Development of a novel NGS methodology for ultrasensitive circulating tumour DNA detection as a tool for early-stage breast cancer diagnosis

Abstract BACKGROUND Breast cancer (BC) is the most prevalent cancer in women. While usually detected when localized, invasive procedures are still required for diagnosis. METHODS Herein, we developed a novel ultrasensitive pipeline to detect circulating tumour DNA (ctDNA) in a series of 75 plasma samples from localized BC patients prior to any medical intervention. We first performed a tumour-informed analysis to correlate the mutations found in tumour tissue and plasma. Disregarding the tumour data next, we developed an approach to detect tumour mutations in plasma. RESULTS We observed a mutation concordance between tumour and plasma of 29.50% with a sensitivity down to 0.03% in mutant allele frequency (AF). We detected mutations in 33.78% of the samples, identifying 8 patients with plasma-only mutations. Altogether, we determined a specificity of 86.36% and a positive predictive value of 88.46% for BC detection. We demonstrated an association between higher ctDNA median AF and higher tumour grade, multiple plasma mutations with likelihood of relapse and more frequent TP53 plasma mutations in hormone receptor-negative tumours. CONCLUSIONS Overall, we have developed a unique ultra-sensitive sequencing workflow with a technology not previously employed in early BC, paving the way for its application in BC screening.