Figure 1

<p>Phylogenetic relationships among the seven <i>Y. pestis</i> SNP genotypes identified in this study. Neighbor-joining tree was created using the data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000220#pone-0000220-t003" target="_blank">Table 3</a> and MEGA2 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000220#pone.0000220-Kumar1" target="_blank">[23]</a>, and was rooted on genotype 0, as this genotype was assigned to the two outgroup isolates (see text). The position of the 19 SNPs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000220#pone-0000220-t001" target="_blank">Table 1</a>) are indicated below the tree. Individual genotypes or nodes were named based upon their position relative to genotype 0.</p

Twenty-four <i>Yersinia</i> spp. strains used to screen FV-1/CO92 SNPs.

a<p>IP, Institute Pasteur; Dugway, Dugway Proving Grounds; CDH, California Department of Health Services; USAMRIID, United States Army Medical Research Institute of Infectious Diseases; ADHS, Arizona Department of Health Services; NAU, Northern Arizona University.</p>b<p>Molecular groups of <i>Y. pestis</i> based upon Achtman <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000220#pone.0000220-Achtman2" target="_blank">[5]</a></p>c<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000220#pone-0000220-t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000220#pone-0000220-g001" target="_blank">Figure 1</a>.</p

19 SNPs discovered in the comparison of the CO92 and FV-1 genome sequences.

<p>Bold = transversion mutation</p

Seven SNP genotypes identified in this study.

a<p>Genotypes are named based upon their phylogenetic position (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000220#pone-0000220-g001" target="_blank">Figure 1</a>).</p>b<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000220#pone-0000220-t001" target="_blank">Table 1</a>.</p>c<p>Number of screening isolates assigned to each genotype (24 total; see text).</p

Integrated Genomic Characterization Reveals Novel, Therapeutically Relevant Drug Targets in FGFR and EGFR Pathways in Sporadic Intrahepatic Cholangiocarcinoma

<div><p>Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the <i>FGFR2</i> locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (<i>in vitro</i> FGFR2 IC₅₀≈350 nM) was noted in a patient with an <i>FGFR2-TACC3</i> fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (<i>in vitro</i>, FGFR2 IC₅₀≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in <i>ERRFI1</i>, a direct negative regulator of <i>EGFR</i> activation. Rapid and robust disease regression was noted in this <i>ERRFI1</i> inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. <i>FGFR2</i> fusions and <i>ERRFI</i> mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.</p></div

Copy number changes and structural rearrangements.

<p>Whole genome data was utilized to determine copy number alterations and structural rearrangements in the genome for Patients 1–5. WGS was not conducted for patient 6. Red indicates copy number gain, green copy number loss and blue lines indicate structural rearrangements. Significant variability between samples was observed for both copy number changes and structural rearrangements. Patient 5 presented with numerous copy number changes and structural rearrangements contrasting with patient 4 who had minimal structural rearrangements and much smaller regions of copy number changes. Patient 3 is characterized by a large number of structural rearrangements with almost no copy number alterations; in contrast, Patient 1 has a moderate number of structural variations, but has large regions of copy number gain and loss. Patient 2 has a moderate number of structural rearrangements with multiple focal amplifications across the genome.</p

Anti-tumor activity of Patient 3, harboring an <i>ERRFI1</i> mutation, to erlotinib, an EGFR inhibitor.

<p><b>A</b>) CT images of patient 3 at baseline and three months demonstrate significant tumor shrinkage (red marks). CT demonstrates right retroperitoneal lymph nodes decreasing from 7.6 cm to 2.9 cm and left retroperitoneal lymph nodes decreasing from 3.3 cm to 1.7 cm. <b>B</b>) PET images of patient 3 at baseline and three months demonstrate significant tumor shrinkage (red arrows). Hypermetabolic areas corresponding to right retroperitoneal lymph nodes demonstrate decrease from 8 cm longest diameter to imperceptible and left retroperitoneal lymph nodes decreasing from 4.2 cm to 1.4 cm. Both regions demonstrated significant reduction in metabolic activity.</p

Summary of mutation type by patient.

<p>Summary of mutation type by patient.</p

<i>FGFR2-IIIb</i> fusion events.

<p>Transcripts and hypothetical protein products are modeled to illustrate the potential functional impact of fusion events involving <i>FGFR2</i> (<b>A–C</b>). The identified fusion events involving <i>MGEA5</i> (patient 4) (<b>A</b>) and <i>BICC1</i> (patient 5, reciprocal event) (<b>C</b>) are chromosome 10 intrachromosomal (<b>D</b>). In addition, patient 6 carried an interchromosomal fusion event (<b>D</b>) involving <i>FGFR2</i> and <i>TACC3</i> (<b>B</b>). The <i>FGFR2</i> gene encodes for several isoforms with eleven representative transcripts and patients 4, 5, and 6 carry fusions involving the epithelial cell specific transcript isoform (<i>FGFR2</i>-IIIb). All identified fusion breakpoints are close in proximity and are predicted to occur within the last intron of the transcript and terminal to a known protein tyrosine kinase domain (<b>A–C</b>, gold domain). Predicted “Other” sites for all of the fusion protein models are the same and include the following: Casein kinase II phosphorylation sites, N-glycosylation sites, Protein kinase C phosphorylation sites, N-myristoylation sites, Tyrosine kinase phosphorylation sites, and cAMP-/cGMP-dependent protein kinase phosphorylation sites (<b>A–C</b>, grey triangle annotations). In all cases, fusions result in a predicted expansion of Casein kinase II phosphorylation and Protein kinase C phosphorylation sites. A protein product model is shown only for one of the reciprocal events involving the <i>FGFR2</i> and <i>BICC1</i> genes (<i>FGFR2</i>→<i>BICC1</i>, <b>C</b>). The fusion breakpoints of the reciprocal events effect Exons 1 and 2 of the BICC1 gene, which translates to a difference of a predicted phosphoserine site within the Casein kinase II phosphorylation region (<b>C</b>, purple triangle within red circle). The FGFR2 gene is located within a fragile site region (FRA10F) and is flanked by two ribosomal protein pseudogenes, RPS15AP5 and RPL19P16 (see D inset (*)), whose repetitive sequence content may also contribute to genomic instability at the <i>FGFR2</i> initiation site.</p

Anti-tumor activity in Patient 6, harboring an <i>FGFR2-TACC3</i> fusion, to FGFR inhibitors.

<p><b>A</b>) CT images of patient 6, whose tumor possessed an <i>FGFR2-TACC3</i> fusion, at baseline and after four months of pazopanib demonstrate significant tumor shrinkage (red arrows), 10.8 mm and 3.1 mm respectively. <b>B</b>) CT images of patient 6 at baseline and two months demonstrate significant tumor shrinkage (red arrows), 41.1 mm and 39.4 mm respectively after subsequent ponatinib treatment, 45 mg/daily, was begun.</p