Patient-derived xenografts (PDXs) have become a prominent cancer model system, as they are presumed to faithfully represent the genomic features of primary tumors. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We observed rapid accumulation of CNAs during PDX passaging, often due to selection of preexisting minor clones. CNA acquisition in PDXs was correlated with the tissue-specific levels of aneuploidy and genetic heterogeneity observed in primary tumors. However, the particular CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients. Several CNAs recurrently observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Notably, the genomic stability of PDXs was associated with their response to chemotherapy and targeted drugs. These findings have major implications for PDX-based modeling of human cancer.
Cancer research relies on interrogating model systems that mirror the biology of human tumors. Cell lines cultured from human tumors have been the workhorse of cancer research, but the marked differences between the in vitro cell culture environment and the in vivo tumor environment raise concerns that these lines may not be fully representative of human tumors. Recently, there have been increasing efforts to use PDXs as models to study drug response1,2,3,4. These in vivo models are assumed to capture the cellular and molecular characteristics of human cancer better than simpler cell-line-based models1,2.
As the value of PDX models depends on their faithful representation of primary tumors, it is important to assess whether PDXs retain their genomic and phenotypic characteristics throughout propagation. Thus far, the genomic stability of PDX models has primarily been evaluated indirectly, leading to the notion that PDXs are highly stable3,5,6. Consistent with this perception, PDX-based studies often involve the analysis of tumors from multiple passages3. However, hints that PDXs may be more genomically unstable than assumed have recently begun to emerge7,8, emphasizing the need for a comprehensive analysis of PDX genomic evolution (Supplementary Note).
Here we systematically analyzed landscapes of aneuploidy and large CNAs in PDX models across multiple human cancers. We generated a comprehensive CNA catalog for 1,110 PDX samples from 24 cancer types and used these data to characterize CNA dynamics during PDX derivation and propagation, to study the origin of passaging-acquired CNAs, and to compare PDX genomic stability across cancer types. We also compared the CNA dynamics observed in PDXs to those of newly derived tumor cell lines and cell-line-derived xenografts (CLDXs). Finally, we compared the CNA landscapes of PDXs to those of human primary and advanced tumors. We found that, despite an overall similarity, the CNA landscapes of PDXs diverge substantially from those of their parental tumors during passaging. We discuss the potential implications of this divergence, including its effect on therapeutic response.
Generating a catalog of aneuploidy and CNAs in PDXs
To enable a comprehensive analysis of aneuploidy and CNAs in PDXs, we created an integrated CNA data set representing 1,110 PDXs. We first assembled data from DNA-based copy number measurements across multiple PDX passages, using published SNP arrays, comparative genomic hybridization (CGH) arrays and DNA sequencing data. Unfortunately, such DNA copy number data were only available for 177 PDX samples from five studies—too few to support a comprehensive analysis of CNA stability (Supplementary Table 1)6,7,9,10,11. In contrast, gene expression profiles were available for 933 PDX samples collected from 511 PDX models across 17 studies (Supplementary Table 1)3,5,6,10,11,12,13,14,15,16,17,18,19,20,21,22,23. To reconstruct chromosomal aneuploidy and large (>5-Mb) CNAs from these expression profiles, we used previously described computational inference algorithms that accurately identify CNAs on the basis of induced coordinated gene expression changes24,25,26. We validated the accuracy of this approach by analyzing PDXs for which both gene expression and SNP array data were available (Supplementary Fig. 1, Supplementary Table 2 and Supplementary Note). Our final data set comprised CNA data for 1,110 PDX samples from 543 unique PDX models across 24 cancer types (Fig. 1a and Supplementary Data 1 and 2). For 342 of these PDX models, data were available from both the primary tumor and its derived PDX model(s), or from multiple PDX passages, thus enabling an analysis of tumor evolution (Fig. 1a and Supplementary Table 1).
We found the CNA landscapes of PDXs to be highly similar to those of their respective tumor types in The Cancer Genome Atlas (TCGA) (mean Pearson's r = 0.79; Fig. 1b and Supplementary Fig. 2), consistent with prior reports6,8,10,11. This confirms that, at the cohort level, PDX models are generally genomically representative of primary human tumors.
Tracking CNA dynamics during PDX derivation and propagation
We set out to follow CNA dynamics in individual PDX models, to assess their stability as well as their similarity to the tumors from which the models were derived. For each model, the earliest passage (in most cases, passage 0 (P0) or P1) was compared to later passages (range, P1–P16; median, P3) to determine the changes that occurred throughout passaging. A representative example of PDX model evolution is shown in Figure 1c.
