Circulating tumour DNA as a specific diagnostic biomarker for ovarian cancer.

Ovarian cancer (OC) is the fifth most common cancer in women in the UK with approximately 7,000 new cases diagnosed in 2009. Owing to the non-specific symptoms of OC women tend to present with advanced disease. 5-year survival rates are 43% overall but are over 90% for tumours diagnosed early. Currently all patients with suspected OC are triaged using a CA125 blood test and pelvic ultrasound scan to calculate a Risk of Malignancy Index (RMI). Due to the current difficulties of early diagnosis of OC and poor outcome in patients diagnosed with advanced disease work is being done to identify new cancer markers that can potentially be used to improve decision making in the investigation and treatment of OC and subsequently be used in population screening for OC. Somatic mutations are highly specific biomarkers of cancer. It has been shown that somatic mutations can be identified in blood samples from patients with relapsed OC. We propose to look at somatic mutations in blood and cervical smear samples from women with OC at the time of diagnosis. If such mutations could be detected this could be used as a non-invasive method for cancer diagnosis that could potentially increase the early detection of such cancers and thereby improve disease outcome.

The overall objective of this project is to develop new non-invasive methods for diagnosis of ovarian cancer that can be utilised earlier in the diagnostic pathway to improve prognosis for women with ovarian cancer.

Primary hypothesis

1.Can ctDNA be detected in plasma from women with OC at the time of diagnosis? Using a tagged-amplicon deep sequencing technique (TAm-Seq), a new method for non-invasive mutation analysis recently developed by the Rosenfeld and Brenton labs, we propose to sequence DNA from matched plasma and tissue samples from the time of diagnosis in women with ovarian cancer. A second phase of the study will look at symptomatic women with elevated CA125 and a pelvic mass on transvaginal ultrasound to explore the ability of TAm-Seq to discriminate between benign and malignant masses when clinical suspicion is equivocal.

Exploratory Endpoints

2.Can mutations detected in plasma samples at diagnosis be detected in cervical smear samples? Using TAm-Seq we propose to compare sequencing of genomic DNA from cervical smear samples and cervical mucous aspirates to plasma and tissue in patients at the time of diagnosis of OC. If these results are promising, future work will explore the development of methods using a self-inserted tampon for collection of tumour DNA that might acceptable for use by patients.

3.Is molecular analysis of routine cervical smear samples a sensitive method for diagnosis of ovarian and endometrial cancer? In a population based study we propose to sequence all routine cervical smear samples classified as showing possible glandular abnormalities for a panel of mutations known to be associated with both OC and endometrial cancer to assess the utility of population based cervical smears in screening for OC.

The first group of people to benefit from this research are women with a diagnosis of ovarian cancer or women being investigated for suspected ovarian cancer. Ovarian cancer is currently the most common cause of death from gynaecologically related cancers. The majority of women have advanced disease at the time of diagnosis which has a significantly negative impact on survival. Difficulties in the diagnosis of OC are related to the non-specific symptoms of the disease resulting in delayed investigations, a significant proportion (>50%) of early stage disease without an elevated CA125 resulting in missed diagnoses and a significant number of benign gynaecological and non-gynaecological conditions, particularly in premenopausal women, with an elevated CA125 leading to inappropriate investigation and referral. By sequencing for mutations in plasma and cervical smear samples from women with ovarian cancer we are looking to develop a method for earlier diagnosis of ovarian cancer. This would significantly benefit women with ovarian cancer as 5-year survival rates are 43% overall but are over 90% for early stage tumours. We aim to improve the specificity of ovarian cancer diagnosis using sequencing techniques. Currently, national guidelines dictate that all patients with a suspected OC are assessed in primary or secondary care using a CA125 serum assay and pelvic ultrasound imaging. These are incorporated into a Risk of Malignancy Index (RMI) and used to triage patients into three different risk categories. The sensitivity and specificity of RMI is relatively low. CA125 is elevated in both benign gynaecological and non-gynaecological conditions. False positive elevations are a particular problem in premenopausal women, the group most likely to present with an adnexal mass. By increasing the specificity of a diagnostic test less women without ovarian cancer would be referred for inappropriate investiagetion and surgery reducing their anxiety and morbidity. This also means that resources can be focused on those with ovarian cancer reducing any potential delays in treatment for these women.

New diagnostic techniques that can be used to improve the diagnosis of ovarian cancer will benefit those working in both primary and secondary care as well as policy makers within the NHS. The 2011 UK National Institute for Clinical Excellence (NICE) clinical guidelines on the recognition and initial management of ovarian cancer state that 'there is a need for greater awareness of ovarian cancer, and for initial investigations in primary and secondary care that enable earlier referral and optimum treatment'. By developing sequencing methods for detection of mutations in both plasma and cervical smear samples from women with ovarian cancer we are aiming to develop cost-effective methods for earlier diagnosis of ovarian cancer that will have direct clinical utility. Improving the sensitivity and specificity of a diagnostic test will help in triaging women from primary to secondary care for further investigation and guide clinicians in secondary care as to subsequent management.