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.

Patient-derived xenografts (PDX) are in vivo preclinical models in which human cancers are engrafted into a mouse for translational cancer research and personalized therapeutic selection (1–4). Prior studies have shown that treatment responses of tumor-bearing mice usually reflect the responses in patients (5, 6). PDXs have been used successfully for preclinical drug screens (4, 5), to facilitate the identification of potential biomarkers of drug response and resistance (4, 7), to select appropriate therapeutic regimens for individual patients (8), and to measure evolutionary processes in cancer in response to treatment (9). At the genomic level, engrafted human tumors have been shown to retain most genomic aberrations from the original patient tumor (8, 10). These successes have led to the development of a number of PDX collections in both academia and industry (5, 11, 12) for use in preclinical testing.

Despite these successes, important questions remain for the use of PDXs as a model system for treatment response. The reproducibility of treatment response has not been well-evaluated because research teams often perform experiments in models that are not used by other groups. Variations in engraftment, dosing, and response assessment protocols also frustrate comparisons of results. Moreover, intratumoral heterogeneity, genetic drift and selection during tumor collection, engraftment, and xenograft passaging can result in genomic variation among primary tumor samples and derived xenografts (10, 13). Whether such variation impacts the accuracy of PDXs as a preclinical model has been unclear. Resolution of this issue requires not only controlled treatment replicates, but also standardized PDX-specific sequence analysis pipelines to robustly identify genomic aberrations. Progress on these topics is important to the overall field of cancer patient–derived models, as analogous concerns pertain for organoids and other three-dimensional culture systems.

To resolve such questions for the use of PDXs in precision medicine, the NCI has supported a consortium of PDX-focused research centers, the NCI PDXNet. Here, we in the PDXNet consortium report the results of experiments to test the robustness of PDX treatment responses across different research centers, using temozolomide treatment on three models because of prior data on their temozolomide responses from the NCI Patient-Derived Models Repository (PDMR). We report on replicate evaluations across four additional PDX Development and Trials Centers (PDTC) using blinded treatment and response evaluation protocols. Simultaneously, we have performed exome (exome-seq) and RNA sequencing (RNA-seq) at each center to determine biological and technical stability of genomic characterizations of samples from each center. These sequence analyses have been performed with optimized analysis pipelines chosen based on an extensive new benchmarking of pipelines from each center on synthetic sequence sets. Finally, we have statistically analyzed the cohort growth curves for each model in each research center using five separate metrics. These studies allow us to answer whether PDXs have sufficiently robust behaviors to withstand variations in experimental procedures, response measurement algorithms, genomic variation among replicates, and alternative sequence analysis protocols. We also report effective standard operating procedures (SOP) for experimental procedures, pipelines for statistical assessment of response, and sequence analysis workflows. We expect these standards to advance the use of PDXs and other in vivo models in cancer precision medicine, a critical need for the evaluation of PDX results in the context of moving novel therapeutics or therapeutic combinations to the clinic.

### Animal models

Three PDX models were selected based solely on their temozolomide responsiveness. They were 625472-104-R (colon adenocarcinoma), 172845-121-T (colon adenocarcinoma), and BL0293-F563 (urothelial/bladder cancer). Cryopreserved PDX tumor fragments were shipped from the PDMR to the individual PDTCs including 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), implanted for initial expansion and then passaged for the preclinical study. Briefly, cryopreserved PDX material was prepared into implantation size pieces as outlined in Table 1. The PDX material plus a drop of Matrigel (BD Biosciences) was then implanted subcutaneously in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) host mice. Mice were housed in sterile, filter-capped polycarbonate cages, maintained in a barrier facility on a 12-hour light/dark cycle, and were provided sterilized food and water, ad libitum. Animals were monitored weekly for tumor growth. The initial passage of material was grown to approximately 1,000–2,000 mm3 calculated using the following formula: tumor volume (mm3) = [tumor length × (tumor width)2]/2 (14). Tumor material was then harvested, a portion cryopreserved, and the remainder implanted into NSG host mice for the preclinical drug study. Related patient data, clinical history, representative histology, and short tandem repeat profiles for the PDX models can be found at https://pdmr.cancer.gov; model BL0293-F563 was originally developed by The Jackson Laboratory (tumor model TM00016, http://tumor.informatics.jax.org/mtbwi/pdxSearch.do).

Table 1.

Comparison of preclinical study set-ups and end-points at the PDMR and individual PDTCs for the temozolomide reproducibility pilot.

PDMRHCI-BCMMDACCWUSTLWIST
Implantation
Implantation type ∼1 mm3 Fragment ∼1 mm3 Fragment ∼1 mm3 Fragment ∼3.0 × 106 cells, dissociateda <1 mm3 fragments in slurry, ∼150 μL of slurry implanted
Implantation site Subcutaneous, single flank Subcutaneous single flank Subcutaneous single flank Subcutaneous single flank Subcutaneous single flank
Staging site (mm3200 100–200 200 200 100
Cohort size 10 10
Dosing and schedule
Temozolomide dose (mg/kg) 50 50 50 50 50 and 100
Schedule QD×5 QD×5 QD×5 QD×5 QD×5
28-day cycle 28-day cycle 28-day cycle 7-day cycle 7-day cycle
Number of cycles of treatment
Route of administration Oral Oral Oral Oral Intraperitoneal
Study endpoints
A Animal health Animal health Animal health Animal health Animal health
B Max. tumor size, 4,000 m3 Max. tumor size, 4,000 m3 Max. tumor size, 1,600–2,000 m3 Max. tumor size, 1,500 m3 Max. tumor size, 1,500 m3
C 300 days, if max. TV not reached 0.5 cycles after last dose When control TV, 1,600–2,000 mm3 4 weeks after last dose When control arm TV, 1,500 mm3
PDMRHCI-BCMMDACCWUSTLWIST
Implantation
Implantation type ∼1 mm3 Fragment ∼1 mm3 Fragment ∼1 mm3 Fragment ∼3.0 × 106 cells, dissociateda <1 mm3 fragments in slurry, ∼150 μL of slurry implanted
Implantation site Subcutaneous, single flank Subcutaneous single flank Subcutaneous single flank Subcutaneous single flank Subcutaneous single flank
Staging site (mm3200 100–200 200 200 100
Cohort size 10 10
Dosing and schedule
Temozolomide dose (mg/kg) 50 50 50 50 50 and 100
Schedule QD×5 QD×5 QD×5 QD×5 QD×5
28-day cycle 28-day cycle 28-day cycle 7-day cycle 7-day cycle
Number of cycles of treatment
Route of administration Oral Oral Oral Oral Intraperitoneal
Study endpoints
A Animal health Animal health Animal health Animal health Animal health
B Max. tumor size, 4,000 m3 Max. tumor size, 4,000 m3 Max. tumor size, 1,600–2,000 m3 Max. tumor size, 1,500 m3 Max. tumor size, 1,500 m3
C 300 days, if max. TV not reached 0.5 cycles after last dose When control TV, 1,600–2,000 mm3 4 weeks after last dose When control arm TV, 1,500 mm3

Abbreviations: QD×5, once daily for 5 days; TV, tumor volume.

aThis is an average across WUSTL models. The numbers of implanted cells per mouse for each model are BL0293-F563: 4.0 × 106; 172845-121-T: 2.6 × 106; and 625472-104-R: 2.5 × 106.