We found that large (>5-Mb) CNAs arose rapidly in PDXs: 60% of the PDX models acquired at least one large chromosomal aberration within a single in vivo passage, and 88% acquired at least one large aberration within four passages (Supplementary Fig. 3a). The CNA landscape of PDX models thus gradually shifted away from those of the original primary tumors, with a median of 12.3% of the genome (range, 0–58.8%) affected by model-acquired CNAs within four passages (Fig. 1d). Of note, similar results were obtained using three different definitions of CNA prevalence—the proportion of the genome affected by CNAs (CNA fraction), the number of discrete events, and the proportion of altered genes (Fig. 1d and Supplementary Fig. 3b,c)—thereby highlighting the robustness of this finding.
There was no significant change in the overall number of CNAs throughout passaging (Supplementary Fig. 3d), indicating equal rates of emergence of new events and disappearance of existing ones. A median of 35.6% of the genome was affected by CNAs, consistent with prior estimates in primary tumors27 (Fig. 1e and Supplementary Fig. 3c). The disappearance of CNAs during passaging was not due to changes in tumor sample purity (for instance, contamination with mouse tissue might dilute the CNA signal), as other primary events were readily detected at similar signal strength. Notably, approximately one of six large CNAs identified in PDX models at P4 was not observed in the respective primary tumor. A similar proportion of primary clonal CNAs could no longer be detected in PDXs by P4. This observed tumor evolution was not limited to large CNAs, but also affected mutations in cancer-related genes (Supplementary Fig. 4 and Supplementary Note). We conclude that individual PDX models can quickly genomically diverge from their parental primary tumors.
Selection of preexisting subclones underlies CNA dynamics
Our observation that CNAs were often gained or lost during PDX passage might be explained by expansion of preexisting subclones, the acquisition and expansion of de novo events, or a combination of these mechanisms. Several lines of evidence suggest that clonal selection of preexisting subclones has a major role in shaping the CNA landscape of PDXs. First, CNAs accumulated with each passage, but their acquisition rate decreased over time (Fig. 2a). Second, apoptosis expression signatures gradually decreased with PDX passage number while signatures of proliferation increased, in line with clonal selection of fitter clones (Fig. 2b). Third, the rates of model-acquired CNAs were similar in PDXs from primary tumors and from metastases (Fig. 2c), despite metastasis-derived PDXs being more aneuploid and exhibiting higher expression of genes associated with chromosomal instability (Supplementary Fig. 5a,b)28; this suggests that model-acquired CNAs predominantly result from clonal dynamics, rather than from genomic instability.
If our hypothesis that acquired CNAs are the result of positive biological selection of existing minor subclones is correct, one would expect that the same minor clones would be enriched in multiple independent grafts of the same tumor (transplanted into 'sibling' P0 mice). Five such PDX pairs (representing breast, lung, pancreas and skin cancer PDXs) were available for analysis (Fig. 2d). The similarity in model-acquired CNAs between sibling PDXs was indeed significantly higher than the similarity between lineage-controlled 'non-sibling' PDXs (P < 1 × 10−5, stratified bootstrap test; Fig. 2e). This finding suggests that clonal evolution occurs through directional selection of preexisting subclones, consistent with observations in breast and hematopoietic cancers7,29.
To further test this idea, we analyzed loss of heterozygosity (LOH) events. Because LOH is an irreversible event at the cellular level, an LOH 'reversion' at the population level can only be explained by expansion of cells that did not undergo LOH in the first place. We queried previously published whole-genome sequencing data from 15 breast cancer pairs of primary tumors and PDXs7 and identified 5 cases of LOH reversion (Fig. 2f and Supplementary Fig. 6). These analyses thus confirm that rare preexisting subclones, which are not readily detected in the primary tumor, can expand and become the dominant clone in PDXs.
We conclude that CNA dynamics are strongest during engraftment and the first few in vivo passages, continue at a reduced rate throughout model propagation, and result primarily from selection of preexisting subclones.
The degree of genomic instability in PDXs mirrors that of primary tumors
As human cancer types differ considerably in their CNA prevalence and acquisition rate (also referred to as the degree of genomic instability, or DGI), we next compared CNA dynamics in PDXs across cancer types. We found that the rate of model-acquired CNAs varied significantly (P = 0.001, Wilcoxon rank-sum test, when comparing the most stable to the least stable tumor type), with brain tumor PDXs being the most stable and gastric tumor PDXs being the least stable (with a median of 0% and 4.2% of the genome affected by CNAs per passage, respectively) (Fig. 3a).
