by Sophie Mumford, research technician working with Prof Sarah Newbury at Brighton and Sussex Medical School (BSMS) 4th December 2017
Recent funding from the Myfanwy Townsend Melanoma Research Fund has provided me with the exciting opportunity to work alongside Professor Sarah Newbury and her research team, to check and validate the microRNA biomarkers identified in these initial experiments.
The importance of early detection
Melanoma remains the most aggressive form of skin cancer with an increasing incidence worldwide. Survival rates depend heavily on the stage of disease at the time of diagnosis; when detected in the early non-metastatic stages (I and II), melanoma has good prognosis with a 5-year survival rate of 100% in stage I melanoma patients and 80%-90% in stage II melanoma patients, as thin local tumours are highly curable by surgical resection.
While surgical resection of a non-metastatic melanoma can result in disease-free intervals, recurrence is common often resulting in progression to locally advanced melanoma and subsequently to metastatic melanoma (stages III and IV), and a 5 year survival rate of ~50% in stage III melanoma patients and 10-25% in stage IV melanoma patients (Cancer Research UK).
This presents a pressing need to establish robust biomarkers that:
(1) Allow detection and diagnosis of melanoma in its earliest stages I and II, and
(2) Allow the identification of patients at high risk of metastatic recurrence after surgical resection of a non-metastatic melanoma.
Early detection will maximise the chances of patient survival, as there are now a number of treatment options available that are effective against certain subtypes of stages I, II and III melanoma.
The role of microRNA biomarkers
Circulating microRNAs are emerging as promising non-invasive cancer biomarkers. Previous work carried out at the Blond McIndoe Research Foundation and the Brighton and Sussex Medical School, identified 3 potential microRNA biomarkers that can distinguish Stage I/II from Stage III/IV melanoma, plus three potential reference controls.
Since our previous report, I have analysed all of the data obtained from the 474 qRT-PCR assays quantifying the relative levels of the three potential biomarkers in RNA extracted from the serum of 20 healthy, 20 stage I/II and 20 stage III/IV donors. I re-extracted RNA from the same patient serum samples using a different miRNA extraction kit, and repeated the qRT-PCR assays to assess whether the RNA extraction method influences the detection of these specific microRNAs from our samples.
Additionally, I added the C. elegans miR-39 spike-in to control for any inconsistencies in the extraction efficiency between samples. I re-analysed the initial Exiqon microRNA Array data obtained at the beginning of this project and identified two additional potential biomarkers. I have analysed the levels of these two microRNAs between our three patient groups, again using qRT-PCR against the RNA extracted using both extraction kits.
I am currently in the process of reviewing the literature on circulating microRNA biomarkers in melanoma, and the limitations of the circulating microRNA biomarker detection and analysis methods. I have designed a spreadsheet to document the microRNAs identified in these papers, as well as the detection and analysis methods used.
The information that I collect will be used to inform the introduction and/or the discussion in our publication. To assess the extraction efficiency of the two miRNA extraction kits used, I will collect serum from healthy donors, add the C.elegans miR-39 spike-in control and run an equal volume of each sample through each kit, followed by comparison using qRT-PCR.
Therefore we will obtain information on the consistency of the two RNA extraction used, which will be very useful for our further work and for other researchers in the field. The data for this experiment will be included in the publication we are currently preparing.
For further information on this report and the relevant melanoma research work contained herein please contact Sarah Newbury at S.Newbury@bsms.ac.uk.