Disease Susceptibility in Women Study Protocol
The Million Women Study: Detailed Investigation into Susceptibility of Disease protocol (2004)
To investigate in detail factors affecting susceptibility to disease within the Million Women Study, including the separate and joint effects of exposure to environmental factors, such as hormone replacement therapy, and of the individual’s genetic constitution. The main focus will be on susceptibility to breast cancer and to cardiovascular disease.
The Million Women Study (Eastern MREC 97/5/01) is a nationwide prospective cohort study set up primarily to investigate the effects of different types of hormone replacement therapy (HRT) on the risk of breast cancer. Over 1.3 million women aged 50-64 (1 in 4 of UK women in this age group) were recruited through NHS breast screening centres between 1996 and 2001. Prevalent and incident disease is monitored through self-reporting on recruitment and follow-up questionnaires and by linkage to the National Health Service Breast Screening Programme, Cancer Registries and the Office of National Statistics. The large scale of the Million Women Study and the detailed prospective information collected on environmental risk factors such as the use of HRT mean that it offers an unprecedented opportunity to study the separate and joint effects of both environmental and genetic risk factors for disease.
Breast cancer is the commonest cancer among women in the UK and is an important cause of morbidity and mortality. There is considerable public and scientific interest in the role of genetic factors in susceptibility to breast cancer. While there is mounting evidence on the substantial risk of breast cancer associated with rare high-risk gene mutations such as BRCA1 and BRCA2, much less is known about the possible effect on breast cancer risk of more common lower-risk variations (polymorphisms) in genes such as those (eg CYP17, CYP19, HSD17B1) involved in the metabolism of sex hormones like oestrogen. Although the risks of breast cancer associated with these lower-risk gene variants are likely to be relatively modest, they are potentially of considerable public health importance as the genetic variations involved are very common (present, on average, in about 20-60% of the population in the UK). Particularly important are the possible joint effects on breast cancer risk of these common genetic variants and equally common environmental risk factors such as the use of hormone replacement therapy (at recruitment a third of women in the Million Women Study were current users of HRT). These joint effects can only be adequately investigated in very large studies. Initial studies in this field have been limited by small study size, overemphasis on marginal findings and failure to replicate positive results; however it is hoped that relevant polymorphisms will be more reliably identified in larger-scale studies currently under way. The Million Women Study has accrued 10 000 incident cases of breast cancer within the first three years of follow-up and over 35 000 incident cases are expected by 2008. We plan to focus our breast cancer susceptibility studies on the investigation of strong candidate polymorphisms once these have been identified, and, unlike previous studies, we will be able to take into account detailed information on tumour histology and on oestrogen and progesterone receptor status as well as prospective data on reproductive factors and HRT use.
Current users of HRT are at approximately three-fold increased risk of venous thromboembolism (deep venous thrombosis and pulmonary embolism). The effect of HRT on the risk of arterial disease, including coronary heart disease and stroke, is less clear; recent results from randomised trials suggest that women using HRT may be at increased risk of stroke (relative risk 1.3) and that the risk of coronary heart disease may be increased, at least in the first year of HRT use. These results contrast with the apparent protective effect of HRT on risk of cardiovascular disease seen in earlier observational studies (and which may have been due to prescribing bias). Venous and arterial disease are common and important causes of morbidity and mortality in post-menopausal women. Genetic factors such as polymorphisms in the genes involved in blood clotting (eg Factor V Leiden and prothrombin 020210A) are known to increase the risk of venous thromboembolism, and may also affect the risk of arterial thrombosis. Evidence from oral contraceptive use suggests that the joint effect of such thrombophilic mutations and exogenous hormone use may be substantial. Compared to non-carriers of the Factor V Leiden mutation who were not using oral contraceptives, the relative risk for venous thromboembolism was 4 for non-carriers using oral contraceptives, 8 for carriers not using oral contraceptives and 35 for carriers using oral contraceptives. In the only published study of polymorphisms of prothrombotic genes and HRT use the risk of venous thromboembolism was increased independently by HRT (relative risk 3) and by Factor V Leiden (relative risk 4); the relative risk in women with both factors was 15. HRT use and thrombophilic mutations are both relatively common and the Million Women Study provides the opportunity to investigate in detail their separate and joint effects on cardiovascular disease risk. It is possible that consideration should be given to screening women for markers of susceptibility before HRT is prescribed; about 5% of women carry the Factor V Leiden mutation, one in thirty carriers of Factor V Leiden may develop venous thromboembolism each year on HRT and this susceptible subgroup of carriers could account for half the thromboembolic events seen in HRT users. incidence rates for non-fatal cardiovascular disease within the Million Women Study are estimated to be about 0.3/100 women/year for ischaemic heart disease and 0.1/100 women/year for both stroke and venous thromboembolism. Follow-up of the entire cohort to the end of 2002 will yield an estimated 5400 non-fatal coronary events, 2700 non-fatal stokes and 3500 venous thromboembolic events.