### Preclinical studies

Specific tumor staging size, implantation method, and cohort size at the PDMR and each PDTC are outlined in Table 1 based on each site's standard practices. In general, tumors were staged to a preselected size (weight = 100–200 mm3). Tumor-bearing mice were randomized before initiation of treatment and assigned to each group. Body weight was monitored 1–2 times weekly and tumor size was assessed 2–3 times weekly by caliper measurement. For all sites, drug studies were performed at passage 3 for 625472-104-R, passage 4 for 172845-121-T, and passage 6 for BL0293-F563 (passage 0 = first implanted host). Temozolomide (NSC 362856) was obtained from the Developmental Therapeutics Program, NCI and administered at the times and doses indicated in Table 1. Animals were sacrificed when the tumors reached an individual PDTC's animal welfare endpoint or a maximum tumor size; if tumor growth delay was observed, a tertiary endpoint was used by some sites (Table 1).

### Ethics statement

The Frederick National Laboratory for Cancer Research (location of the PDMR) is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and follows the USPHS Policy for the Care and Use of Laboratory Animals. All the studies were conducted according to an approved animal care and use committee protocol in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council; 1996; National Academy Press; Washington, D.C.).

All patients and healthy donors gave written informed consent for study inclusion and were enrolled on institutional review board–approved protocols of record for the sites that developed the PDX models (DCTD, NCI, and The Jackson Laboratory). The study was performed in accordance with the precepts established by the Helsinki Declaration. The study design and conduct complied with all applicable regulations, guidances, and local policies and was approved by the institutional review board of record for each PDTC.

### Statistical analysis of tumor growth data

There is not a single consensus in literature in terms of which endpoint to use to measure tumor response in PDX models. There are a number of potential options. Rather than considering just one, our strategy was to consider a wide range of potential analytic strategies, each of which captures different aspects of the response and has its own strengths and weaknesses. Analytic strategies for evaluating tumor growth data include Percent Change in tumor volume (⁠{\Delta _{{{\bm{V}}_{{\bm{t}}{\rm{\ }}}}}}$⁠, normalized relative to starting volume before treatment), Area under the tumor growth curve up to time t (⁠aAU{C_t}$⁠), Adjusted area under the curve (⁠aAU{C_{max}}$⁠), RECIST criteria (⁠RECIS{T_{t,c}}$⁠). Metrics computed to evaluate antitumor activity of the treatment group compared with the control group include tumor growth inhibition (⁠TG{I_t}$⁠) and progression-free survival (⁠PF{S_\delta }$⁠; See Supplementary Materials S1 and S2; Supplementary Table S1; Supplementary Figs. S1–S10 for details, including percentages and parameters used to classify tumor response). Here, we compare and contrast these metrics in this pilot study and assess the robustness of sensitivity assessments across different analytic strategies, with the goal of making recommendations for the broader community. Toward this goal, we built an R analysis pipeline that computes all of the following measures as well as generates a set of useful graphical summaries.

One-way ANOVA or two-sample t tests were performed to test the difference of tumor volume changes (⁠{\Delta _{{V_t}}}\$⁠) at day t = 21 between treatment and control groups as appropriate, and similar analyses were done for the AUC measures. Fisher exact test was performed to test the association between treatment and drug response (non-PD vs. PD). The logrank test was used to compare PFS distributions between treatment and control groups. All of the analysis was implemented using R.

We have developed an R markdown script that can be used to automatically run these analyses and produce summary plots given the input data is formatted as described in Supplementary Material S1 (E-mail cgc@sbgenomics.com to request the R script that we freely share with this publication for other researchers to use to analyze their PDX data).

### Computational workflows

All analyses were performed on the Cancer Genomics Cloud (CGC, https://cgc.sbgenomics.com/; ref. 15) with workflows and tools implemented using Common Workflow Language. Human and mouse data were aligned to GRCh38 and mm10 assemblies, respectively. All workflows are available in the Temozolomide Pilot Workflows Project on the CGC). CGC users can request access to the workflows by e-mailing cgc@sbgenomics.com.

We compared several tools for mouse-human read deconvolution. These were Xenome (v1.0.0; ref. 16), BBSplit (v37.93; https://sourceforge.net/projects/bbmap/), Disambiguate (v1.0; commit c52402a; https://sourceforge.net/projects/bbmap), ICRG (17), and XenofilteR (v1.5; ref. 18). For the WES data benchmark and the RNA-seq benchmark, we, respectively, used experimental WES series and RNA-seq data to simulate human-mouse mixture for evaluation. For tools requiring aligned data inputs (BAM Files), BWA-Mem was used for alignment. Only reads unambiguously classified as human by a tool were labeled “human.” All other reads were considered “not human” for the true/false positive/negative calling. See Supplementary Materials S3 for additional details.

### Tumor-normal WES variant calling

Five tumor-normal WES data analysis workflows from PDXNet research groups were tested on the benchmark sets, as detailed in (Supplementary Tables S2 and S3; Supplementary Figs. S11–S13), with the goal of evaluating the accuracy in the presence of variable mouse contamination, coverage, and variant allele frequency (VAF). Starting from FASTQ data the workflows performed mouse-human disambiguation, alignment, and variant calling with one or more somatic variant callers [Mutect (19, 20), VarScan (21), Strelka (9), Manta (22), and Pindel (23)]. Two simulated whole exome-seq (WES) datasets were used in the benchmark for the tumor-normal variant calling workflow. The first dataset (DN) was prepared by researchers from HCI-BCM and consisted of data based on two normal samples, with variants from ClinVar spiked in and with 10% and 50% mouse contamination. The second dataset (BS) was NA12878 WES data contaminated with 10% mouse reads, which was spiked with BamSurgeon [i] at 0.05, 0.1, 0.2, and 0.3 VAF using both the ClinVar variant set used for DN, variants from The Cancer Genome Atlas BRCA SNPs combined, and with indels from the ClinVar set (BS-BRCA). For all the submitted workflows, default parameters were used as specified by the workflow authors (see Supplementary Materials S4–S6 for additional details). All workflows are accessible through the CGC upon request.

### Tumor-only WES variant and CNV calling

Because a substantial number of PDXs among the broader research community lack matched normal DNA, we also developed a workflow for tumor-only mutation calling (Supplementary Fig. 14). Preprocessing steps include quality control filtering, removing adaptors, mouse reads were removed with xenome, trimmed reads were aligned to human genome (build GRCh38.p5), duplicate reads were removed with PicardTools, and BaseRecalibrator from the Genome Analysis Tool Kit (GATK) v4.0.5.1 (24, 25) was used to adjust the quality of raw reads. Variants were called in Mutect2 using the Exome Aggregation Consortium (26) database lifted over to GRCh38 as a germline reference with the allele frequency of samples not in reference set to 0.0000082364. Variant calls were then filtered using GATK FilterMutectCalls v 4.0.5.1. See Supplementary Materials 6 for additional details. Workflow is available from the CGC upon request.

To call copy number, we built a pooled normal reference using CNVkit v0.9.3 (27) from the three samples that used the same exome-seq capture kit and with sex matching. Afterward we used CNVkit to call the CNV segments from each sample using the pooled normal reference. MDACC samples exhibited low mean target coverage so we turned on the –drop-low-coverage option in CNVkit to reduce the noise in the CNV profile.

### RNA-seq expression calling

Because the disambiguation of mouse and human reads was sharp for both DNA and RNA data, we did not expect expression calling workflows to have issues specific to PDXs. Therefore, we dockerized only one PDX RNA-seq expression workflow (Supplementary Material S7; Supplementary Fig. S14) that was submitted by The Jackson Laboratory (JAX). The transcriptomes of hg38 and NOD (based on the mm10 mouse genome) were used to construct the xenome (version 1.0.0; ref. 16) indices (k = 25), and then reads were classified as human, mouse, both, neither or ambiguous at default xenome parameters. Reference indices for the alignment were built by rsem-prepare-reference using ENSEMBL annotation (version GRCh38.91) for STAR aligner (version 2.5.1b; ref. 28). Human-specific reads were mapped to reference indices using STAR, and expression estimates were computed using rsem-calculate-expression v1.2.31 (29) at default parameters. Picard CollectRnaSeqMetrics: (broadinstitute.github.io/picard/picard-metric-definitions.html) was used to calculate the post-alignment mapping statistics. An implementation of this workflow has been deployed on the CGC.

