TC chart-assisted digital PCR analysis of clinical gastric cancer biopsy specimens.

<p>Each case was evaluated by HER2-IHC and digital PCR, and then validated by <i>HER2</i>-DISH (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.s002" target="_blank">S1 Table</a>). The results are plotted on the TC-chart. The findings of HER2-IHC are represented by symbols as follows; 3+ (positive) as rhombi; 2+ (equivocal) as triangles; 0~1+ (negative) as cross-mark. The results of <i>HER2</i>-DISH are depicted by colors as follows; positive, red; equivocal, purple; negative, blue. Multiple cases taken from the same patients (Pt) are highlighted. The vertical axis between 0.5 and 2.5 was linearly ordered and the upper area was logarithmically ordered. There were no positive cases (red rhombi) in the negative area, demonstrating that digital PCR can screen out negative cases with high specificity. On the other hand, cases plotted in the equivocal area were not always positive, as determined by CEP17 copy number. Three cases (#25, #26, and #42) showed markedly high scores and a clustering pattern in <i>HER2</i>-DISH (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.g003" target="_blank">Fig 3C</a>).</p

Histological analysis of clinical gastric cancer biopsy specimens and estimation of TCR using Tissue Studio.

<p>(A–C) Representative cases are shown with HE staining, HER2-IHC, and <i>HER2</i>-DISH. (A) Case #37: HER2-IHC score = 0, <i>HER2</i>-DISH ratio = 1.09. (B) Case #6: HER2-IHC score = 2+, <i>HER2</i>-DISH ratio = 2.83. (C) Case #25: HER2-IHC score = 3+, <i>HER2</i>-DISH ratio = 6.56. (D) TC rate was calculated based on the ratio of nuclei counts of whole nucleated and cancerous cells, which were semi-automatically distinguished by nucleus size—with cancerous cells showing enlarged nuclei—by means of Tissue Studio image analysis software.</p

Summary of correlations between immunohistochemistry and TC chart-assisted digital PCR data.

<p>Summary of correlations between immunohistochemistry and TC chart-assisted digital PCR data.</p

Primer and TaqMan probe sequences for digital PCR.

<p>Primer and TaqMan probe sequences for digital PCR.</p

Tumor Content Chart-Assisted <i>HER2/</i>CEP17 Digital PCR Analysis of Gastric Cancer Biopsy Specimens

<div><p>Evaluating <i>HER2</i> gene amplification is an essential component of therapeutic decision-making for advanced or metastatic gastric cancer. A simple method that is applicable to small, formalin-fixed, paraffin-embedded biopsy specimens is desirable as an adjunct to or as a substitute for currently used HER2 immunohistochemistry and in situ hybridization protocols. In this study, we developed a microfluidics-based digital PCR method for determining <i>HER2</i> and chromosome 17 centromere (CEP17) copy numbers and estimating tumor content ratio (TCR). The <i>HER2</i>/CEP17 ratio is determined by three variables—TCR and absolute copy numbers of <i>HER2</i> and CEP17—by examining tumor cells; only the ratio of the latter two can be obtained by digital PCR using the whole specimen without purifying tumor cells. TCR was determined by semi-automatic image analysis. We developed a Tumor Content chart, which is a plane of rectangular coordinates consisting of <i>HER2</i>/CEP17 digital PCR data and TCR that delineates amplified, non-amplified, and equivocal areas. By applying this method, 44 clinical gastric cancer biopsy samples were classified as amplified (n = 13), non-amplified (n = 25), or equivocal (n = 6). By comparison, 11 samples were positive, 11 were negative, and 22 were equivocally immunohistochemistry. Thus, our novel method reduced the number of equivocal samples from 22 to 6, thereby obviating the need for confirmation by fluorescence or dual-probe in situ hybridization to < 30% of cases. Tumor content chart-assisted digital PCR analysis is also applicable to multiple sites in surgically resected tissues. These results indicate that this analysis is a useful alternative to HER2 immunohistochemistry in gastric cancers that can serve as a basis for the automated evaluation of <i>HER2</i> status.</p></div

<i>HER2</i>-DISH in cell blocks and TC chart in the stepwise mixture system.

