Scatter diagram of plasma amphiregulin and heregulin in 50 patients with colorectal cancer.

<p>Patients were categorized into four groups based on median levels of amphiregulin (16.8 pg/mL) or heregulin (1,292 pg/mL). Red dots are patients who responded to anti-EGFR therapy.</p

Kaplan-Meier progression-free survival curves.

<p>Plasma levels of amphiregulin and heregulin were measured by ELISA. Patients are divided into subgroups based on amphiregulin (AR) or heregulin (HRG) abundance, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143132#pone.0143132.g002" target="_blank">Fig 2</a>. The red curve represents survival in patients with high amphiregulin and low heregulin (n = 12), and the green line represents survival in all other patients (n = 38).</p

Circulating amphiregulin and heregulin in 50 patients with colorectal cancer.

<p>Plasma amphiregulin (A) and heregulin (B) were measured by enzyme-linked immunosorbent assay. Each bar represents a single patient. Receiver operating characteristic (ROC) curves are shown for the ability of amphiregulin (C) and heregulin (D) to predict response and non-response to cetuximab.</p

Patient characteristics.

<p>ECOG PS, Eastern Cooperative Oncology Group Performance Status</p><p>PR, partial response; SD, stable disease; PD, progressive disease.</p><p>Patient characteristics.</p

Plasma amphiregulin and heregulin stratified by patient characteristics.

<p>ECOG PS, Eastern Cooperative Oncology Group Performance Status; CPT-11, irinotecan.</p><p>Plasma amphiregulin and heregulin stratified by patient characteristics.</p

Kaplan-Meier overall survival curves.

<p>Plasma levels of amphiregulin and heregulin were measured by ELISA. Patients are divided into subgroups based on amphiregulin (AR) or heregulin (HRG) abundance, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143132#pone.0143132.g002" target="_blank">Fig 2</a>. Survival in patients with high amphiregulin and low heregulin (n = 12) is plotted in red. Survival in all other patients (n = 38) is plotted in green.</p

Extended <i>RAS</i> and <i>BRAF</i> Mutation Analysis Using Next-Generation Sequencing

<div><p>Somatic mutations in <i>KRAS</i>, <i>NRAS</i>, and <i>BRAF</i> genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended <i>RAS</i> and <i>BRAF</i> mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within <i>KRAS</i>, <i>NRAS</i>, and <i>BRAF</i>, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10^-5). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). <i>KRAS</i> mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. <i>KRAS</i> and <i>BRAF</i> mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant <i>RAS</i> and <i>BRAF</i> mutations using FFPE and liquid samples.</p></div

Concordance of plasma and tumor results for <i>RAS</i> and <i>BRAF</i> mutations.

<p>Concordance of plasma and tumor results for <i>RAS</i> and <i>BRAF</i> mutations.</p

Read number and error rate of the NGS assay.

<p>(A) Average read number of 9 amplicons in 10 normal DNA samples (average with standard deviation). Horizontal axis, number of reads; vertical axis, PCR amplicon. (B) Error rate of position detection in 10 normal DNA samples (average with standard deviation). Horizontal axis, base position; vertical axis, error rate. (C) Average read number of 9 amplicons in 20 colorectal cancer FFPE DNA samples (average with standard deviation). Horizontal axis, number of reads; vertical axis, PCR amplicon.</p

Minimum detection limit of the NGS mutation assay.

<p>Minimum detection limit of the NGS mutation assay.</p