A Robust Protocol for Using Multiplexed Droplet Digital PCR to Quantify Somatic Copy Number Alterations in Clinical Tissue Specimens

<div><p>The ability of droplet digital PCR (ddPCR) to accurately determine the concentrations of amplifiable targets makes it a promising platform for measuring copy number alterations (CNAs) in genomic biomarkers. However, its application to clinical samples, particularly formalin-fixed paraffin-embedded specimens, will require strategies to reliably determine CNAs in DNA of limited quantity and quality. When applied to cancerous tissue, those methods must also account for global genetic instability and the associated probability that the abundance(s) of one or more chosen reference loci do not represent the average ploidy of cells comprising the specimen. Here we present an experimental design strategy and associated data analysis tool that enables accurate determination of CNAs in a panel of biomarkers using multiplexed ddPCR. The method includes strategies to optimize primer and probes design to cleanly segregate droplets in the data output from reaction wells amplifying multiple independent templates, and to correct for bias from artifacts such as DNA fragmentation. We demonstrate how a panel of reference loci can be used to determine a stable CNA-neutral benchmark. These innovations, when taken together, provide a comprehensive strategy that can be used to reliably detect biomarker CNAs in DNA extracted from either frozen or FFPE tissue biopsies.</p></div

Comparison of CNAs in the same set of biomarkers in matched frozen and FFPE tissue specimens measured by our multiplexed ddPCR method.

<p>Mean normalized <i>R</i>_<i>i/b</i> values were determined for 15 biomarkers in DNA extracted either frozen or FFPE tissue biopsies of SCC in Area 1 () or the dysplasia in Area 2 (○). In general good agreement of normalized <i>R</i>_<i>i/b</i> values is observed between the matched samples when the fragmentation correction is applied (<b>A</b>), while a much poorer agreement is observed when the fragmentation correction is not applied (<b>B</b>). Error bars represent a 95% confidence interval.</p

Overlayed output data for a set of <i>n</i> = 4 duplex ddPCR experiments amplifying the <i>CPT2</i> (HEX) reference on 1p32 and the <i>D4S1652</i> (FAM) micro-satellite biomarker, a ATCT tetranucleotide repeat found on 4q35.

<p>(<b>A</b>) gDNA from normal frozen blood amplified using primer/probe sets designed using standard Primer 3 software. A disperse secondary cluster and dense rain between clusters are observed. (<b>B</b>) Amplification of the same sample using primer/probe sets optimized according to the guidelines in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161274#pone.0161274.s009" target="_blank">S2 Table</a>. More tightly focused clusters and a reduction in rain are achieved. (<b>C</b>) gDNA recovered from a normal FFPE sample amplified using the same primer/probe sets as in (A). Significant levels of rain and poorly focused clusters are observed. (<b>D</b>) gDNA recovered from FFPE tissue amplified using the optimized primer/probe sets. Rain levels are greatly reduced and the clusters are tightly focused.</p

Comparison of CNAs measured within frozen tissue specimens by our multiplexed ddPCR method and by aCGH.

<p>15 biomarkers within DNA extracted from four frozen tissue biopsies collected in a field study of the oral cavity of an oral cancer patient: <b>A</b> = Area 4 (normal connective tissue); <b>B</b> = Area 1 (OSCC-positive tissue); <b>C</b> = Area 2 (moderate to severe dysplasia); and <b>D</b> = Area 3 (OSCC-positive tissue). The log₂ ratios from aCGH are the averaged values for the three or four probes that map closest to the biomarker interrogated by ddPCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161274#sec002" target="_blank">Materials and Methods</a>), with the error bars showing the respective high and low log₂ ratios for these probes. Purple-filled bars are <i>R</i>_<i>i/b</i>—1 (normalized) values computed using our CNA-neutral benchmark as reference and orange-filled bars are <i>R</i>_{<i>i/RPP30</i>}<i>−</i>1 values computed using <i>RPP30</i> as a single reference locus. Error bars represent a 95% confidence interval. Biomarkers have been sorted by chromosomal location (x-axis).</p

Analysis of DNA fragmentation as a function of template length and type of sample.

