Expression levels and mutation status of tumor suppressor pathway genes.

<p>(A) Integrated analysis of <i>mRNA Expression</i>, <i>Mutations</i>, and <i>Copy Number</i> was performed for genes in the <i>TP53</i> and <i>RB1</i> tumor suppressor pathways. Gene-level expression (FPKM) values for pathway member genes are presented and relative gene expression for each gene across the panel of PDX models is indicated by the heatmap (green = lower than the median, red = greater than the median). Mutations (amino acid changes) in <i>TP53</i> and <i>RB1</i> are indicated. Gene copy numbers are presented, and variants indicated as losses (<i>i</i>.<i>e</i>., < 2) or gains (<i>i</i>.<i>e</i>., > 2) shown by darker shades of red and green, respectively. (B) Expression levels (FPKM) of tumor suppressor pathway genes in each PDX are shown in the bar graph.</p

Conservation of genomic variants between patient bladder cancer specimens and PDXs.

<p>WES analysis was performed on genomic DNA samples isolated from parental patient tumors and engrafted PDX P0 tumors. WES data was filtered for variants occurring at a frequency of >0.5 and then compared between samples on the basis of variant types. The number and type of variants occurring in each parental tumor and in the P0 PDX tumor were quantified and depicted as percentage of conservation in the graph. For all variants, 91.8% and 97.6% were conserved in BL0429 and BL0440, respectively.</p

Efficacy test of molecularly guided targeted therapy matched with aberrations.

<p>(A) Efficacy studies of molecularly guided targeted therapy. BL0269 overexpressed HER2 and Src. Compared to the control group of median progression-free survival (PFS) of 13 days, a HER2 inhibitor lapatinib and a Src inhibitor ponatinib had little effect in suppressing tumor growth with a median PFS of 12 (p = 0.16) and 18 (p = 0.11) days, respectively. In contrast, lapatinib was very effective in BL0440 that expressed both HER2 and HER3 with PFS of 25.4 days versus 18.4 days in the control group (p = 0.0007). In BL0269 which also harbored a PIK3CA activation mutation H1047R, a PIK3CA inhibitor BEZ235 significantly suppressed tumor growth (p<0.0001). (B) Studies of efficacy and secondary resistance mechanisms of BGJ398 in BL0293. BL0293 overexpressed FGFR3. Compared to the control group of PFS at 9.5 days, an FGFR3 inhibitor BGJ398 significantly prolonged PFS to 18.5 days (p = 2. 61 X 10⁻⁶). Mice were sacrificed and PDXs were harvested (red arrows) before treatment, at Day 3 and at Day 17 (time of resistance) for western blot. Low levels of p-Akt and p-Erk at the baseline and at Day 3, suggesting low downstream signaling activity of FGFR3. Upon the development of resistance to BGJ398 at Day 17, both p-Akt and p-Erk levels increased, suggesting re-activation of the downstream signaling activity. BGJ398-resistant PDX BL0293 was re-implanted in NSG mice to form xenografts. Mice carrying BGJ398-resistant BL0293 were treated with PBS control, BGJ398, or a Raf inhibitor sorafenib plus a PIK3CA inhibitor BEZ235 combination. Compared to the BGJ398 group, treatment of BGJ398-resistant PDX with sorafenib and BEZ235 significantly prolonged PFS from 12 days to 22 days (p = 0.001). (C) Screening for effective chemotherapeutic agents. The first six PDXs were tested for sensitivity to cisplatin, gemcitabine or combination of both drugs. Only BL0440 was sensitive to cisplatin while only BL0269 and BL0479 were resistant to gemcitabine. Resistance to cisplatin could be overcome by gemcitabine, leaving four of these six PDXs sensitive to the combination therapy.</p

Morphology of PDX.

