Introduction
Bladder cancer is the most common urinary tract cancer with 10,000 new UK diagnoses annually. The current diagnostic test for Non muscle-invasive Bladder Cancer (NMIBC) patients requires cystoscopy which is invasive, uncomfortable and time consuming. This requires lifelong monitoring for many patients thus being one of the most expensive cancers to manage.
The major clinical problem is the 40% risk of recurrence within 3 years.
Herein, lies the unique translational opportunity of mitochondrial DNA (mtDNA). During ageing mtDNA accumulates mutations that can lead to disease. Tumour cells are readily shed into urine, suggesting a urine sample could be used for non-invasive testing, to detect bladder cancer recurrence. We therefore propose to identify a tumour biomarker, or “barcode” to be used to detect tumour cells within urine.
Patient Cohort
Clinical samples were received from twelve NMIBC patients within Urology at Freeman Hospital, Newcastle, UK undergoing Radical Cystectomy. Fresh resected NMIBC tissue: tumour, adjacent normal, matched blood and voided urine were collected. In cases of tumour multifocality a separate biopsy of the distinct lesion was taken.
Methods
Urine was separated by centrifugation into cell-free and cell pellet. Blood was separated by centrifugation into plasma, buffy coat and red blood cells. Primary tissue was cut in half and two workflows performed. Half of the tissue was snap frozen and the other half was fixed in formalin, processed and embedded into a formalin fixed paraffin embedded block. Bladder tissue sections were also cut, stained and tumour content scored by a Pathologist. DNA was extracted from each derivative and the whole mtDNA genome amplified by long range PCR for Next Generation Sequencing to detect mtDNA variants.
Results
mtDNA mutations are tumour specific and absent from cells in the patient’s normal bladder lining. Tumour specific mtDNA variants were identified from patient’s bladder tumour tissue and confirmed within the patient’s cellular urine and cell-free urine compartment.
Conclusions
Currently there are no bladder urinary biomarkers in routine clinical use to detect tumour cells. Urine however, provides a non-invasive liquid biopsy of bladder tumour mtDNA. The mitochondrial genome can be detected, reliably amplified and sequenced from both cellular and non-cellular components of urine.
mtDNA variants are credibly confirmed in patients matched urine and bladder tumour. The absence of mtDNA variants can be identified from germline DNA and normal urothelial allowing lineage tracing of mtDNA mutations to determine Bladder Cancer recurrence.