### Comparisons of xenograft sequence data across PDTCs

Each PDTC submitted WES and RNA-seq data from untreated xenografts that had been successfully grown in mice at the respective sites (Supplementary Tables S4–S7; Supplementary Figs. S15–S22). These data are available through the Sequencing Read Archive at accession number PRJNA608267. Groups were asked to submit xenograft sequence data according to their standard practices, without prespecification of the sample passage number or the sequencing protocol. In the intersection analysis, only variants with allele frequency > 0.2 were retained. We note that MDACC had fewer calls that passed the allele frequency filter in comparison with other centers. This is because MDACC-provided samples had mean target coverage approximately 30×, whereas samples from other centers were sequenced to a depth of approximately 150× (Supplementary Table S7). We also analyze mutational differences in cancer-related genes, using the CancerMine database: http://bionlp.bcgsc.ca/cancermine/. We listed the top 15 genes, by citation count, associated with each of the terms cancer driver, oncogene and tumor suppressor from the database and then combined these to get 33 unique cancer genes (Supplementary Figs. S17–S19).

For the copy number comparisons, the copy number alteration (CNA) segments obtained from CNVkit using a pooled normal were median-centered and visualized in IGV v2.4.13 (30). To determine the overall concordance of the CNA between each pair of samples, we first intersected the CNA segments for each pair of samples and then binned them into 100 kb windows using Bedtools v2.26.0 (31).

RNA-seq data provided by each center were generated using different kits and protocols, and the data from HCI-BCM was sequenced in single end mode (Supplementary Table 6). Sequencing data were analyzed with the “PDXnet RNA Expression Estimation” and the “PDXnet RNA Expression Estimation – SE” workflows on the CGC. RNA expression estimates were downloaded from CGC for additional analyses. The single-end data provided by HCI-BCM yielded estimates of RNA expression that were twice as high when compared with the paired-ended sample provided by other centers due to differential handling of paired-end and single-end data by RSEM (29) tool. To eliminate the biases in the count estimation across centers, HCI-BCM–estimated transcript counts were divided in half. From the mapping stats and from automatic library type detection algorithm in the tool Salmon, we noted that RNA-Seq library generated at MDACC are nondirectional although the sequencing protocol used is for directional library, thus we decided to consider MDACC library as nondirectional during the analysis.

### Study design and treatment results

A critical, yet unresolved, question that motivated the inception of the PDXNet was what the interlaboratory reproducibility of PDX drug studies would be across centers with independently established practices for preclinical testing, that is, how much standardization would be needed to run large-scale, multicenter preclinical studies. To address this question, the NCI Patient Derived Models Repository (PDMR) reviewed preclinical studies performed by the Biological Testing Branch (BTB/DCTD/NCI), which has performed numerous in vivo studies with PDX models. The PDMR selected three PDX models with nonpublished known responses to temozolomide for an inter-laboratory reproducibility pilot. The three PDX models selected were 625472-104-R (colon adenocarcinoma, nonresponsive model), 172845-121-T (colon adenocarcinoma, intermediate response), and BL0293-F563 (urothelial/bladder cancer, complete response). Patient data, clinical history, and representative histology and sequence data can be found at https://pdmr.cancer.gov.

For the study set-up (Fig. 1A), the four PDTCs – HCI-BCM, MDACC, WUSTL, and WIST – were directed to use their standard preclinical study set-up (Fig. 1B) and monitoring SOPs (Fig. 1C) and to use literature searches to determine temozolomide dosing and schedule. Each group also performed exome and RNA-seq of untreated tumors that had been successfully engrafted (Fig. 1D). All PDTCs were kept blinded to which models were temozolomide sensitive or resistant and to all other groups' preclinical study set-ups. In addition, none of the PDTCs had previous experience with temozolomide; so the reference doses/schedules would need to be determined independently at each center. The exceptions to blinding were that all PDTCs were required to use NSG host mice and implant PDX material subcutaneously. In addition, the PDTCs used drug prepared by the Developmental Therapeutics Clinic (DTP/NCI) to ensure that there were no variations in manufacture.

Figure 1.

A, Three models were distributed for experimentation to four centers: HCI-BCM, MDACC, WUSTL, and WIST. These three centers were chosen based on prior results on temozolomide treatment response obtained by the NCI Patient-Derived Models Repository. B, Each of the three models were treated with temozolomide by the four 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 1.

A, Three models were distributed for experimentation to four centers: HCI-BCM, MDACC, WUSTL, and WIST. These three centers were chosen based on prior results on temozolomide treatment response obtained by the NCI Patient-Derived Models Repository. B, Each of the three models were treated with temozolomide by the four centers under blinded protocols. C, Treatment responses were comparatively assessed under several biostatistical protocols. D, Sequence data were collected by each center and assessed.

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The laboratory SOPs for the preclinical study set-ups were collated by the PDMR (Table 1). While all centers staged tumors to between 100 and 200 mm3, implantation methodologies varied. Three groups directly implanted approximately 1 mm3 PDX fragments into each host mouse, one group minced an approximately 1 mm3 PDX fragment into a slurry for implantation, and one dissociated PDX material and implanted 3–5 × 106 cells per host (for each model all hosts had the same number of cells injected in all control and treated animals; variation was only across models). Comparison of vehicle control growth curves for all groups demonstrated overall similar growth kinetics of the models at each site irrespective of the implantation methodology used (Supplementary Fig. S1).

Each PDTC independently researched published literature to select a temozolomide dosing and schedule for its site, with key references noted: HCI-BCM (32–34), MDACC (35), WUSTL (34, 36–39), and WIST (40, 41). While diverse literature was considered, all sites selected a 50 mg/kg dose and one of two different dosing schedules. These schedules were either daily temozolomide treatment for 5 days followed by 23 days of rest (28-day cycle) or 5 days of treatment followed by 2 days of rest (7-day cycle); 1–4 cycles were used (Table 1).

Overall, all sites reported similar responses irrespective of the methodology, dosing, or schedule used (Fig. 2AO), with especially strong concordance in the nonresponsive and complete response model results, as detailed quantitatively below. If the drug × model combination had been performed as part of an exploratory study, these independent experiments would likely yield similar decisions about treatment efficacy. The intermediate response models showed more variation in growth across centers. The intermediate cases were also more clearly affected by the variability in SOP endpoint times, one of the biggest variations among methodologies (Table 1). For example, some groups sacrificed all mice once the vehicle control group reached a threshold volume, while other groups ended after a defined length of time after the last dosing. This resulted in some studies observing strong tumor inhibition through the end of study, while others observed regrowth after initial inhibition (Supplementary Fig. S2). Nevertheless, the similarities in response indicated that the existing range of methodologies is sufficient and robust enough to capture the critical cases of strong response and nonresponse. After discussion of these results, the PDXNet Consortium has agreed on a standard of continued monitoring of all cohorts where response is observed for at least 1.5–2 cycle lengths beyond the last dosing cycle, provided animal health end-points are not reached. Detailed quantitative comparisons and statistical analysis are addressed in the next section.

Figure 2.

Comparison of PDX tumor volume control and temozolomide (TMZ) treatment arms at the PDMR (A–C), HCI-BCM (D–F), MDACC (GI), WIST (J–L), and WUSTL (M–O). Model 625472-104-R (A, D, G, J, and M), 172845-121-T (B, E, H, K, and N), and BL0293-F563 (C, F, I, L, and 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 Fig. 3 and Table 2.

Figure 2.