The susceptibility study will be a case-control study nested within the Million Women Study cohort. The study will include approximately 20 000 cases (women who have reported incident breast cancer (breast cancer diagnosed after recruitment to the Million Women Study) (10 000), or incident cardiovascular disease (or other diseases of interest)(10 000)) and 40 000 controls (women who have not reported the onset of breast cancer, cardiovascular disease or other diseases of interest since recruitment to the Million Women Study).
This study will involve the collection of biological samples for the analysis of DNA and for biochemical tests (such as measures of endogenous hormones). Blood samples would provide some advantages; plasma and/or serum would be available for biochemical analyses, and methods of DNA extraction and analysis and of sample storage are well established. if however it proves difficult to obtain blood samples from such a large number of women throughout the UK, self-administered buccal swabs provide a possible alternative. While DNA yields from buccal smears are generally lower than those from blood, and biochemical analyses on saliva samples may be more difficult than on plasma or serum, buccal smears have been used successfully for genetic testing in large epidemiological studies and methods for long¬term storage are currently being tested with encouraging results. We will include both blood and buccal smear sampling in our pilot studies to ascertain which is most appropriate for the main study.
Women eligible for the susceptibility study will be those who have completed the primary follow-up questionnaire three years after recruitment (and in the case of women who have reported breast cancer, who have also completed a secondary breast cancer follow-up questionnaire). All women taking part in the MWS have given signed consent for follow-up.
Approach to participants
Eligible women will be sent a follow-up/susceptibility study questionnaire. The questionnaires will request further details of the woman’s health and of disease risk factors such as use of hormone replacement therapy and family history of disease; questionnaires sent to case women will also ask for details of disease diagnosis and treatment. Two versions of the questionnaire will be used in the initial pilot study.
The version used to recruit women for blood collection will include a question asking if the woman might be willing in principle to supply a blood sample for the susceptibility study. The questionnaire will then be posted back to the Million Women Study co-ordinating centre. If the woman is willing, a letter and information sheet about the Million Women Study and the susceptibility study will then be sent to her general practitioner asking if they are willing to take the blood sample at their GP practice. If the GP does not object, the woman will be sent a letter and information sheet giving details of the susceptibility study, a blood collection kit with full instructions for the GP or Practice Nurse, and two copies of the consent form. She will be asked to attend the GP practice for blood collection and to post back the blood sample and one signed consent form to the Million Women Study co-ordinating centre in Oxford. The other copy of the consent form will be kept by the participant for her records.
With the other version of the questionnaire, used to recruit women for buccal smear collection, women will be sent a letter and information sheet explaining the susceptibility study and asking if they would consider providing a buccal cell/saliva sample for biochemical analyses and analysis of DNA, a kit for collecting the buccal swab, with full instructions, and two copies of the consent form. Women who decide to take part in the susceptibility study will be asked to take the buccal swab sample themselves at home and to post the sample and signed consent forms, with the follow-up questionnaire, to the Million Women Study co-ordinating centre in Oxford. The consent forms will be countersigned by a study investigator and one copy returned to the participant for her records.
Those who prefer not to provide a blood or buccal sample will be able to return the questionnaire alone, as part of continuing follow-up within the Million Women Study.
Processing and storage of data and of biological samples
Data from questionnaires will be entered onto a secure database at the Million Women Study co-ordinating centre. Initial processing and storage of blood and of buccal cell and saliva samples will take place at the Million Women Study co-ordinating centre laboratory. Further analyses, including the extraction of DNA and the analysis of genetic polymorphisms, will be carried out either at the Million Women Centre laboratory or at other secure specialist laboratories.
