Lactate dehydrogenase (LDH) is the only blood-based biomarker that has been incorporated in the American Joint Committee on Cancer (AJCC) staging system for melanoma. Elevated LDH levels are associated with higher disease burden, decreased survival and as a stratification parameter in many clinical trials. However, LDH is not sensitive or specific, as conditions besides malignancies can result in elevation of LDH.
An ideal substitute for a blood-based biomarker test is circulating tumour DNA (ctDNA). It is released from tumour cells via various mechanisms including necrosis and apoptosis enabling real-time measurement of changes in tumour status resultant of therapy or recurrent disease, or predicting recurrence.
The use of plasma ctDNA requires tumour-specific mutations to be used as markers. In melanoma, V600EBRAF mutation is the most common mutation. Other common mutations are V600KBRAF and several different NRAS codon 61 mutations which occur mutually exclusively to BRAF mutations.
We employed droplet digital PCR (ddPCR) technology, which can be readily used to quantify low levels mutant DNA copies in patient plasma across multiple time-points with detection sensitivity that can approach 0.01%. Firstly, melanoma cell lines with known mutational profiles were utilised to test primer-probe specificity. The ddPCR system accurately detected mutant DNA from all cell lines. No cross-reactivity to either the wild type or other mutations was detected, including V600EBRAF/V600KBRAF. There were no false positive events detected using mutant probes on wild type DNA.
Metastatic melanoma patient plasma samples were analysed for mutational ctDNA levels. Our data has shown in multiple patients that ctDNA level anticipates the dynamic changes in disease burden as monitored by RECIST criteria throughout treatment. Based on these results we hypothesise that melanoma ctDNA is a sensitive and specific tumour marker, which can be used to monitor treatment response in melanoma patients in a non-invasive and cost-effective way.