Comparison of PDX tumor volume control and temozolomide (TMZ) treatment arms at the PDMR (A–C), HCI-BCM (D–F), MDACC (GI), WIST (J–L), and WUSTL (M–O). Model 625472-104-R (A, D, G, J, and M), 172845-121-T (B, E, H, K, and N), and BL0293-F563 (C, F, I, L, and 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 Fig. 3 and Table 2.

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### Statistical robustness of PDX treatment response

#### Statistical approaches for evaluating cohort drug response

A challenge of evaluation of PDX response is that there is still no standard statistical approach for analysis of tumor response for PDX growth data. Common measures of tumor size include percent change in volume from baseline to a fixed time endpoint; area under the tumor growth curve; tumor growth inhibition, defined as the ratio of the average tumor size at a given time point relative to control; and time to progression, a potentially censored endpoint measuring time from baseline until growth to a certain multiple of baseline. Classification of growing PDX tumors into RECIST-like categories (complete response, CR; partial response, PR; stable disease, SD; and progressive disease, PD; ref. 42) is another assessment that has the advantage of congruence with clinical trials, but it can be strongly dependent on category thresholds that do not analogize straightforwardly with patient primary tumors. Each of these measures has their own strengths and limitations. For example, the percent change from baseline is intuitive, interpretable, and unlike RECIST does not require specification of a cut-off point. In contrast to the AUC approaches, it does not use all of the tumor time course information but only the first and last points. Here we consider all of these measures and assess concordance of results across analytic strategies as well as across growth data from each center.

#### PDX tumor volume analysis software

We have devised an automated analysis script in R that, given data in a prespecified format and a time point of interest, will automatically plot the tumor growth curves and group mean curves, compute all of these statistical measures and their associated plots, and produce an annotated .html report in R markdown that serves as a complete summary of the results (see Methods). In the supplementary materials (Supplementary Materials S1; Supplementary Table S1), we describe a standard format for the recorded data that is compatible with our analysis scripts and we also provide instructions for researchers to use this script to analyze their own data. We believe that this automated script can enhance reproducibility and transparency of analyses and can be revised and adapted as a standard analysis script for general use.

#### Comparisons across statistical methods

We statistically assessed drug response for the measures mentioned above across all research groups. Table 2 contains the P values for assessing treatment versus control differences for each of the statistical tests (see Materials and Methods). Figure 3 shows associated plots from the HCI-BCM studies for each of the three models (Fig. 3, columns) for several data representations and statistical evaluation approaches (Fig. 3, rows). Associated plots for drug response at other sites, that is, MDACC, WUSTL, PDMR, and WIST are shown in Supplementary Figs. S3–S6, respectively. Overall, we found that assessments of drug response were robust across research groups, with particularly decisive evaluations for the nonresponsive and responsive models. The various analytic methods (Supplementary Figs. S7–S10) also gave results consistent with one another, with a few exceptions noted below. However, the intermediate group was difficult to classify. For the intermediate group, most of the statistical measures showed clear difference from control, but the results were inconsistent for RECIST criteria.

Table 2.

Statistical tests of treatment versus control difference.

PD (Model 625472-104-R)
RECIST21
SiteΔV21aAUC21aAUCmaxTGI21-95,-50,10-95,-30,20-95,-30,50-95,-30,100-95,-50,50-95,-50,100
MDACC 0.163 0.236 0.448 0.067 1.000 1.000 1.000 1.000 1.000 1.000
WUSTL 0.918 0.376 0.470 0.538 1.000 1.000 1.000 1.000 1.000 1.000
HCI-BCM 0.143 0.072 0.177 0.814 1.000 1.000 1.000 1.000 1.000 1.000
PDMR 0.404 0.756 0.501 0.751 1.000 1.000 1.000 1.000 1.000 1.000
SD (Model 172845-121-T)
RECIST21
Site ΔV21 aAUC21 aAUCmax TGI21 -95,-50,10 -95,-30,20 -95,-30,50 -95,-30,100 -95,-50,50 -95,-50,100
MDACC <0.001 0.003 <0.001 <0.001 0.048 0.048 0.008 0.048 0.008 0.048
WUSTL <0.001 <0.001 <0.001 <0.001 1.000 0.474 0.211 <0.001 0.211 <0.001
HCI-BCM <0.001 <0.001 <0.001 <0.001 0.200 0.026 <0.001 <0.001 <0.001 <0.001
PDMR <0.001 <0.001 <0.001 <0.001 0.003 <0.001 <0.001 <0.001 <0.001 <0.001
WISTa <0.001 <0.001 <0.001 <0.001 1.000 1.000 1.000 0.200 1.000 0.200
<0.001 <0.001 <0.001 <0.001 1.000 1.000 0.200 0.026 0.200 0.026
CR (Model BL0293-F563)
RECIST21
Site ΔV21 aAUC21 aAUCmax TGI21 -95,-50,10 -95,-30,20 -95,-30,50 -95,-30,100 -95,-50,50 -95,-50,100
MDACC <0.001 <0.001 <0.001 <0.001 <0.001 0.008 0.008 0.008 0.008 0.008
WUSTL <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
HCI-BCM <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
PDMR <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
WISTa <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
PD (Model 625472-104-R)
RECIST21
SiteΔV21aAUC21aAUCmaxTGI21-95,-50,10-95,-30,20-95,-30,50-95,-30,100-95,-50,50-95,-50,100
MDACC 0.163 0.236 0.448 0.067 1.000 1.000 1.000 1.000 1.000 1.000
WUSTL 0.918 0.376 0.470 0.538 1.000 1.000 1.000 1.000 1.000 1.000
HCI-BCM 0.143 0.072 0.177 0.814 1.000 1.000 1.000 1.000 1.000 1.000
PDMR 0.404 0.756 0.501 0.751 1.000 1.000 1.000 1.000 1.000 1.000
SD (Model 172845-121-T)
RECIST21
Site ΔV21 aAUC21 aAUCmax TGI21 -95,-50,10 -95,-30,20 -95,-30,50 -95,-30,100 -95,-50,50 -95,-50,100
MDACC <0.001 0.003 <0.001 <0.001 0.048 0.048 0.008 0.048 0.008 0.048
WUSTL <0.001 <0.001 <0.001 <0.001 1.000 0.474 0.211 <0.001 0.211 <0.001
HCI-BCM <0.001 <0.001 <0.001 <0.001 0.200 0.026 <0.001 <0.001 <0.001 <0.001
PDMR <0.001 <0.001 <0.001 <0.001 0.003 <0.001 <0.001 <0.001 <0.001 <0.001
WISTa <0.001 <0.001 <0.001 <0.001 1.000 1.000 1.000 0.200 1.000 0.200
<0.001 <0.001 <0.001 <0.001 1.000 1.000 0.200 0.026 0.200 0.026
CR (Model BL0293-F563)
RECIST21
Site ΔV21 aAUC21 aAUCmax TGI21 -95,-50,10 -95,-30,20 -95,-30,50 -95,-30,100 -95,-50,50 -95,-50,100
MDACC <0.001 <0.001 <0.001 <0.001 <0.001 0.008 0.008 0.008 0.008 0.008
WUSTL <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
HCI-BCM <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
PDMR <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
WISTa <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Note: This table presents the P values reported for various analytic measures, including change from baseline to 21 days (ΔV21), adjusted AUC for 21 days (aAUC21), adjusted AUC until last measurement (aAUCmax), tumor growth inhibition at day 21 (TGI21), and RECIST criteria for various choices of boundaries between CR/PR, PR/SD, and SD/PD given by (c1, c2, c3). For RECIST, P values are testing PD vs. not PD. Bold text indicates statistical significance (P < 0.05).

aFor WIST, the first row is for temozolomide treated at 50 mg/kg and the second row is 100 mg/kg.

Figure 3.

