MICA: Using circulating tumour DNA to identify and predict mechanisms of resistance to targeted cancer therapies.

If a person has a cancer, there are a number of ways in which the cancer cells differ from the normal cells that make up their body. Some of the most important differences are between the genes of the cancer cells compared to the normal cells; these genes are described by the DNA in the cells. Mutations in the DNA can accumulate and eventually lead to cells becoming cancerous. Increasingly it appears that each person's cancer is very different, and although cancers in different people may originate from the same part of the body, e.g. the breast, they are very different from each other when viewed in terms of the mutations that have caused the cancer to grow. Currently much work is being done to match specific treatments to specific cancers, known as 'targeted therapy'. This field has shown exciting results and has become the basis of 'personalised medicine' in cancer.

To target specific treatment at the mutations in a persons cancer, we must be able to identify and monitor the mutations. This is commonly done by biopsying the tumour. This process has a number of disadvantages. It is invasive, with a low but not negligible risk of complications. There is also some evidence that mutations in cancer cells in one part of the body can be different from cancer cells elsewhere, so that a biopsy from one place might not give the whole picture. Finally, mutations in cancer can change over time, yet only rarely are biopsies repeated. There is a need for a less invasive and easily repeatable method of identifying mutations that could in some circumstances replace biopsies.

Recently, advances in technology have meant it has become possible to identify mutations in DNA that comes from the cancer in a simple blood test. This is called 'circulating tumour DNA' or 'ctDNA' for short. It is possible that this test could form the basis of a 'liquid biopsy', where a patient's cancer is analysed for monitoring and treatment with a blood test. It could also be more representative of a patient's cancer than a biopsy of a single site.

This research project is examining the blood samples of patients with metastatic breast cancer who participated in a clinical trials testing a promising new anti-cancer drug called palbociclib. Palbociclib is called a 'targeted therapy', as it specifically targets a particular part of cells called CDK4/6. As part of this research the blood samples from these patients will be analysed for ctDNA to see whether there were any particular mutations that meant people did particularly well or badly on palbociclib. It will also examine whether early changes in the level of ctDNA could be indicators of how the cancer will respond to the drug. In this way the study hopes to identify future ways of matching this targeted treatment to the patients whose cancers are most likely to respond, and find a means of checking early on in treatment whether it is working. The research will also serve as an example of whether this approach with ctDNA could be useful across a wide range of cancers and targeted treatments, potentially benefiting many more patients than just those taking palbociclib.

Aims: To investigate circulating tumour DNA (ctDNA) as a biomarker to predict sensitivity and primary or acquired targeted therapy, focusing on advanced breast cancer treated with the CDK4/6 inhibitor palbociclib. CDK4/6 inhibitors are one of the most promising targeted therapies in clinical development with evidence of activity in multiple tumour types.
Objectives:
1) Identify genetic aberrations in baseline ctDNA that predict for sensitivity to CDK4/6 inhibition, to select the most appropriate patients for therapy.
2) Assess whether change in abundance of ctDNA following 2 weeks of treatment can identify cancers that are resistant to treatment early.
3) Assess whether analysis of ctDNA can identify the emergence of resistant clones on disease progression.

Methodology: The project will utilise samples collected at baseline and day 15 of treatment, then again on progression from two trials treating breast cancer patients with palbociclib, PEARL (350 samples) and PALOMA-3 (500 samples).
1) Identify genetic aberrations in baseline ctDNA that predict for sensitivity to CDK4/6 inhibition by sequencing plasma DNA using a custom targeted Ampliseq panel on the Ion Torrent Personal Genome Machine. Validation will be by using droplet digital polymerase chain reaction assays run on the BioRad QX200 platform.
2) Sequence day 15 plasma, and establish criteria for fractional change in abundance of ctDNA that predict for outcome.
3) Paired plasma DNA samples, baseline and progression, with a high proportion of ctDNA will undergo exploratory whole-exome sequencing. Acquired mutations will be validated, the function of recurrent acquired mutations investigated in vitro.
Scientific and medical opportunities: This will be largest cohort of plasma sequenced for ctDNA in the literature, offering an unprecedented opportunity to link prospectively collected clinical data to ctDNA, validating it as a biomarker for targeted therapy potentially for routine clinical practice

Given the proposed project's promise of direct clinical improvements using emerging technologies, there is the potential to generate significant impact in a variety of ways for a wide number of different groups. These include breast cancer patients, the health service, commercial providers of technology used in the research, and further potential for considerable impact on a wider scale at a policy making and service planning level through strengthening the position of circulating tumour DNA (ctDNA) as the cancer biomarker for delivery of targeted therapy.

The first group to benefit from the research are breast cancer patients potentially eligible for treatment with palbociclib. The proposed research will elucidate those groups who gain the most benefit from treatment with palbociclib, who can be identified through a ctDNA test prior to treatment. In addition this will reduce the number of patients who would otherwise be needlessly treated with a medication that was unsuitable for them, reducing risk of harm through side-effects and reduction in quality of life. Furthermore, the research may potentially reduce the number of biopsies that are required from patients by establishing a practical, non-invasive alternative. This will further reduce potential harm from investigations required for therapy. These specific impacts will have effects at a service provision level for the NHS and other providers of oncology care, helping to focus increasingly limited resources on the delivery of care that is likely to yield the best results, minimising resource consumption on care with the potential to harm.

The NHS will potentially benefit through enhanced selection of which patients will benefit from therapy. New therapies are required to demonstrate suitable cost/benefit ratios before they will be incorporated within many providers of healthcare, most notably the NHS in the UK. Novel targeted therapies can fail commercially if the appropriate group for targeting is not identified. The development of ctDNA biomarkers to guide palbociclib reduces the risk of this occurring.

There is also potential for this research to produce impact at a national and international level with regards policy decisions concerning the diagnosis, treatment and monitoring of cancer. There is a growing body of evidence that ctDNA will become a key biomarker for the future delivery of oncology therapies. The proposed research involves the examination of a large number of longitudinal plasma samples from patients in a phase III study of targeted therapy, something not yet reported in the scientific literature. Case series describing the use of ctDNA as a biomarker for treatment selection and the development of resistance have been small, but very promising. In confirming these findings on a larger scale within the context of a trial, the proposed research offers the prospect of yielding high-quality data that could make the case at a national or health provider level for routine inclusion of ctDNA testing in certain areas of clinical practice.