<p>(A) Comparison of morphology between patient specimens and PDXs, subcutaneous and orthotopic PDXs, of PDX BL0293 and BL0440. Hematoxylin and eosin stain (H & E stain) showed that cell morphology was maintained during establishment of PDX and during passaging in mice, both at the subcutaneous site (S.C., at passage 6) and at the orthotopic bladder wall (at passage 4). However, more mitotic cells were observed in PDXs, especially at passage six. (B) Staining of human Ki67. Both PDXs were stained positive with anti-human Ki67, supporting that these PDX cells were indeed of human origin. In PDX BL0440, more cells were stained positive with Ki67 at passage six PDX compared to the human bladder cancer specimen, suggesting more cells were in cell proliferation, and this finding was consistent with the observation of more mitotic cells in Panel A of H & E staining. (C) Staining of human vimentin. Some human bladder cancer cells (left panel) and stromal cells (middle panel) were stained positive for human vimentin in the patient specimens. In the PDX specimen at passage 0, only a few bladder cancer cells were stained positive for human vimentin, suggesting that the stromal cells in PDX were not derived from human stromal cells.</p

An integrated PDX para-clinical platform to improve molecularly guided targeted therapy.

<p>① Patients with muscle-invasive or advanced bladder cancer undergo transurethral or surgery resection. Some tumor specimens are implanted into NSG mice to establish PDXs. ② When the first PDXs (Passage 0 or P0) are established, some PDX specimens are implanted to more NSG mice to establish more PDXs (P1) while some other specimens are submitted for deep sequencing, including whole genome, exome and transcriptome sequencing. ③ Patients and mice carrying P1 PDXs receive the same primary treatment. ④ Computational biology is used to identify genetic aberrations in cancer cells. ⑤ Validation tests, such as direct sequencing, western blot, IHC and immunofluorescence staining, are performed to validate the genetic aberrations as identified in Step ④. ⑥ Because of the subcutaneous location and fast tumor growth in mice, tumor relapses in PDXs will occur sooner than that in human patients. Upon relapse, mice carrying PDXs will be treated with regimens A-D targeting 4 genetic alterations identified in Steps ④ and ⑤. In addition, serial biopsies can be performed during treatment. These biopsy specimens can be studied to decipher the secondary resistance mechanisms and guide the development of further therapy to overcome resistance. ⑦ Since only some of the genetic alterations are driver ones, some medications (Drug B and C) can induce response while PDXs do not respond to some other treatments (Drug A and D). ⑧ Upon cancer relapse in human patients, biopsy can be performed to validate the existence of genetic aberrations in the relapsed cancer, then the effective drug (Drug C) matching to the genetic aberrations in human cancers is used to treat relapsed cancer in patients. ⑨ Drug C can potentially be used while patients are in remission to cure or delay cancer recurrence.</p

Mutation status of known bladder cancer functionally active genes and significantly mutated genes in PDXs.

<p>WES was performed on P0 tumors from each PDX model. Subsequently, the GATK was utilized for variant detection and functional annotation. The results were filtered for somatic mutations occurring in a consolidated list of 130 known bladder cancer functionally active genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134346#pone.0134346.ref027" target="_blank">27</a>] and significantly mutated genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134346#pone.0134346.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134346#pone.0134346.ref025" target="_blank">25</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134346#pone.0134346.ref026" target="_blank">26</a>]; these are indicated in the right side panel as <i>BLCA Driver (IntOGen)</i>, <i>TCGA Significantly Mutated</i>, or <i>BGI Significantly Mutated</i>. Somatic mutations were found in 51 different genes (indicated along the side of the table) <b>(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134346#pone.0134346.s006" target="_blank">S3 Table</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134346#pone.0134346.s007" target="_blank">S4 Table</a>)</b>. The PDX model number is indicated across the top of the grid. Frequencies (0.3–1.0) for each mutant allele are indicated for each variant and represented by increasing color intensities.</p

Expression levels and mutation status of tumor suppressor pathway genes.

<p>(A) Integrated analysis of <i>mRNA Expression</i>, <i>Mutations</i>, and <i>Copy Number</i> was performed for genes in the <i>TP53</i> and <i>RB1</i> tumor suppressor pathways. Gene-level expression (FPKM) values for pathway member genes are presented and relative gene expression for each gene across the panel of PDX models is indicated by the heatmap (green = lower than the median, red = greater than the median). Mutations (amino acid changes) in <i>TP53</i> and <i>RB1</i> are indicated. Gene copy numbers are presented, and variants indicated as losses (<i>i</i>.<i>e</i>., < 2) or gains (<i>i</i>.<i>e</i>., > 2) shown by darker shades of red and green, respectively. (B) Expression levels (FPKM) of tumor suppressor pathway genes in each PDX are shown in the bar graph.</p