We therefore asked whether this spectrum of PDX aneuploidy reflects the aneuploidy levels of human cancer types. We measured aneuploidy in TCGA data according to two metrics. First, we used the percentage of samples with whole-genome duplication (WGD)27. Across seven tissues for which data were available from both TCGA and PDX data sets, the CNA acquisition rate in PDXs correlated strongly with WGD prevalence in TCGA samples (Spearman's ρ = 093, P = 0.003; Fig. 3b and Supplementary Fig. 7a). Second, we found that the median number of PDX-acquired arm-level CNAs and the median number of arm-level CNAs acquired during tumor development in TCGA samples were correlated across ten different cancer types (Spearman's ρ = 0.76, P = 0.010; Fig. 3c). We thus conclude that DGI variation among PDX tumor types parallels that of primary tumors.
As we found that expansion of preexisting subclones had a major role in shaping the CNA landscape of PDXs, we next examined whether the tissue-specific rate of CNA dynamics correlates with the degree of heterogeneity that characterizes each cancer type. Indeed, the CNA acquisition rate in PDXs correlated well with the median number of clones of the respective primary tumor type30 across six cancer types that could be matched (Spearman's ρ = 0.82, P = 0.044; Supplementary Fig. 7b). Interestingly, melanoma had the highest degree of intratumoral heterogeneity, but only a moderate level of DGI in PDXs, and was therefore the only cancer type that substantially deviated from the observed correlation; the correlation became even stronger when melanoma was removed from the analysis (Spearman's ρ = 1, P < 2.2 × 10−16; Fig. 3d and Supplementary Note).
The combined results of these analyses suggest that PDX models have characteristic tissue-specific levels of CNA dynamics that correspond both to the DGI and to the degree of heterogeneity of the respective primary tumor type. As genetic heterogeneity is closely associated with aneuploidy levels and DGI in primary tumors30,31,32, either of these factors—or both together—might explain the observed correlations.
CNA recurrence analysis identifies distinct selection pressures in PDXs and in primary tumors
A key question is whether the clonal dynamics observed in PDXs mimic those observed in human patients. To address this question, we asked whether recurrent arm-level genetic events that are observed in human tumors remain under selection pressure when the tumors are transplanted into mice; loss of these signature events would signal key differences in selection pressures between human and mouse hosts.
We identified 61 recurrent arm-level CNAs across TCGA tumor types and followed them in PDXs. Surprisingly, events that were recurrent in the TCGA data set (reflecting positive selection in humans) tended to disappear throughout PDX passaging. Specifically, among lineage-matched PDXs, we observed 116 model-acquired events that were in the opposite direction (gain instead or loss, or vice versa) to the recurrent TCGA CNAs, and only 79 model-acquired events were in the same direction (P = 0.01, McNemar's test Fig. 4a). We identified 12 recurrent events in TCGA samples that were preferentially lost throughout PDX passaging across five cancer types (glioblastoma multiforme (GBM), breast, lung, colon and pancreatic cancers; Fig. 4b and Supplementary Fig. 8). Events that tend to disappear throughout PDX propagation should be less prevalent in high-passage than in low-passage PDXs. Indeed, 9 of the 12 events that tended to disappear in PDXs, including the hallmark gains of chromosomes 1q and 8q in breast cancer and chromosome 7 in GBM and the hallmark losses of chromosome 10 in GBM and chromosome 4q in non-small-cell lung cancer, were less common in high-passage PDXs (Fig. 4c and Supplementary Fig. 9).
Taken together, these data demonstrate that PDXs can lose recurrent chromosomal aberrations that are believed to have causal roles in the development of human tumors. This suggests that the selection pressures that led to the acquisition and retention of these hallmark CNAs in patients may no longer exist in the mouse model environment.
Distinct CNA dynamics during tumor progression in PDXs and in human patients
To further assess whether the clonal dynamics observed in PDXs are indeed fundamentally different from those occurring during tumor evolution in patients, we next analyzed CNA dynamics during the progression of primary tumors into advanced disease (metastases and recurrences). We predicted that, if recurrent CNAs tend to disappear in PDXs owing to mouse-specific selection pressures, this trend should not be found during tumor evolution in humans.
To address this question, we compiled CNA data from 306 tumor samples of matched primary tumors (n = 132) and their derived metastases or recurrences (n = 174) across five cancer types represented in our PDX data set (colon, lung, endometrial, brain, and head and neck)33,34,35,36,37,38,39,40 (Supplementary Table 3). By comparing each metastasis or recurrence to its matched primary tumor, we found that tumor progression in patients was associated with a dramatic shift in CNA landscape, with a median of 18.2% of the genome (range, 0–95.4%) affected by progression-acquired CNAs (Fig. 4d). This change was greater, on average, than the change observed in PDXs (P < 1 × 10−5, stratified bootstrap test; Fig. 1d), likely reflecting the much longer time periods (often years) between paired resections and the strong treatment-associated selection pressures.