<p>ln (<i>R</i>_<i>i/cr</i>)_<i>M</i> values determined from triplex ddPCR experiments on gDNA recovered from normal blood (open symbols) or FFPE tissue specimens (filled symbols). Each triplex experiment co-amplifies the <i>HER2 cr</i> and one length <i>i</i> each of the <i>CPT2</i> length-based control (circles) and the <i>KCNS3</i> length-based control (triangles). Error bars represent a 95% confidence interval.</p

Primer/probe sets must be optimized to produce unbiased high-quality data from multiplexed ddPCR experiments.

<p><i>R</i>_<i>i/r</i> (<i>i</i> = <i>D4S1652</i>, <i>r</i> = <i>CPT2</i>) values and low and high 95% confidence intervals (CIs) computed from each ddPCR dataset shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161274#pone.0161274.g001" target="_blank">Fig 1</a>.</p

Multiplex (4-plex) ddPCR output generated using either a standard orthogonal layout of droplet clusters or our staggered layout technique.

<p>Standard orthogonal layout of droplet data for DNA extracted from (<b>A</b>) normal blood or (<b>B</b>) normal FFPE tissue. Staggered layout of droplet data for DNA extracted from (<b>C</b>) normal blood or (<b>D</b>) normal FFPE tissue. The droplets shown in each plot are a combination of <i>n</i> = 4 replicates and the four primary clusters are labelled. The four loci amplified were 1 = <i>ACADM</i>; 2 = <i>KCNS3</i>; 3 = <i>SLC25A12</i> and 4 = <i>HFE2</i>. The orthogonal layout was achieved using the following concentrations of labelled probes (1 = 600 nM FAM; 2 = 300 nM FAM; 3 = 300 nM HEX; 4 = 600 nM HEX). The staggered layout was achieved using the recipe described in main text. Each plot was created by the overlayed output data for a set of <i>n</i> = 4 multiplex ddPCR experiments.</p

Selection of reference loci comprising a CNA-neutral benchmark.

<p>The ln (<i>R</i>_<i>i/cr</i>)_<i>A</i> distributions for all 13 reference loci are reported for DNA extracted from (<b>A</b>) Area 4 (normal tissue) and (<b>B</b>) Area 2 (tissue displaying moderate to severe dysplasia (D3)). (<b>C</b>) The centroid reference locus <i>i</i> (<i>RPP30</i>) for the Area 4 sample and the |<i>z</i>_<i>ij</i>| values comparing the centroid locus to each other reference loci. For this normal tissue specimen, the ln (<i>R</i>_<i>j/cr</i>)_<i>A</i> for all reference loci <i>j</i> (≠ <i>i</i>) were statistically indistinguishable from that of the centroid locus <i>i</i> (all |<i>z</i>_<i>ij</i>| < <i>z</i>_<i>c</i>). (<b>D</b>) The same analysis applied to the Area 2 specimen, for which <i>CPT2</i> was determined to be the centroid locus and the ln (<i>R</i>_<i>j/cr</i>)_<i>A</i> for a subset of 7 reference loci were found to be statistically indistinguishable from that of <i>CPT2</i>. For each sample <i>z</i>_<i>c</i> was computed assuming α = 0.05 and then correcting for multiple comparisons using the Bonferroni method.</p

Initial Diagnosis of <i>ALK</i>-Positive Non-Small-Cell Lung Cancer Based on Analysis of <i>ALK</i> Status Utilizing Droplet Digital PCR

We describe a novel droplet digital PCR (ddPCR) assay capable of detecting genomic alterations associated with inversion translocations. It is applied here to detection of rearrangements in the anaplastic lymphoma kinase (<i>ALK</i>) gene associated with <i>ALK</i>-positive non-small-cell lung cancer (NSCLC). NSCLC patients may carry a nonreciprocal translocation on human chromosome 2, in which synchronized double stranded breaks (DSB) within the echinoderm microtubule-associated protein-like 4 (<i>EML4</i>) gene and <i>ALK</i> lead to an inversion of genetic material that forms the non-natural gene fusion <i>EML4-ALK</i> encoding a constitutively active tyrosine kinase that is associated with 3 to 7% of all NSCLCs. Detection of <i>ALK</i> rearrangements is currently achieved in clinics through direct visualization via a fluorescent <i>in situ</i> hybridization (FISH) assay, which can detect those rearrangements to a limit of detection (LOD) of ca. 15%. We show that the ddPCR assay presented here provides a LOD of 0.25% at lower cost and with faster turnaround times