methods 30
2.2.1 DNA Extraction Protocol 30
2.2.2 Real-Time PCR Protocol 32
3. Results 35
3.1 NanoVueâ„¢ Results 35
3.2 Real-Time PCR Results 35
4. Discussion 41
4.1 General Discussion 41
4.2 Future Work 45
5. References 48
6. Appendices 59
List of Figures
Figure 1 The main causes of death in the UK 10
Figure 2 Comparison of the five most common cancers in the USA
and China 11
Figure 3 Genetic model for cancer progression 12
Figure 4 The Hallmarks of Cancer 12
Figure 5 The cell cycle 14
Figure 6 The 10 most common causes of female cancer related deaths in
the UK 15
Figure 7 Cross section of the breast 16
Figure 8 HPV genome 20
Figure 9 Amplify 96 well PCR plate 29
Figure 10 qRT-PCR amplification of positive control for HPV 16/18/31 36
Figure 11 qRT-PCR amplification of sample 19 and the control for HPV 16/18/31 38
Figure 12 qRT-PCR amplification of sample 59 and the control for HPV
Figure 13 qRT-PCR amplification of positive control for HPV 33/35/56 40
Figure 14 qRT-PCR amplification of sample 19 and the control for HPV 33/35/56 40
List of Tables
Table 1 Examples of tumour suppressor genes and oncogenes 13
Table 2 Variants of IDC 17
Table 3 Some of the risk factors associated with breast cancer 17
Table 4 Viruses associated with cancer development 18
Table 5 The functions of papillomavirus proteins 21
Table 6 Some examples of studies carried out investigating the association
between HPV infection and breast cancer 26
Table 7 Breast tissue sample and weight 32
Table 8 Reaction volume for HPV 16/18/31 33
Table 9 Reaction volume for HPV 33/35/56 33
Table 10 qRT-PCR application program 34
Table 11 DNA concentration and purity of each sample 35
Table 12 Ct mean values for HPV types 16, 18 and 31 36
Table 13 Ct mean values for HPV types 33, 35 and 56 39
bp Base Pairs.
BPV Bovine Papillomavirus.
Ct Cycle Threshold.
DCIS Ductal carcinoma In Situ.
DNA Deoxyribonucleic Acid.
FDA Food and Drug Administration.
FFPE Formalin-Fixed Paraffin-Embedded.
HPV Human Papillomavirus.
IDC Invasive Ductal Carcinoma.
ILC Invasive lobular Carcinoma.
LCIS Lobular Carcinoma in Situ.
MHC Major Histocompatibility.
PC Positive Control.
PCR Polymerase Chain Reaction.
qRT-PCR Quantitative Real-Time Polymerase Chain Reaction.
RNA Ribonucleic Acid.
TSG Tumour Suppressor Gene.
Cancer is a global burden responsible for over 7.5 million deaths a year (Jemalet al. 2011) and thus a leading cause of death, second only to cardiovascular disease (Fig. 1) (King et al. 2006; Kochaneket al. 2011; Murphy et al. 2012). In the UK alone there were over 320,000 new cases diagnosed in 2010 and approximately 157,000 deaths (Cancer Research UK. 2012).
Figure : The main causes of death in the UK (King et al. 2006).
Canceris not a modern disease. In fact the earliest evidence of cancer has been found among human mummies of ancient Egypt (American Cancer Society. 2011). Despite this ancient historical context it is the aging populations and the modern lifestyles of smoking, physical inactivity and poor diet that have greatly impacted on the growing number of cancers encountered (King et al. 2006). The impact lifestyle choices have on cancer can be seen in the prevalence of certain types of cancers in different parts of the world (Fig.2) (King et al. 2006; Tobias et al. 2010a).It is now known that there are more than 200 different types of cancers, each with their own risk factors, symptoms, treatments and outcomes.
Figure : Comparison of the five most common cancers in the USA and China (King et al. 2006).
Carcinogenesis is a multi-step process (Fig. 3) which usually develops over many decades (Tobias et al. 2010b; Weinberg. 2006a). During this process normal cells acquire six biological capabilities known as the hallmarks of cancer (Hanahan and Weinberg. 2000). These hallmarks are; sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis and activating invasion and metastasis (Fig. 4)(Hanahan and Weinberg. 2011). Carcinogenesis has been divided into three stages based on experimental observations; initiation, promotion and progression. Initiation occurs with DNA damage due to exposure to carcinogens which can be biological, physical or chemical. Promotion stimulates cell proliferation of these initiated cellsresulting in the formation of hyperplastic lesions. The final stage, progression provides the catalyst,through further genetic alterations, for these cells to become invasive(McKinnellet al. 2006).
Figure : Genetic model for cancer progression: A genetic model for carcinogenesis. The letters A, B and C indicate genetic events resulting in DNA changes (Adapted from Aspinall et al. 2001).
Figure : The Hallmarks of Cancer (Hanahan and Weinberg 2011).
Genes associated with cancer
There are two main types of genes that are associated with cancer and these are oncogenes and tumour suppressor genes (TSG) (Table 1).
Table : Examples of Tumour suppressor genes and oncogenes (Adapted from Cassidy et al. 2010)
Tumour Suppressor Genes
G1-S checkpoint control and triggers apoptosis.
Breast, lung, colon, sarcoma.
Cell cycle control.
Retinoblastoma, small-cell lung cancer.
Involved in DNA repair.
Glioma, melanoma, lung, bladder.
