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. 2016 Oct 19;11(10):e0165023.
doi: 10.1371/journal.pone.0165023. eCollection 2016.

Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

Sarah Hrebien  1 , Ben O'Leary  1   2 , Matthew Beaney  1 , Gaia Schiavon  3 , Charlotte Fribbens  1   2 , Amarjit Bhambra  2 , Richard Johnson  2 , Isaac Garcia-Murillas  1 , Nicholas Turner  1   2
Affiliations

Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

Sarah Hrebien et al. PLoS One. .

Abstract

Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48-72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77-0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Comparison of total free plasma DNA levels between immediate processed EDTA samples and delayed processed Streck samples.
A. Correlation plasma DNA levels of immediate processed EDTA samples and delayed processed Streck samples. Pearson correlation coefficient. B. Bland-Altman plot of data in part A with dashed lines representing 95% CI. C. Overall survival with plasma DNA quantified in immediate EDTA tubes divided on high plasma DNA levels above the upper quartile versus low plasma DNA below the upper quartile. Log rank test with hazard ratio (HR) and 95% confidence intervals (95% CI). D. Overall survival with plasma DNA quantified in delayed Streck tubes divided on high plasma DNA levels above the upper quartile versus low plasma DNA below the upper quartile. Log rank test with hazard ratio.
Fig 2
Fig 2. Agreement in mutation calling between immediate EDTA and delayed Streck tubes.
A. Contingency table for mutation detection on immediately processed tubes versus delayed processing tubes,. B. Scatter plot of mutation allele frequency in concordant vs discordant samples. Mann Whitney U test. C. Correlation of mutational allele frequent frequency on immediate and delayed processing tubes. Pearson correlation coefficient. D. Correlation of mutant copies per ml of plasma in immediate and delayed processing tubes. Pearson correlation coefficient.
Fig 3
Fig 3. Mutation frequency observed in advanced breast cancer.
A. Mutation frequency observed in plasma of patients with advanced cancer. Only samples with concordant mutations in both samples are assessed as having a mutation. Individual mutations observed for B. PIK3CA and C. ESR1 mutations detected.
Fig 4
Fig 4. Agreement in change in mutation abundance in sequential samples between immediate EDTA and delayed Streck samples.
A. Agreement in fold change in mutation allele frequency between immediate and delayed samples in sequential samples from 6 patients (r = 0.85, p = 0.0002). B. Agreement in fold change in mutant copies per ml between immediate and delayed samples in sequential samples from 6 patients (r = 0.84, p = 0.0003).
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Grants and funding

This work was funded by Breast Cancer Now (http://www.breastcancernow.org/, NT), National Institute for Health Research (http://www.nihr.ac.uk/, NT), Cancer Research UK, C30746/A16642 (http://www.cancerresearchuk.org/, NT), Le Cure (http://lecuredefrance.org/, NT), and Cridlan trust (http://opencharities.org/charities/1112652, GS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.