Systematic Establishment of Robustness and Standards in Patient-Derived Xenograft Experiments and Analysis

Abstract

Patient-derived xenografts (PDX) are tumor-in-mouse models for cancer. PDX collections, such as the NCI PDXNet, are powerful resources for preclinical therapeutic testing. However, variations in experimental and analysis procedures have limited interpretability. To determine the robustness of PDX studies, the PDXNet tested temozolomide drug response for three prevalidated PDX models (sensitive, resistant, and intermediate) across four blinded PDX Development and Trial Centers using independently selected standard operating procedures. Each PDTC was able to correctly identify the sensitive, resistant, and intermediate models, and statistical evaluations were concordant across all groups. We also developed and benchmarked optimized PDX informatics pipelines, and these yielded robust assessments across xenograft biological replicates. These studies show that PDX drug responses and sequence results are reproducible across diverse experimental protocols. In addition, we share the range of experimental procedures that maintained robustness, as well as standardized cloud-based workflows for PDX exome-sequencing and RNA-sequencing analyses and for evaluating growth. SIGNIFICANCE: The PDXNet Consortium shows that PDX drug responses and sequencing results are reproducible across diverse experimental protocols, establishing the potential for multisite preclinical studies to translate into clinical trials.

Figure 1.

a) Three models were distributed for experimentation to 4 centers: Huntsman Cancer Institute/Baylor College of Medicine (HCI-BCM), MD Anderson Cancer Center (MDACC), Washington University-St. Louis (WUSTL), and The Wistar Institute/University of Pennsylvania/MDACC (WIST). These three centers were chosen based on prior results on temozolomide treatment response obtained by the NCI Patient-Derived Models Repository (PDMR). b) Each of the three models were treated with temozolomide by the 4 centers under blinded protocols. c) Treatment responses were comparatively assessed under several biostatistical protocols. d) Sequence data were collected by each center and assessed.

Figure 2.

Comparison of PDX tumor volume control and temozolomide treatment arms at the PDMR (a-c), HCI-BCM (d-f), MDACC (g-i), WIST (j-l), and WUSTL (m-o). Model 625472–104-R (a, d, g, j, m), 172845–121-T (b, e, h, k, n), and BL0293-F563 (c, f, i, l, o). Axes are held constant for comparison between studies. Dashed lines, vehicle control groups, Solid lines, temozolomide treatment groups. Median ± SD. For statistical assessments, see Figure 3 and Table 2. (PDMR - NCI Patient-Derived Models Repository, HCI-BCM - Huntsman Cancer Institute/Baylor College of Medicine, MDACC - MD Anderson Cancer Center, WUSTL - Washington University-St. Louis, and WIST - The Wistar Institute/University of Pennsylvania/MDACC.)

Figure 3.

Analytical Summaries, HCI-BCM Study. Analytical results from HCI-BCM study for progressive model (625472–104-R), stable disease model (172845–121-T) and complete response model (BL0293-F563) (columns 1, 2, and 3 respectively), with interpolated individual curves (row 1), mean curves for treatment and control with 95% confidence bands (row 2), waterfall plots demonstrating ΔV₂₁(row 3), boxplots of aAUC₂₁(row 4) and aAUC_max(row 5) for treatment and control, and a boxplot of TGI₂₁(row 6), along with p-values comparing treatment to control for each measure.

Figure 4.

Workflow Benchmarking and Analysis Summary. a) Panel A shows results of the evaluation of mouse-human disambiguation tools (Xenome, BBSplit, Disambiguate, ICRG, XenofilteR). Each figure shows precision (blue) and recall (green) for a simulated data. Left figure shows results of mouse disambiguation for whole exome data. Right figure shows results of mouse disambiguation for RNA-seq data. b) The panel shows the wiring diagram for the whole exome workflow selected to process data for this study. The selected workflow was selected from 5 workflows submitted by the PDTCs. Wiring diagrams for submitted whole exome workflows submitted by the PDX Development and Trials Centers. Wiring diagrams include nodes and connections. Nodes depict inputs - , outputs - , tools - , and workflows - . Connections between nodes depict that input to a node is from the output of another node. Orange nodes - identify a tool or a workflow with an available update. c) Panel shows performance evaluations of five workflows submitted by the PDTC. Each workflow was evaluated by SNP (top), INS (middle), and DEL (bottom) with a range of variant allele frequencies (0.025, 0.05, 0.3, 0.2, 0.3). Each plot shows recall and precision respectively on the x and y axis. Results for each of the workflow are shown with the same color: Workflow 1- blue, Worfklow 2 – green, Workflow 3- light blue, Workflow 4 – purple, and Workflow 5 – black d) A Venn diagram showing the overlap in high-quality variant calls for model BL0293-F563 by model using intersected array and removing lower allele frequency (AF) calls. e) Dendrogram of median polish by center (by MBatch) using TMM normalized count per million values. Foe d) and e), HCI-BCM -Huntsman Cancer Institute/Baylor College of Medicine, MDACC -MD Anderson Cancer Center, WUSTL -Washington University-St. Louis, and WIST -The Wistar Institute/University of Pennsylvania/MDACC.