Detection of circulating cell free tumour DNA in renal cancer patients for the prediction and detection of early disease recurrence

The number of patients diagnosed with kidney cancer (RCC) has been increasing over the last 10 years and accounts for 4% of all cancers in the UK. Clear cell RCC (ccRCC) is the predominate subtype accounting for approximately 75% of all cases. Surgery is often curative if the disease is identified at an early stage. However approximately 1/3 of patients who undergo surgery with the aim of removing all the kidney cancer, develop recurrence of their disease in the future, which is often incurable. Because of this, RCC is recognised as the most lethal cancer of the urinary tract with up to 50% of patients dying of their disease.

Cancer cells shed DNA into the blood of patients with the disease. It is anticipated that liquid biopsy, through study of the patients' blood and identification of this circulating cell free tumour DNA (ctDNA) will provide useful information for predicting disease recurrence and monitoring for this disease recurrence. This would then allow tailored interventions to improve patient outcomes. However, this ctDNA is a small fraction of the overall cell free DNA found in the circulation, which also has cell free DNA derived from healthy cells within the body. The small fraction of cell-free tumour DNA makes this difficult to detect. Nonetheless, studies in other cancers, such as lung, breast and colon cancer, have shown promising results for the detection of tumour specific DNA changes in the blood of patients with those respective cancers, which could have clinically meaningful implications. Unfortunately using the same approaches to identify ctDNA in RCC has been more difficult and disappointing.

Previous work in our own lab has shown significant changes in a DNA modification called methylation, in ccRCC. Furthermore, very recent small-scale studies have demonstrated much higher levels of ctDNA in ccRCC patients than previously reported, when methylation changes are used to interrogate the cfDNA in both blood and urine. We therefore hypothesis that using RCC specific methylation changes (rather than DNA mutational changes) to identify ctDNA in the blood of patients with RCC will improve the detection of ctDNA in RCC patients.

We have performed a large-scale study with publicly available data to develop a ccRCC specific methylated ctDNA profile, which will be validated in tumour samples from our own cohort. Thereafter using innovative technology, that allows the detection of up to 10000 methylation changes in the blood or urine of patients, we will study the blood from a large number of patients with ccRCC both at diagnosis and throughout follow-up as well as healthy controls. Patients will have RCC representative of the spectrum of disease. All samples have high quality well annotated clinical data which will be correlated with ctDNA findings to identify the utility of this profile for predicting and detecting disease recurrence following surgery. This will potentially lead to the development of clinically applicable tests to improve patient outcomes.

We aim to use clear cell renal cell carcinoma (ccRCC) specific differentially methylated regions to identify ccRCC circulating cell-free tumour DNA (ctDNA) in ccRCC patients' plasma and assess whether this ctDNA can be used for predicting disease recurrence and monitoring for minimal residual disease following apparently curative surgery. To do this a synthetic ccRCC specific ctDNA profile has been constructed from publicly available methylation data from ccRCC tumours, leucocytes, erythrocytes and healthy tissue. This profile will be validated using the illumina methylation EPIC array in ccRCC tissues from primary and metastatic tumours available in the Edinburgh Renal Cancer Biobank, using the illumina EPIC array. Plasma from ccRCC patients and healthy controls, available from the Edinburgh Renal Cancer Biobank, will be interrogated to identify this methylated ctDNA profile. This will be performed using innovative and locally developed Nonacus technology that allows the identification of up to 10K methylated CpGs in a sample. Correlation between ctDNA findings and primary tumour tissue will be made with selected re-profiling of the primary tumours using the illumina EPIC array. Plasma samples across the spectrum of RCC stages at presentation and then during follow-up after surgery will be assessed for the presence of methylated ctDNA and linked with high-quality, well annotated clinical data. Correlation between ctDNA findings and clinical and outcome data in the ccRCC patients and healthy control cohorts detailed will allow us to assess the utility of plasma ctDNA for the prediction of outcome following surgery and monitoring for disease recurrence.