Analytic summaries, HCI-BCM Study. Analytic results from HCI-BCM study for progressive model (625472-104-R), SD model (172845-121-T), and CR 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 aAUCmax (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 3.

Analytic summaries, HCI-BCM Study. Analytic results from HCI-BCM study for progressive model (625472-104-R), SD model (172845-121-T), and CR 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 aAUCmax (row 5) for treatment and control, and a boxplot of TGI21 (row 6), along with P values comparing treatment to control for each measure.

Close modal

RECIST yielded qualitatively similar ordering of the models as the other methods, but it had the lowest power and showed considerable variability across cut-off points, complicating its use. The percent change in tumor size and AUC measures largely agreed and showed good statistical power. The tumor growth inhibition measure also yielded consistent results. The natural statistical test is whether this ratio is less than 1, but this should be accompanied by an assessment of the clinical significance of the effect size, because it is possible to have a small P value with minimal inhibition in a preclinical study, for example, 10% or 20%, that might not ultimately correspond to a clinical response. We recommend statistical testing versus control while accompanied by an assessment of clinical significance that may depend on the context.

### Cloud workflows for PDX sequence analysis

Robust sequence analysis pipelines are essential for understanding cancer genetics from PDX models. While prior PDX pipelines have been published, for example, (13, 43), it can be time-consuming for researchers to implement and evaluate other groups' methods. To address this problem, five PDXNet teams provided sequence analysis workflows for PDX exome-seq mutation calling, and the PDXNet Data Commons and Coordinating Center (PDCCC) dockerized these for colocalized application and sharing with the research community via the National Cancer Institute Cancer Genomics Cloud (CGC). The Seven Bridges Genomics team in the PDCCC also independently evaluated each of these pipelines. Each submitting group also specified parameters as part of the workflow submission. Evaluations were performed on simulated benchmark mixtures of human and mouse reads with various mouse/human read ratios and variant allele frequencies (see Materials and Methods).

#### Benchmarking of human-mouse read disambiguation

We first compared the efficacy of the five pipelines (Supplementary Table S2) for human-mouse read disambiguation using a series of simulated benchmark WES and RNA-Seq datasets. The simulated WES and RNA-Seq datasets were used to test the five commonly used human-mouse read deconvolution tools: BBSplit, Xenome, Disambiguate, Xenofilter, and ICRG. All tools achieved >99% precision for both WES and RNA-Seq benchmarks (Fig. 4A). Xenofilter showed the lowest recall (96.60% and 89.63% recall in WES and RNA-seq benchmarks, respectively), whereas BBSplit showed the best overall performance, that is, highest precision without any loss in recall (99.87 % and 99.64 % precision in WES and RNA-seq benchmarks, respectively.

Figure 4.

Workflow benchmarking and analysis summary. A, 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, results of mouse disambiguation for whole exome data. Right, results of mouse disambiguation for RNA-seq data. B, 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, ; workflows, . Connections between nodes depict that input to a node is from the output of another node. On the Seven Bridges platform, orange nodes () identify a tool or a workflow with an available update. C, 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 VAFs (0.025, 0.05, 0.3, 0.2, 0.3). Each plot shows recall and precision respectively on the x and y axes. 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 AF calls. E, Dendrogram of median polish by center (by MBatch) using TMM normalized count per million values.

Figure 4.

Workflow benchmarking and analysis summary. A, 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, results of mouse disambiguation for whole exome data. Right, results of mouse disambiguation for RNA-seq data. B, 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, ; workflows, . Connections between nodes depict that input to a node is from the output of another node. On the Seven Bridges platform, orange nodes () identify a tool or a workflow with an available update. C, 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 VAFs (0.025, 0.05, 0.3, 0.2, 0.3). Each plot shows recall and precision respectively on the x and y axes. 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 AF calls. E, Dendrogram of median polish by center (by MBatch) using TMM normalized count per million values.

Close modal

#### Benchmarking of WES analysis pipelines

We next compared WES results generated by the five pipelines including variant calling and the effectiveness of mouse-human disambiguation. For this analysis, two simulated benchmark datasets were created, with two levels of mouse contamination (10% and 50%) and a range of VAFs, 0.025, 0.05, 0.1, 0.2, and 0.3, with spike-ins of point mutations and indels (see Materials and Methods). For performance metrics, we used precision/recall (across SNPs, INS, DELs) and pseudo-ROC curves (see Materials and Methods). We observed minimal impact of different percentages of mouse contamination on the performance of the five workflows (Supplementary Table S3). The overall best performing workflow, Workflow 2, is shown in Fig. 4B and performance results across workflows are shown in Fig. 4C. When analyzing variant caller performances, we observed that MuTect2 (used in Workflows 2 and 4) performed consistently well across all samples for all the tested VAF levels. Supplementary Figure S11 shows SNP performance across 0.05 and 0.3 VAFs for BS-DN dataset across different coverage values (although we only show 2 VAF levels, the caller performed well across all VAF levels tested, that is, 0.05–0.3); however, indel recall decreased at lower VAFs. VarScan2 (used in Workflows 3 and 4) called only a small number of variants at lower VAFs as evident from the very low recall values. We also observed marked differences in performance of two VarScan2 PDTC workflows, for example, the DN dataset when processed through workflow 3 at low VAFs, that is, at 0.025 and 0.05 VAF had SNP precision values of 0% and 1.71%, respectively, and when processed through workflow 4 had SNP precision values of 2.16% and 12.4%. The difference in performance between workflows 3 and 4 is possibly due to the fact that in workflow 3 Varscan2 was run independently, whereas in workflow 4 the final calls are a union of VarScan2 and Mutect2 calls. Recall was good at higher VAFs, but precision varied. For example, the DN dataset when processed through workflow 3 at 0.2 and 0.3 VAF had SNP precision values of 98.43% and 99.13%, respectively, and when processed through workflow 4 had SNP precision values of 33.04% and 45.03. Strelka2 (part of workflows 1 and 5) was the most aggressive caller, achieving considerable recall even at the lowest VAFs tested. However, Strelka2 performance varied between the two workflows that used it, that is, workflow 1 and workflow 5, possibly because workflow 1 used the recommended settings for running Strelka (combining it with Manta), whereas workflow 5 ran Strelka independently. We observed similar trends in the pseudo-ROC curves consistent with results described above.

#### PDXNet Exome, RNA-seq, and CNV workflows

According to the achieved precision and recall values across SNPs, INS, and DELs (F1 statistic), Workflow 2 was the best performing WES workflow for PDX data. Consequently, we recommend using Workflow 2 for somatic calling in PDX tumor-normal paired WES samples. As the other workflows (Supplementary Figs. S12 and S13) may be suited for other datasets, we are releasing all workflows on the CGC. In addition, we are releasing a tumor-only exome-seq variant calling pipeline, an RNA-seq expression pipeline, and a CNV calling from exome-seq pipeline (see Materials and Methods; Supplementary Fig. S14). The tumor-only exome-seq, RNA-seq, and CNV calling pipelines were used to analyze samples from each PDTC in the temozolomide experiments.

#### Robustness of PDX sequence evaluations

To test the robustness of these sequence analysis workflows, we applied them to PDX samples from the temozolomide study. Each PDTC generated an independent biological sample of an untreated PDX for each of the three patient models. They then sequenced these independently and submitted the sequence data to the coordinating center.

#### Variant calls from exome-seq

FASTQ files from WES were obtained from the four PDTCs (MDACC, HCI-BCM, WUSTL, and WIST). Each center provided WES and RNA sequencing data from the PDX models: 625472-104-R, 172845-121-T, and BL0293-F563. No matched normal data were available for these models.