However, in contrast to the disappearance of recurrent CNAs during PDX passaging, the opposite was observed during tumor evolution in patients: recurrent CNAs more often emerged than disappeared during tumor progression. We observed 158 progression-acquired events that were in the same direction as the recurrent TCGA CNAs, and only 101 model-acquired events were in the opposite direction (P = 0.0005, McNemar's test; Fig. 4a). The relative proportion of recurrent CNAs that emerged, in comparison to the proportion that disappeared, during tumor evolution was significantly different between PDXs and advanced human disease (P = 0.0001 Chi-squared test; Fig. 4a). Therefore, these data further demonstrate that distinct selection pressures shape the CNA landscapes of tumors during their evolution in humans and in mouse hosts.
Genomic instability of PDXs is comparable to that of cell lines and CLDXs
PDXs are generally considered to reflect primary human tumors more faithfully than cell lines, owing to the artificial cell culture environment1,41. However, the subcutaneous environment in immunodeficient mice also differs considerably from the natural human host. To address the assumption that PDXs better preserve the fidelity to human tumors, we compared the CNA dynamics of PDXs in vivo to those of cell lines in vitro.
We found that the prevalence of model-acquired CNAs in newly derived cell lines was similar to that in PDXs. We analyzed the CNA landscapes of 38 samples of nine new cell lines derived in our laboratory for five cancer types (colon, GBM, pancreas, esophagus and thyroid; Supplementary Table 4). Similar to our observations with PDXs, newly derived cell lines acquired CNAs with passaging, and their CNA landscape gradually shifted away from that of the earliest passage (Supplementary Fig. 10a). As seen in PDXs, changes occurred mostly during the first few passages, and the rate of model-acquired CNAs decreased throughout propagation in culture (Fig. 5a). Notably, while CNA rates (defined as the fraction of the genome affected by model-acquired CNAs per passage) varied considerably among cell lines, they fell well within the range seen in PDXs, in a lineage-matched comparison (P = 0.55, stratified bootstrap test; Fig. 5b). These results were recapitulated with newly derived cell lines from three independent studies of GBM42,43, kidney44, and head and neck22 cancers (n = 31; Supplementary Fig. 10b and Supplementary Table 4), suggesting that clonal selection is not unique to a particular cell line propagation method.
Next, we compared CNA dynamics between PDXs and CLDXs. To assess CNA dynamics during the in vivo propagation of established cancer cell lines, we turned to the National Cancer Institute (NCI) MicroXeno project, which profiled gene expression of 49 human cancer cell lines across multiple in vivo passages45. We used the same computational algorithms24,25,26 that we applied to the PDX models to infer aneuploidy and CNAs from these gene expression profiles, resulting in 823 copy number profiles (Supplementary Data 3 and 4). We found that CNAs accumulated with in vivo passaging of CLDXs (Fig. 5c) and that the DGI of CLDXs correlated with the karyotypic complexity of their parental cell lines (Fig. 5d and Supplementary Fig. 10c), in line with what we observed in PDXs. However, the rate of CNA acquisition was lower in CLDXs: within four passages, the median model-acquired CNA fraction was 2.2% in CLDXs, as compared to 12.3% in PDXs (P = 1.6 × 10−6, Wilcoxon rank-sum test), likely reflecting the reduced heterogeneity of established cell lines in comparison to primary tumors at the time of xenograft initiation46.
Taken together, our data from three types of cancer models (PDXs, cell lines and CLDXs) demonstrate that switching the environment in which cancer cells are propagated results in CNA dynamics that gradually alter the CNA landscape. All cancer models are subject to such clonal selection. PDXs do not appear to be spared.
CNA dynamics in PDXs and cell lines may affect drug response
It is conceivable that, while PDXs undergo selection in the mouse, such selection is unimportant with respect to modeling therapeutic response. To address this possibility, we turned to a data set of PDXs with accompanying data on responses to both genotoxic chemotherapies and targeted therapeutics3.
Both very low and very high levels of aneuploidy have been associated with response to genotoxic drugs and improved patient survival28,30,47,48. Notably, CNA acquisition rate (DGI), rather than absolute levels of aneuploidy, determines sensitivity to further perturbation of chromosome segregation49. We therefore determined the DGI of PDX models and asked whether it similarly predicts response to chemotherapies. For three of five chemotherapies tested, extreme (either very low or very high) levels of DGI—but not overall aneuploidy levels—were associated with favorable therapeutic response (Fig. 6a): dacarbazine in skin cancer PDXs, paclitaxel in lung cancer PDXs, and combined abraxane and gemcitabine therapy in pancreatic cancer PDXs (P = 0.04, 0.014, and 0.006, respectively, Wilcoxon rank-sum test). The biological activity and clinical efficacy of these drugs were previously linked to chromosomal instability50,51,52,53,54. PDXs thus recapitulate the correlation observed in patients between genomic instability and response to cytotoxic chemotherapies.