<p>(A–C) Micrographs of <i>HER2</i>-DISH of LCL, H522, and SK-BR-3 FFPE cell blocks. <i>HER2</i> signals are shown as black dots or clusters, and CEP17 signals are shown as red dots. <i>HER2</i> and CEP17 copy numbers measured by <i>HER2</i>-DISH and digital PCR are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.t002" target="_blank">Table 2</a>. (A) The LCL was genomically stable with two pairs of signals corresponding to <i>HER2</i> and CEP17, mimicking non-cancerous stromal or inflammatory cells. (B) In H522 cells, both <i>HER2</i> and CEP17 signals appeared as dots and individual signals were discernible. (C) In SK-BR-3 cells, <i>HER2</i> signals formed clusters and exact measurements were difficult. CEP17 signals varied between 3 and 7, with an average score of 83/20 = 4.15. (D, E) Genomic DNA of H522 and SK-BR-3 cells was mixed with the LCL genome in a stepwise manner and analyzed by digital PCR. The horizontal axis represents the ratio of H522 or SK-BR-3 to LCL, corresponding to TCR [<i>x</i>] in clinical cases; the vertical axis represents the ratio of HER2 to CEP17 in digital PCR [<i>r</i>]. [<i>A</i>] and [<i>B</i>] were determined by <i>HER2</i>-DISH (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.t001" target="_blank">Table 1</a>). (D) In H522, [<i>A</i> = 2.05] and [<i>B</i> = 4.25], yielding [<i>r</i> = (4.25<i>x</i> + 2(1 − <i>x</i>))/(2.05<i>x</i> + 2(1 − <i>x</i>))] (solid gray line). Plotted data were approximated by the theoretical line. (E) In SK-BR-3 cells, [<i>A</i> = 4.15], but <i>HER2</i> copy number [<i>B</i>] was difficult to determine due to cluster formation. <i>HER2</i> copy number [<i>B′</i>] was predicted [<i>B′</i> = 5.69 × 4.15 = 23.61] based on digital PCR data [<i>r</i>] and CEP17 copy number [<i>A</i>]. The equation [<i>r</i> = (23.61<i>x</i> + 2(1 − <i>x</i>))/(4.15<i>x</i> + 2(1 − <i>x</i>))] is represented by the solid gray line. Plotted data were approximated by the theoretical line.</p

Information on HER2 status and clinicopathological factors for representative cases.

<p>Information on HER2 status and clinicopathological factors for representative cases.</p

<i>HER2</i>/CEP17 ratios from <i>HER2</i>-DISH and digital PCR in H522 and SK-BR-3 cells and LCL.

<p><i>HER2</i>/CEP17 ratios from <i>HER2</i>-DISH and digital PCR in H522 and SK-BR-3 cells and LCL.</p

Equations and TC chart.

<p>(A) Mathematical representation of the correlation between <i>HER2</i>/CEP17 ratios obtained by digital PCR [<i>r</i>] and TCR [<i>x</i>] (upper). When [<i>B</i>/<i>A</i> = 2], [<i>r</i>] is represented by the right side of the equation (indicated by an arrow), and the right-hand member of the equation indicates a monotonic increase. A schematic model is shown for [<i>A</i> = 2] and [<i>B</i> = 4] (lower right). (B) TC chart demonstrated by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154430#pone.0154430.e001" target="_blank">Eq (1)</a>. The relationship can be plotted as a straight line (red) represented by [<i>r</i> = <i>x</i> + 1], which is the threshold between negative and equivocal areas. As the value of [<i>A</i>] exceeds 2.0, lines become increasingly convex and curve upwards according to this equation. Lines converge at the coordinates (0,1) and (1,2). In clinical samples, [<i>A</i>] was limited to a value between 2 and 8. The area enclosed by the straight red line [<i>r</i> = <i>x</i> + 1](<i>A</i> = 2) and the purple line curving upwards [<i>r</i> = (7<i>x</i> + 1)/(3<i>x</i> + 1)](<i>A</i> = 8) was designated as the equivocal area, and the area above the purple line was designated as the positive area.</p