The WES data were analyzed with the optimal WES pipeline that was modified to take into account the lack of normal DNA, that is, the “PDX WES Tumor-Only: Mutect2” workflow. The exome capture kits used by each center covered different regions and total amounts of the genome (Supplementary Table S4), resulting in disparate variant calls among centers. The length of the genome covered by the intersection of the capture loci across all groups was 33.71 Mb. Filtering out variants from nonintersecting regions or with low allele frequencies (AF < 5%) made the average number of variant calls across centers for each model comparable (Fig. 4D; Supplementary Table S5; Supplementary Fig. S15), although centers with lower sequencing depth had fewer calls meeting the quality control threshold. A distribution of allele frequencies for calls meeting the quality control threshold for each sample across each center is shown in Supplementary Fig. S16. Mutations in cancer genes showed similarities across centers (Supplementary Figs. S17–S19), although there were variations related to sequencing depth and allele frequency, for example, the lower depth of the MDACC samples resulted in fewer variant calls. When found, mutations appeared at similar AFs across centers, and shared mutations tended to have higher AFs. These results indicate that, although our chosen pipeline is an improvement over prior ones, increased sequencing depth would still be valuable.

#### Copy number calls from exome-seq

We called the copy number for each sample using CNVkit with a pooled normal approach (Supplementary Fig. S20; ref. 27). Overall, we observed similar profiles among samples from the same model. The most apparent difference between samples was an overall shift relative to the baseline. As such, comparing absolute copy number gain and loss calls between samples remains challenging. Supplementary Figure S21 shows the Pearson correlation coefficients between samples. We observed higher Pearson coefficients (>0.746) for pairwise comparisons for samples of the same tumor among the HCI-BCM, WUSTL, and WIST PDTCs, compared with samples of different tumors. On the other hand, the MDACC profiles were noisier due to lower coverage, despite using the “drop low coverage” option in CNVkit, and we were unable to identify strong correlations between samples of the same tumor for MDACC.

#### Expression calls from RNA-seq

Data provided by each PDTC were generated using different RNA-seq protocols (Supplementary Table S6) and were analyzed with the rsem-1-2-31-workflow-with-star-aligner (single-end data) and rsem-1-2-31-workflow-with-star-aligner-pe (paired-end data) workflows on the CGC, with small adjustments based on single versus paired end sequencing or directionality parameters (see Materials and Methods). To account for differences in library size, data were normalized by trimmed mean of M-values (TMM), and further converted to count per million (CPM) with the R package edgeR (29, 44). Following normalization and CPM conversion, significant batch effects were still present in these data (Supplementary Fig. S22). To correct for batch effect among centers, median polish by center was applied to TMM normalized CPM data as implemented in the MBatch R package (github.com/MD-Anderson-Bioinformatics/MBatch). Following batch correction, samples tended to cluster by model rather than sample, though with some exceptions (Fig. 4E).

Our work demonstrates the robustness of PDXs as a model system for studying cancer drug response. In particular, we have demonstrated the experimental robustness of PDX response for three different models even among research groups blinded to the expected response and who followed independently developed preclinical protocols. As has been published numerous times, reproducibility of experimental results is a confounding factor in the ability to build on previously published data (45–47). These results demonstrate that in the context of a cytotoxic agent, even when groups are not told what experimental protocol to use, PDXs can yield accurate and consistent treatment responses. Even given these results we feel that it is important to standardize preclinical methodologies and analysis tools so that data can be compared across the PDTCs over time. For example, one change that will be implemented at all sites will be to monitor tumor volume changes for at least 1.5–2 cycle lengths beyond the last dosing cycle to assess durability of response. It is also important to recognize that different classes of drugs, more heterogeneous tumors, as well as some histologies may have wider variation in reproducibility or response; standardization of methodologies will help minimize the experimental variables that may affect interpretation of the data.

While prior studies have also investigated the robustness of PDX drug response, they have not included comparisons across research groups. For example, Izumchenko and colleagues (6) demonstrated similar responses between 92 patients and matched xenografts, and Gao and colleagues (5) and Townsend and colleagues (48) showed that 1 × 1 × 1 (animal, model, treatment) xenograft experiments were predictive of response in larger cohorts, including for resistance mechanisms to MAPK inhibition in melanoma (5) and to MDM2 inhibition in hematologic malignancies (48). However, such results may depend on the chosen treatment protocols. Our findings further show that PDX treatment results can be robust enough to withstand protocol variations and blinding. Moreover, this work extends prior investigations to standardize statistical analysis of PDX growth data (49) by showing that statistical analyses can tolerate a wide range of variations in experimental protocols and statistical parameters.

In addition, we have developed standardized PDX sequence analysis pipelines for tumor-normal variant calling, tumor-only variant calling, and RNA-seq expression calling. We have provided these as public tools on the CGC, making them easily accessible for other researchers and applicable to the broad data collections shared on the CGC. Not only have these pipelines been tested on extensive benchmark datasets, but we have also applied the tumor-only variant calling and RNA-seq pipelines to sequence data generated across the PDTCs in the temozolomide study. These give similar results across the groups, demonstrating both the efficacy of the pipelines and the minor sequence evolution from PDX to PDX during the process of generating test cohorts across groups.

Importantly, we have also developed biostatistical analysis workflows for tumor volume data, which we are releasing here as well. Our results show a high level of concordance among the various biostatistical analysis strategies, but with some caveats. The RECIST criteria is heavily threshold dependent, has lower statistical power, and less consistent with results from the other strategies. Because each strategy has its own strengths and weaknesses, we recommend testing multiple strategies for PDX analyses. It is also important to consider clinical as well as statistical significance, considering effect sizes to be sure any effect is of sufficient magnitude to be meaningful, a determination that may depend on the clinical context. Classifying PDX volume data into meaningful patient-analogous categories of CR, SD, and PR remains challenging, although this may become possible as datasets with paired clinical and PDX response data increase. In the meantime, our automated analysis scripts, which collate the results and analytical steps into an automated report, provide a standard tool for the PDX field, and future PDXNet volume data will be released in a data format consistent with these scripts. We encourage others to follow the volume data standards we have developed here, which will assist in the quantitative application of PDX treatment data for predicting the efficacy of drugs in patients.

F. Meric-Bernstam reports receiving commercial research grants from Novartis, AstraZeneca, Calithera, Aileron, Bayer, Jounce, CytoMx, eFFECTOR, Zymeworks, PUMA Biotechnology, Curis, Millennium, Daiichi Sankyo, Abbvie, Guardant Health, Takeda, Seattle Genetics, and GlaxoSmithKline as well as grants and travel related fees from Taiho, Genentech, Debiopharm Group, and Pfizer. She also served as a consultant to Pieris, Dialectica, Sumitomo Dainippon, Samsung Bioepis, Aduro, OrigiMed, Xencor, Jackson Laboratory, Zymeworks, Kolon Life Science, and Parexel International, and advisor to Inflection Biosciences, GRAIL, Darwin Health, Spectrum, Mersana, and Seattle Genetics. A.L. Welm and B.E. Welm receive a portion of royalties of University of Utah licenses for certain PDX models to for-profit entities. M.T. Lewis is a founder of and equity stakeholder in Tvardi Therapeutics Inc., a founder of and limited partner in StemMed Ltd., and a manager in StemMed Holdings LLC. He also receives a portion of royalties of Baylor College of Medicine licenses for certain PDX models to for-profit entities. No potential conflicts of interest were disclosed by the other authors.