Validation of disease diagnoses
Diagnoses of breast cancer are validated within the existing structure of the Million Women Study by record linkage to participating National Health Service Breast Screening Programme centres, cancer registries and the Office of National Statistics. It will be necessary to validate self-reported diagnoses of cardiovascular disease by comparison with hospital records. This will initially be done for a sample of cases in a pilot study (see below); all case women taking part in the susceptibility study will be asked to give signed consent for us to contact their hospital consultant for further information if necessary.
Standard statistical methods, including Mantel-Haenszel calculation of odds ratios and logistic regression modelling, will be used to obtain relative risks for the development of breast cancer, ischaemic heart disease, stroke and venous thromboembolism in relation to the separate and joint effects of HRT use and of relevant genetic polymorphisms. Analyses will take into account potential confounding factors such as previous medical history, tobacco and alcohol use; and such biochemical data as are available from the susceptibility study.
Assuming a 60% response rate, we expect approximately 6000 cases of breast cancer and 1800 cases each of ischaemic heart disease, stroke and thromboembolism. With 3 controls per case, and minimum population prevalences of 30% for HRT use and between 2 and 20% for relevant genetic polymorphisms, the study will have 95 % power at 99% significance to detect the following minimum relative risks: for breast cancer, for the effect of HRT exposure in the various genotype subgroups, 1.1-1.4; for the effect of each genotype in the various exposure subgroups, 1.2-1.4; and an interaction ratio (the extra effect of joint exposure to HRT and a genetic factor, above that expected under a multiplicative model) of 1.4; and for each cardiovascular disease, for effect of HRT exposure 1.3-6.0, for genotype 1.7-3.0, and an interaction ratio for joint exposure to HRT and Factor V Leiden of 2.0.
Consent, confidentiality and ethical issues
The study will conform to current ethical and legal guidelines regarding consent, confidentiality and the use of human biological samples, and accords with the protocol outlined for the UK Biobank study (www.ukbiobank.ac.uk/protocol.htm). It will be conducted in accordance with relevant aspects of the Data Protection Act, the Human Rights Act 1998, the General Medical Council’s Guidance on Confidentiality and the Council of Europe’s Recommendation on the Protection of Medical Data. It will follow the guidance outlined in the Medical Research Council’s documents on Personal Information in Medical Research and on Collections of Human Tissue and Biological Samples for Use in Medical Research. Each biological sample will be physically identified only by a code number. The information needed to link this number with questionnaire data, which may include personal identifying data where necessary for future follow-up within the Million Women Study, will be held securely and separately from other databases. All data will be analysed only in anonymised form and future publications will not identify individual women taking part.
Participation in the susceptibility study will be entirely voluntary. Potential participants will be informed in writing that:
- their decision whether or not to participate will not affect their future health care in any way
- they are free to withdraw from the study at any time, and if they do so their biological sample will be destroyed and neither the sample nor the information derived from it used in further analyses
- all information will be treated with absolute confidentiality in accordance with the Data Protection Act, used for medical research purposes only and will never be used in a way which could identify them personally
- they will not receive individual feedback about the results of genetic or other tests
- the study is important for future research and many of the tests and analyses which will be conducted in the future cannot be specified at present
- the study has been approved by the appropriate Multi- Research Ethics Committee.
Participants will be asked to give written consent for the storage of their blood or buccal cell/saliva sample and for the use of the sample for unspecified biochemical and genetic tests now and in the future.
Participants will be encouraged to ask about or comment on any aspect of the study either in writing or by telephoning a study Freephone number.
Pilot studies will be carried out on a small sample (about 200 – 400 women with disease and a similar number of control women). The results, including comments from participants, will be used to test methods of collection, storage, processing and analysis of biological samples and to confirm acceptability and usefulness of the study methods for the questionnaires. A major feature of the pilot study will be to compare the feasibility of collection and analysis of blood samples and of buccal smears. As part of the pilot studies information from the hospital records of a sample of women reporting incident cardiovascular disease will be compared with self-reported information from questionnaires to assess diagnostic validity of questionnaire reports.
Publication of results
The Million Women Study is a joint research project of the NHS Breast Screening Programme, the Medical Research Council and Cancer Research UK. Results from the study will be published in peer-reviewed journals and appropriate authorities (eg the Department of Health and the Medicines Control Agency) will be notified of relevant results where necessary.
A number of minor amendments to this protocol have been approved. Details are available on request.