<i>HER2:EFTUD2</i> digital PCR for determinant of <i>HER2</i> status.

<p>Representative droplet digital plots from a tumour with high level amplification (left panel), low level amplification (middle panel) and a non-amplified tumour (right panel). The four quadrants represent top left: droplets with <i>HER2</i> DNA only, top right: droplets with both <i>HER2</i> and <i>EFTUD2</i> DNA, bottom right: droplets with <i>EFTUD2</i> DNA only, and bottom left: droplets with no DNA.</p

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

<div><p>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 <i>PIK3CA</i>, <i>ESR1</i> and <i>ERBB2</i>, and for <i>AKT1</i> 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.</p></div

Mutation frequency observed in advanced breast cancer.

<p>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. <i>PIK3CA</i> and C. <i>ESR1</i> mutations detected.</p

Agreement in change in mutation abundance in sequential samples between immediate EDTA and delayed Streck samples.

<p>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).</p

Comparison of total free plasma DNA levels between immediate processed EDTA samples and delayed processed Streck samples.

<p>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.</p

Agreement in mutation calling between immediate EDTA and delayed Streck tubes.

<p>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.</p

Clinical and pathological characteristics of study patients.

<p>Clinical and pathological characteristics of study patients.</p

<i>KRAS</i> mutations observed in human NSCLC in order of decreasing frequency (http://www.sanger.co.uk/cosmic; accessed 14^th July 2015).

<p><i>KRAS</i> mutations observed in human NSCLC in order of decreasing frequency (<a href="http://www.sanger.co.uk/cosmic" target="_blank">http://www.sanger.co.uk/cosmic</a>; accessed 14^th July 2015).</p

<i>KRAS</i> multiplex digital PCR assays A-C and corresponding duplex assays.

<p>Multiplex A (top left panel) is an assay combination of 900 nM primers and 500 nM G13C probe (red dashed square), 450 nM primers and 250 nM G12C probe (blue dashed square) and 225 nM primers and 125 nM G12V probe (yellow dashed square). Multiplex B (top middle panel) is an assay combination of 675 nM primers and 375 nM G12S probe (red dashed square), 450 nM primers and 250 nM G12D probe (blue dashed square) and 225 nM primers and 125 nM G13D probe (yellow dashed square). Multiplex C (top right panel) is an assay combination of 675 nM primers and 375 nM G12R probe (red dashed square), 450 nM primers and 250 nM G12A probe (blue dashed square) and 900 nM primers and 500 nM Q61H probe (yellow dashed square). Multiplex C has 900 nM primers and 500 nM Q61H wild-type probe in addition to a G12C wild-type assay. All other wild-type droplet populations shown, except in the Q61H duplex assay, are 450 nM primers and 250 nM G12C wild-type probe. All panels in the left and centre columns show a FAM amplitude up to 18000 and an HEX amplitude up to 6000. Panels in the right column have a FAM amplitude up to 18000 and a HEX amplitude up to 11000.</p

<i>KRAS</i> mutant FFPE tissue DNA analysis using multiplex and duplex assays to detect <i>KRAS</i> mutant clones.

<p>All samples, except for S011, were analysed with multiplexes A, B and C (upper panels) and the <i>KRAS</i> mutation detected was subsequently confirmed with the appropriate duplex assay (lower panels). Mutant DNA droplet populations are highlighted with a red dashed square. Droplet populations caused by cross-reactivity with a <i>KRAS</i> mutant DNA species not present in the multiplex are indicated by a yellow dashed square.</p