Conception and design: Y.A. Evrard, J.H. Chuang

Development of methodology: Y.A. Evrard, J.S. Morris

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y.A. Evrard, J.H. Doroshow

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y.A. Evrard, A. Srivastava, J. Randjelovic, NCI PDXNet Consortium, D.A. Dean, J.S. Morris, J.H. Chuang

Writing, review, and/or revision of the manuscript: Y.A. Evrard, A. Srivastava, J. Randjelovic, NCI PDXNet Consortium, J.H. Doroshow, D.A. Dean, J.S. Morris, J.H. Chuang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y.A. Evrard, NCI PDXNet Consortium, J.H. Doroshow, D.A. Dean

Study supervision: Y.A. Evrard, D.A. Dean, J.H. Chuang

Other (led the analytic procedures including development of software underlying the analyses in the paper): J.S. Morris

This work was supported by the NIH to the PDXNet Data Commons and Coordination Center (NCI U24-CA224067), to the PDX Development and Trial Centers (NCI U54-CA224083, NCI U54-CA224070, NCI U54-CA224065, NCI U54-CA224076, NCI U54-CA233223, NCI U54-CA233306), and to the Cancer Genomics Cloud (HHSN261201400008C and HHSN261201500003I). All authors in this publication are part of the NCI PDXNet Consortium. Additional contributing members are: Baylor College of Medicine, Houston, TX (Salma Kaochar, Michael T. Lewis, Nicolas Mitsiades); Frederick National Laboratory for Cancer Research, Frederick, MD (Li Chen, Rajesh Patidar); The Jackson Laboratory for Genomic Medicine, Farmington, CT (Peter N. Robinson, Zi-Ming Zhao); The Jackson Laboratory, Bar Harbor, ME (Carol J. Bult, Michael Lloyd, Steven Neuhauser, Xing Yi Woo); National Cancer Institute, Investigational Drug Branch, Bethesda, MD (Jeffrey A. Moscow); Seven Bridges Genomics, Inc., Cambridge, Charlestown, MA (Brandi Davis-Dusenbery, Jack DiGiovanna, Christian Frech, Ryan Jeon, Nevena Miletic, Jacqueline Rosains, Isheeta Seth, Tamara Stankovic, Adam Stanojevic); University of California School of Medicine, Davis, CA (Luis Carvajal-Carmona, Moon Chen, Chong-Xian Pan); The University of Texas M.D. Anderson Cancer Center, Houston, TX (Huiqin Chen, Michael Davies, Bingliang Fang, Min Jin Ha, Funda Meric-Bernstam, Jack Roth); University of Utah Huntsman Cancer Institute, Salt Lake City, UT (Sasi Arunachalam, David Nix, Alana L. Welm, Bryan E. Welm); Washington University School of Medicine in St. Louis, St. Louis, MO (Sherri Davies, Li Ding, Ramaswamy Govindan, Shunqiang Li, Cynthia Ma, Brian A. Van Tine); The Wistar Institute, Philadelphia, PA (Meenhard Herlyn, Andrew Kossenkov, Vito Rebecca, Jayamanna Wickramasinghe, Min Xiao).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Tentler
JJ
,
Tan
AC
,
Weekes
CD
,
Jimeno
A
,
Leong
S
,
Pitts
TM
, et al
Patient-derived tumour xenografts as models for oncology drug development
.
Nat Rev Clin Oncol
2012
;
96
:
338
50
.
2.
Cho
S
,
Kang
W
,
Han
JY
,
Min
S
,
Kang
J
,
Lee
A
, et al
An integrative approach to precision cancer medicine using patient-derived xenografts
.
Mol Cells
2016
;
39
:
77
86
.
3.
Byrne
AT
,
Alférez
DG
,
Amant
F
,
Annibali
D
,
Arribas
J
,
Biankin
AV
, et al
Interrogating open issues in cancer precision medicine with patient-derived xenografts
.
Nat Rev Cancer
2017
;
17
:
254
68
.
4.
Krepler
C
,
Sproesser
K
,
Brafford
P
,
Beqiri
M
,
Garman
B
,
Xiao
M
, et al
A comprehensive patient-derived xenograft collection representing the heterogeneity of melanoma
.
Cell Rep
2017
;
21
:
1953
67
.
5.
Gao
H
,
Korn
JM
,
Ferretti
S
,
Monahan
JE
,
Wang
Y
,
Singh
M
, et al
High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response
.
Nat Med
2015
;
21
:
1318
25
.
6.
Izumchenko
E
,
Paz
K
,
D
,
Sloma
I
,
Katz
A
,
Vasquez-Dunddel
D
, et al
Patient-derived xenografts effectively capture responses to oncology therapy in a heterogeneous cohort of patients with solid tumors
.
Ann Oncol
2017
;
28
:
2595
605
.
7.
Dong
G
,
Mao
Q
,
Yu
D
,
Zhang
Y
,
Qiu
M
,
Dong
G
, et al
Integrative analysis of copy number and transcriptional expression profiles in esophageal cancer to identify a novel driver gene for therapy
.
Sci Rep
2017
;
7
:
42060
.
DOI: 10.1038/srep42060
.
8.
Garralda
E
,
Paz
K
,
López-Casas
PP
,
Jones
S
,
Katz
A
,
Kann
LM
, et al
Integrated next-generation sequencing and avatar mouse models for personalized cancer treatment
.
Clin Cancer Res
2014
;
20
:
2476
84
.
9.
Kim
S
,
Scheffler
K
,
Halpern
AL
,
Bekritsky
MA
,
Noh
E
,
Källberg
M
, et al
Strelka2: fast and accurate calling of germline and somatic variants
.
Nat Methods
2018
;
15
:
591
4
.
10.
Ben-David
U
,
Ha
G
,
Tseng
Y-Y
,
Greenwald
NF
,
Oh
C
,
Shih
J
, et al
Patient-derived xenografts undergo mouse-specific tumor evolution
.
Nat Genet
2017
;
49
:
1567
75
.
11.
Doroshow
J
.
Abstract IA12: NCI's patient-derived cancer models repository
.
Clin Cancer Res
2016
;
22
:
IA12
IA12
.
12.
Krupke
DM
,
Begley
DA
,
Sundberg
JP
,
Richardson
JE
,
Neuhauser
SB
,
Bult
CJ
.
The mouse tumor biology database: A comprehensive resource for mouse models of human cancer
.
Cancer Res
2017
;
77
:
e67
70
.
13.
Bruna
A
,
Rueda
OM
,
Greenwood
W
,
Batra
AS
,
Callari
M
,
Batra
RN
, et al
A biobank of breast cancer explants with preserved intra-tumor heterogeneity to screen anticancer compounds
.
Cell
2016
;
167
:
260
274.e22
.
14.
Teicher
B
,
Plowman
J
,
Dykes
D
,
M
,
Simpson-Herren
L
,
Alley
M
.
Human tumor xenograft models in NCI drug development
.
Totowa, NJ
:
Humana Press Inc.
;
1997
.
15.
Lau
JW
,
Lehnert
E
,
Sethi
A
,
Malhotra
R
,
Kaushik
G
,
Onder
Z
, et al
The cancer genomics cloud: collaborative, reproducible, and democratized - a new paradigm in large-scale computational research
.
Cancer Res
2017
;
77
:
e3
6
.
16.
Conway
T
,
Wazny
J
,
Bromage
A
,
Tymms
M
,
Sooraj
D
,
Williams
ED
, et al
Xenome-a tool for classifying reads from xenograft samples
.
Bioinformatics
2012
;
28
:
172
8
.
17.
Callari
M
,
Batra
AS
,
Batra
RN
,
Sammut
SJ
,
Greenwood
W
,
Clifford
H
, et al
Computational approach to discriminate human and mouse sequences in patient-derived tumour xenografts
.
BMC Genomics. BMC Genomics
;
2018
;
19
:
19
.
18.
Kluin
