The treatment interval strongly influences the probability of tumor extinction.

<p>(<b>A</b>) Schematic representation of the modeled surgery time point followed by intravesical treatment time points (arrows). The symbol * indicates the time of transurethral resection (TUR) followed by the 2 weeks interval before BCG therapy starts. Three weekly instillations are given to initiate innate and adaptive immune responses. The time interval between the third through sixth treatment dose was varied and designated by <i>N</i> (as measured in weeks). (<b>B</b>) Probability of tumor extinction as a function of <i>N</i>, the inter-instillation interval during the 3^rd – 6^th treatment doses. The dotted line marks the outcome after the recommended interval of <i>N</i> = 1 week.</p

Simplified diagrammatic representation of our mathematical model.

<p>The dynamic model represents the interaction between healthy urothelial tissue, tumor cells, and their association with BCG (interaction depicted by green line), which in turn results in the generation of BCG-associated healthy tissue and tumor cells. The latter cell populations possess the capacity to trigger the activation of innate immune effector cells and in turn the priming of adaptive immune effector cells. Red lines ending in circles indicate the input stimuli that influence the recruitment of the indicated immune cell populations. Black arrows indicate either increase (e.g., recruitment or proliferation) or decrease (e.g., death or cell turn-over) of the respective cell population. Lines with blunt ends indicate direct killing of target cell populations (solid lines) or bystander death (dashed lines). The model was parameterized using available clinical and <i>in vivo</i> data and tuned to achieve 50% probability of tumor extinction after six weekly intravesical instillations of BCG. A more detailed version of this figure is provided by the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056327#pone.0056327.s001" target="_blank"><b>Figure S1</b></a> of the supplemental material.</p

Timing of BCG therapy, BCG dose and dwell time influence probability of tumor extinction.

<p>(<b>A</b>) Probability of tumor extinction with varying the time from surgery to the start of BCG therapy. The grey shaded area represents the time from surgery to the typical initiation of BCG therapy (i.e., 2 weeks). The cancer growth rate used is based on the clinical observation that high-grade lesions become visible by 3 months in the absence of treatment (detailed definition of tumor dynamics is provided in the supplementary information and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056327#pone.0056327.s002" target="_blank"><b>Figure S2</b></a>). The probability of tumor extinction after six weekly BCG instillations was modeled as a function of (<b>B</b>) BCG dose, and (<b>C</b>) BCG dwell time.</p

Association between (A) the maximum urinary calprotectin increase and ischaemic time and (B) the maximum urinary NGAL increase and ischaemic time in Group 1 (NSS with renal ischaemia) in a linear regression model, as assessed by Spearman’s correlation test.

<p>Regression lines are plotted. Linear regression model: A: R = 0.16, B = 103.61 (95% CI -219.96–427.19), p = 0.51; B: R = 0.09, B = 1.71 (95% CI -7.88–11.31), p = 0.71. Spearman’s correlation: A: R = 0.29 (95% CI -0.19–0.66), p = 0.21; B: R = 0.26 (95% CI -0.22–0.64), p = 0.26. NSS, nephron sparing surgery for kidney tumours; B, regression co-efficient, slope of the regression line; R, correlation co-efficient; CI, confidence interval.</p

Concentrations of (A) urinary calprotectin, (B) urinary neutrophil gelatinase—associated lipocalin (NGAL), (C) plasma creatinine, and (D) the estimated glomerular filtration rate (eGFR) in patients undergoing nephron sparing surgery (NSS) for kidney tumours with renal pedicle clamping (renal ischaemia, Group 1) or without renal ischaemia (Group 2), and in patients undergoing radical nephrectomy for living kidney donation (Group 3).

<p>Data are presented as median and interquartile range. Asterisks indicate significant changes from baseline in a non-parametric analysis of variance (Kruskal-Wallis test, corrected for multiple comparisons by Dunn’s test as a post hoc pairwise multiple comparison). p.o., post-operative. * p < 0.05, ** p < 0.01, *** p < 0.001.</p

Deep Clonal Profiling of Formalin Fixed Paraffin Embedded Clinical Samples

<div><p>Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.</p> </div

Whole exome sequencing of matching cell line and sorted FF and FFPE PDA tissues.

<p>A) Flow cytometry histograms and the detection of a 3.0N tumor population in FF (A10-46) and FFPE (A10_AT) tissues. B) Exomic sequencing coverage for 3.0N PDA genome from flow sorted FF (A10-46) and FFPE (A10_AT) tissues, and the matched tumor derived cell line A10_74. Left <u>></u>20×coverage, right <u>></u>30×coverage. The % coverage is based on Agilent SureSelect target regions.</p

Combined aCGH and whole exome analysis.

<p>Whole exome sequencing of sorted fresh frozen (FF) and formalin fixed paraffin embedded (FFPE) pancreatic ductal adenocarcinoma (PDA) tissue. A) Flow sorted 3.0N tumor population from PDA tissue. B) Homozygous <i>TP53</i> mutation in sorted FF and FFPE tissues, and matching cell line. C) Chromosome 17 CGH plot of 3.0N population from sorted FF sample. D) Heterozygous <i>KRAS</i> mutation in sorted FF and FFPE tissues, and matching cell line. E) Chromosome 12 aCGH plot of 3.0N population from sorted FF sample.</p

Whole genome comparison of aCGH results with matching sorted FFPE and FF samples.

<p>Flow sorting and aCGH analysis of matching fresh frozen (FF) and formalin fixed paraffin embedded (FFPE) samples from a pancreatic ductal adenocarcinoma. Flow sorting histogram and detection of 3.2N tumor population in A) FF tissue 11164 and B) FFPE tissue B3733. C–D) Whole genome aCGH plots of 3.2N population sorted from each tissue. Red arrow denotes highly aberrant region on chromosome 2. Black arrow denotes highly aberrant region on chromosome 9. Light blue shaded areas in lower left of A and B show sample and cell debris in flow data.</p

Gene-specific comparison of aCGH results with matching sorted FFPE and FF samples.

<p>Gene view comparison of ADM2 calls in matched fresh frozen (FF) and formalin fixed paraffin embedded (FFPE) pancreatic ductal adenocarcinoma sample. A) Chromosome 2p14 region includes a focal amplicon with the <i>MEIS1</i> gene (arrow). B) Chromosome 9p22.2 region includes a homozygous deletion of <i>CDKN2A</i> (black arrow) and a focal amplicon of <i>SH3GL2</i> (blue arrow). Shaded areas denote ADM2 defined copy number aberrant region.</p