Colon, lung, melanoma.
Lung, breast, cervix.
Growth factor receptor.
Breast, lung, stomach.
Oncogenes are mutations of normal genes called proto-oncogenes. In normal cells these proto-oncogenes promote cellular proliferation however when mutated these genes become improperly activated and resistant to inactivation. These oncogenes can become activated by point mutation, gene amplification, chromosomal rearrangement or viral mutagenesis. Activationresults in cancer formation by producing an abnormal product such as mutated Ras or over producing normal products such as Her-2(Cassidy et al. 2010a; Peedell.2005a).
Tumour suppressor genes are genes that inhibit cell proliferation in normal cells. However in cancer cells these genes become inactivated due to mutations or deletion and therefore lose the ability to induce apoptosis. Inactivation of tumour suppressor genes occurs with two hits. Firstly the TSG is inactivated due to a mutation. A second hit results in the complete loss of an entire region of the chromosomes containing the remaining normal allele. Examples of TSG's include p53 and Rb genes (Cassidy et al. 2010a; Peedell. 2005a). The p53 TSG is involved in the regulation of both the G1/S and G2/M checkpoints during the cell cycle (Fig.5). p53 induces cell cycle arrest and apoptosis in response to DNA damage (Hebneret al. 2006). The Rb TSG is involved regulation of the cell cycle by preventing progression into the S phase (Hebneret al. 2006).
Figure : The cell cycle:During the G1 phase (gap phase) the production of enzymes which are necessary for DNA synthesis takes place.During the S phase (synthesis) DNA replication occurs. The G2 phase sees the synthesis of specialised proteins and RNA. The M phase (mitosis) is when cell division takes place. All dividing cells pass through this process and careful control of the cell cycle is necessary to protect against DNA damage (Adapted from Weinberg. 2006a)
1.2 Breast Cancer
Breast cancer is the most commonly diagnosed cancer in women and isthesecond leading cause of cancer related deaths among womenworldwide (DeSantiset al. 2011; Jemalet al. 2011; Siegel et al. 2012). Globally there are over one million new cases diagnosed each year resulting in approximately half a million deaths (Jemalet al. 2011; Tobias et al. 2010c). In the UK alone there are approximately 45,000 new cases of breast cancer diagnosed each year and approximately 12,000 deaths a year, accounting for approximately 15.5% of all female cancer related deaths (Neal et al. 2009).Furthermore current statistics estimate one in eight women will develop breast cancer in her lifetime (Tobias et al. 2010c).
Figure 6: The 10 most common causes of female cancer related deaths in the UK (Cancer Research UK).
Pathologically, breast cancer is classified as either lobular or ductal depending on their site of origin (Fig. 7) (Neal et al. 2009). Invasive ductal carcinomas (IDC) which account for 90% of breast cancers can be further divided into subtypes including those listed below (Table 2) (Cassidy et al. 2010b; Peedell. 2005b).Invasive lobular carcinomas (ILC) account for most of the other cases of breast cancer. Pre-invasive carcinomas can also occur in either the ducts or the lobes of the breast and these are referred to as ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS) (Peedell. 2005b).
Figure 7: Cross section of the breast (NHS choices)
In most cases the aetiology of breast cancer remains unknown, however certain factors (Table 3) which increase the risk of developing the diseasehave been established through epidemiological studies (Cassidy et al. 2010b; McPherson et al. 2000). Of these,sex and age have been identified as the most important. Over 99% of all breast cancer cases are diagnosed in women. Furthermore more than 75% of these cases occur in women over the age of 50 (Tobias et al. 2010c).The risk of breast cancer also increases 3-fold for those with a first degree relative affected by the disease. In geographical terms, the Western World has a 5-fold increase in risk compared to the Far East. Moreover studies have found that within two generations migrants to the Western World adopt the same rates of breast cancer as those of the host country (McPherson et al. 2000; Tobias et al. 2010).Reproductive factors include menarche before the age of 11, menopause after the age of 54, nulliparity and older age at first pregnancy. Despite the number of recognised risk factors 50-80% of cases present with no known riskfactors resulting in the attemptto identify newpossible causes such as viral infections(De Leon et al. 2009; Klug et al. 2005).
Table : Variants of IDC (adapted from Cassidy et al. 2010).
Her2- positive breast cancer
Triple negative breast cancer
Table : Some of the risk factors associated breast cancer (adapted from Cassidy et al. 2010).
High fat diet
Benign breast disease
Family history of breast cancer
Hormone replacement therapy
1.3 Viruses and Cancer
An estimated 10-15% of all cancers worldwide have been associated with viral infection (Moore et al. 2010).The first human tumour virus, the Epstein-Barr virus, was identified in 1964; fifty three years after Francis Peyton Rous began his landmark cancer virus transmission experiments (Moore et al. 2010). Since then a total of seven viruses have been found to be capable of causing cancer development (Table 4). Moreover both DNA and RNA viruses have been identified as cancer causing (Liao. 2006). Many years of study have also revealed that virus infection alone is generally not enough to cause cancer and that other factors including immunosuppression, somatic mutations and exposure to carcinogens play a role (Liao. 2006; Moore et al. 2010). Viral carcinogenesis have been divided into two categories: direct carcinogens that express viral oncogenes whichdirectlypromote cancer cell transformation and indirect carcinogens that cause carcinogenic mutations through chronic infection and inflammation (Moore et al. 2010).
Table : Viruses associated with cancer development (Adapted from Morreet al. 2010)