RJC
,
Kemper
K
,
Kuilman
T
,
de Ruiter
JR
,
Iyer
V
,
Forment
J V.
, et al
XenofilteR: computational deconvolution of mouse and human reads in tumor xenograft sequence data. BMC bioinformatics
.
BMC Bioinformatics
;
2018
;
19
:
1
15
.
19.
Auwera
GA van der
.
Somatic variation discovery with GATK4 [abstract]
. In:
Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1–5; Washington, DC
.
:
AACR
;
2017
.
Abstract nr 3590
.
20.
Cibulskis
K
,
Lawrence
MS
,
Carter
SL
,
Sivachenko
A
,
Jaffe
D
,
Sougnez
C
, et al
Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples
.
Nat Biotechnol
2013
;
31
:
213
9
.
21.
Wilson
RK
,
Mardis
ER
,
McLellan
MD
,
Koboldt
DC
,
Shen
D
,
Zhang
Q
, et al
VarScan 2: Somatic mutation and copy number alteration discovery in cancer by exome sequencing
.
Genome Res
2012
;
22
:
568
76
.
22.
Chen
X
,
Schulz-Trieglaff
O
,
Shaw
R
,
Barnes
B
,
Schlesinger
F
,
Källberg
M
, et al
Manta: rapid detection of structural variants and indels for germline and cancer sequencing applications
.
Bioinformatics
2016
;
32
:
1220
2
.
23.
Ye
K
,
Schulz
MH
,
Long
Q
,
Apweiler
R
,
Ning
Z
.
Pindel: A pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads
.
Bioinformatics
2009
;
25
:
2865
71
.
24.
DePristo
MA
,
Banks
E
,
Poplin
R
,
Garimella K
V
,
Maguire
JR
,
Hartl
C
, et al
A framework for variation discovery and genotyping using next-generation DNA sequencing data
.
Nat Genet
2011
;
43
:
491
8
.
25.
McKenna
A
,
Hanna
M
,
Banks
E
,
Sivachenko
A
,
Cibulskis
K
,
Kernytsky
A
, et al
The genome analysis toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data
.
Genome Res
2010
;
20
:
1297
303
.
26.
Lek
M
,
Karczewski
KJ
,
Minikel
EV
,
Samocha
KE
,
Banks
E
,
Fennell
T
, et al
Analysis of protein-coding genetic variation in 60,706 humans
.
Nature
2017
;
536
:
285
91
.
27.
Talevich
E
,
Shain
AH
,
Botton
T
,
Bastian
BC
.
CNVkit: genome-wide copy number detection and visualization from targeted DNA sequencing
.
PLoS Comput Biol
2016
;
12
:
1
18
.
28.
Dobin
A
,
Davis
CA
,
Schlesinger
F
,
Drenkow
J
,
Zaleski
C
,
Jha
S
, et al
STAR: ultrafast universal RNA-seq aligner
.
Bioinformatics
2013
;
29
:
15
21
.
29.
Li
B
,
Dewey
CN
.
RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome
.
BMC Bioinformatics
2014
;
41
74
.
30.
Thorvaldsdóttir
H
,
Robinson
JT
,
Mesirov
JP
.
Integrative genomics viewer (IGV): high-performance genomics data visualization and exploration
.
Brief Bioinform
2013
;
14
:
178
92
.
31.
Quinlan
AR
,
Hall
IM
.
BEDTools: a flexible suite of utilities for comparing genomic features
.
Bioinformatics
2010
;
26
:
841
2
.
32.
.
Temozolomide (Temodar) [new drug approval package #21–029]
. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/99/21029_Temodar.cfm.
33.
Hirst
TC
,
Vesterinen
HM
,
Sena
ES
,
Egan
KJ
,
MacLeod
MR
,
Whittle
IR
.
Systematic review and meta-analysis of temozolomide in animal models of glioma: was clinical efficacy predicted
.
Br J Cancer
2013
;
108
:
64
71
.
34.
Keir
ST
,
Maris
JM
,
Reynolds
CP
,
Kang
MH
,
Kolb
EA
,
Gorlick
R
, et al
Initial testing (stage 1) of temozolomide by the pediatric preclinical testing program
.
Pediatr Blood Cancer
2013
;
60
:
783
90
.
35.
Middlemas
DS
,
Stewart
CF
,
Kirstein
MN
,
Poquette
C
,
Friedman
HS
,
Houghton
PJ
, et al
Biochemical correlates of temozolomide sensitivity in pediatric solid tumor xenograft models
.
Clin Cancer Res
2000
;
6
:
998
1007
.
36.
Kitange
GJ
,
Carlson
BL
,
Schroeder
MA
,
Grogan
PT
,
Lamont
JD
,
Decker
PA
, et al
Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts
.
Neuro Oncol
2009
;
11
:
281
91
.
37.
Stacchiotti
S
,
Tortoreto
M
,
Bozzi
F
,
Tamborini
E
,
Morosi
C
,
Messina
A
, et al
Dacarbazine in solitary fibrous tumor: a case series analysis and preclinical evidence vis-à-vis temozolomide and antiangiogenics
.
Clin Cancer Res
2013
;
19
:
5192
201
.
38.
Stevens
MFG
.
Chapter 5 - temozolomide: from cytotoxic to molecularly targeted agent
. In:
Neidle
S
,
editor.
Cancer Drug Design and Discovery
,
second edition
.
San Diego, CA
:
;
2014
.
p.
145
64
.
39.
Nair
AB
,
Jacob
S
.
A simple practice guide for dose conversion between animals and human
.
J Basic Clin Pharm
2016
;
7
:
27
31
.
40.
Plowman
J
,
Waud
W
,
Koutsoukos
A
,
Rubinstein
L
,
Moore
T
,
Grever
M
.
Preclinical antitumor activity of temozolomide in mice: efficacy against human brain tumor xenografts
.
Cancer Res
1994
;
4
:
3793
9
.
41.
Viel
T
,
Schelhaas
S
,
Wagner
S
,
Wachsmuth
L
,
Schwegmann
K
,
Kuhlmann
M
, et al
Early assessment of the efficacy of temozolomide chemotherapy in experimental glioblastoma using [18F]FLT-PET imaging
.
PLoS One
2013
;
8
:
e67911
.
42.
Eisenhauer
EA
,
Therasse
P
,
Bogaerts
J
,
Schwartz
LH
,
Sargent
D
,
Ford
R
, et al
New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)
.
Eur J Cancer
2009
;
45
:
228
47
.
43.
Woo
XY
,
Srivastava
A
,
Graber
JH
,
V
,
Sarsani
VK
,
Simons
A
, et al
Bioinformatics workflows for genomic analysis of tumors from patient derived xenografts (PDX): challenges and guidelines
.
BMC Med Genomics
2019
;
12
;
92
.
44.
Robinson
MD
,
McCarthy
DJ
,
Smyth
GK
.
edgeR: A Bioconductor package for differential expression analysis of digital gene expression data
.
Bioinformatics
2009
;
26
:
139
40
.
45.
Prinz
F
,
Schlange
T
,
K
.
Believe it or not: How much can we rely on published data on potential drug targets?
Nat Rev Drug Discov
2011
;
10
:
712
3
.
46.
Ioannidis
JPA
.
Why most clinical research is not useful
.
PLoS Med
2016
;
13
:
1
10
.
47.
Collins
AT
,
Lang
SH
.
A systematic review of the validity of patient derived xenograft (PDX) models: the implications for translational research and personalised medicine
.
PeerJ
2018
;
2018
:
1
22
.
48.
Townsend
EC
,
Murakami
MA
,
Christodoulou
A
,
Christie
AL
,
Köster
J
,
DeSouza
TA
, et al
The public repository of xenografts (ProXe) enables discovery and randomized phase II-like trials in mice
.
Cancer Cell
2016
;
29
:
574
86
.
49.
Mer
AS
,
Ba-alawi
W
,
Smirnov
P
,
Wang
YX
,
Brew
B
,
Ortmann
J
, et al
Integrative pharmacogenomics analysis of patient-derived xenografts
.
Cancer Res
2019
;
79
:
4539
50
.