43P - Development and validation of multiplex digital PCR assays on circulating tumor DNA in advanced breast cancer: Implementation for clinical routine use

Date 07 May 2015
Event IMPAKT 2015
Session Welcome reception and Poster Walk
Topics Breast Cancer, Metastatic
Translational Research
Presenter Isaac Garcia-Murillas
Citation Annals of Oncology (2015) 26 (suppl_3): 15-24. 10.1093/annonc/mdv117
Authors I. Garcia-Murillas1, G. Schiavon1, S. Hrebien1, B. Oleary1, J. Morden2, I.E. Smith3, N. Turner1
  • 1The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research ICR, SW3 6JB - London/UK
  • 2Clinical Trials & Statistics Unit, Institute of Cancer Research ICR, SM2 5NG - London/UK
  • 3Department Of Medicine, Royal Marsden Hospital NHS Foundation Trust, SW3 6JJ - London/UK



Circulating tumor DNA (ctDNA) analysis allows non-invasive detection of tumor mutations in advanced cancer. In this study we assessed the robustness of multiplex digital PCR (mdPCR) assays on ctDNA in advanced breast cancer. We developed and optimized mdPCR assays for hotspot actionable mutations in 4 known drivers in breast cancer: PIK3CA exon 9 and 20, ESR1 ligand binding domain, AKT1 (c.49G > A; p.E17K) and HER2. For this study we recruited a cohort of 42 women with advanced breast cancer with a contemporaneous biopsy of metastatic disease and blood samples for extraction of plasma ctDNA (cohort 1). We also recruited a second cohort of 49 women with advanced breast cancer who had two separate blood samples processed immediately or after posting in preservative Streck tubes to a central lab with processing 48-72 hours after venipuncture (cohort 2). In cohort 1, there was 95% agreement between mutation profile assessment by mdPCR in plasma ctDNA and tumour DNA (Kappa 0.89, 95% CI 0.7604 to 1.000). Considering tumour tissue testing to be the gold standard, the sensitivity of ctDNA screening was 87.5% and the specificity 100%. There was 100% agreement between mdPCR and repeat uniplex assay results. In cohort 2, assessment of technical reproducibility between immediately processed samples and posted samples demonstrated 100% agreement in all assays tested. There was very high correlation between mutation abundance (mutant copies per ml) between samples (r2 = 0.98). There was a lower correlation for total amount of free plasma DNA between pairs (r2 = 0.86), with posting samples shown in 4/49 cases to increase total free plasma DNA likely as a result of white blood cell lysis. Multiplex digital PCR assays to screen non-invasively for hotspot actionable mutations in breast cancer has very high agreement with contemporaneous tumour biopsy. Posting samples in preservative Streck tubes to a central laboratory is a robust alternative to on-site processing in patients with metastatic breast cancer, where digital PCR analysis is planned. We demonstrate that digital PCR with centralized analysis has the potential for widespread, multi-center screening in advanced breast cancer.

Disclosure: All authors have declared no conflicts of interest.