We next asked whether particular model-acquired CNAs might affect PDX responses to targeted therapies, given that specific recurrent arm-level or whole-chromosome aberrations have been reported to alter the cellular response to certain drugs55,56,57. We evaluated the association between PDX response to targeted therapies and the presence or absence of individual arm-level CNAs, focusing on the 12 driver CNAs found to be selected against during PDX passaging. We identified three statistically significant drug response–CNA associations (Fig. 6b): chromosome 4 loss was associated with increased response of colon cancer PDXs to the TNKS inhibitor LCJ049 (P = 0.005, q = 0.04 for 4p loss and P = 0.00002, q = 0.0003 for 4q loss); chromosome 20q gain was associated with increased response of pancreatic cancer PDXs to combination of the PI3K inhibitor BKM120 and the SMO inhibitor LDE225 (P = 0.024, q = 0.19); and chromosome 1q gain was associated with increased response of breast cancer PDXs to the ERBB3 inhibitor LJM716 (P = 0.013, Wilcoxon rank-sum test, q = 0.23). These results indicate that it is not unusual for CNAs (and presumably other genomic events) that undergo negative selection in the mouse host to be associated with changes in sensitivity to specific targeted agents. Such associations may affect the stability of PDX drug response.
To further assess the potential functional relevance of model-acquired chromosomal changes, we turned to the Cancer Cell Line Encyclopedia (CCLE) project and its associated data sets of genomic features, genetic dependencies and drug responses58,59,60. For the 12 recurrent CNAs, we compared cell lines with and without the aberration with regard to gene expression profiles, genetic dependencies and drug sensitivity (controlling for cell lineage). In line with the PDX drug response data, colon cancer cell lines with chromosome 4q loss were more sensitive to knockdown of TNKS (P = 0.077), breast cancer cell lines with chromosome 1q gain were more sensitive to knockdown of ERBB3 (P = 0.048) and pancreatic cancer cell lines with chromosome 20q gain were more sensitive to knockdown of multiple PI3K genes (P = 0.020, 0.076, 0.005 and 0.014 for PIK3C2A, PIK3CD, PIK3CG and PIK3R2, respectively, Wilcoxon rank-sum test; Fig. 6c and Supplementary Fig. 11a). The analysis of cell lines also showed that arm-level CNAs were commonly associated with significant up- or downregulation of genes residing within the aberrant arm and that these changes were significantly associated with cell line genetic dependencies and pharmacological responses (Supplementary Figs. 11 and 12, Supplementary Tables 5 and 6, and Supplementary Note). We conclude that recurrent arm-level CNAs are associated with drug response, and their gradual disappearance throughout PDX propagation may therefore be functionally important.
The ability to directly transfer human tumors into mice and propagate them for multiple passages in vivo offers unique opportunities for cancer research and drug discovery, making PDXs a valuable cancer model. Like any other model system, however, understanding its limitations—and the ways in which it differs from human tumors in their natural environment—is required for optimal application. Our findings suggest that the genomic instability of PDXs has been underappreciated: the CNA landscapes of PDXs change continuously, and so their propagation distances them from the primary tumors from which they were derived (Supplementary Note). Indeed, comparison of PDXs to newly derived cell lines showed that PDXs do not necessarily capture the genomic landscape of primary tumors better than cell lines, in contrast to common belief1 (Supplementary Note). The similar CNA rates suggest that multiple cell line models from a single primary tumor may capture more of the original genomic landscape and its heterogeneity than a single PDX model. Moreover, the acquisition of genetic alterations throughout model propagation is unlikely to be restricted to CNAs (Supplementary Fig. 13 and Supplementary Note).
As our analysis was based on bulk-population measurements, the cellular origin of each model-acquired event could not be definitively determined. Our study strongly suggests that preexisting alterations have a major role in model-acquired CNAs, especially at the early stages of PDX derivation and propagation, but that de novo events also contribute to genomic instability (Supplementary Fig. 14 and Supplementary Note). Regardless of their origin, we found that CNAs often quickly became fixed in the population; a single in vivo passage sometimes rendered an undetected chromosomal aberration readily detectable at the population level. Such strong clonal dynamics suggest that distinct selection pressures between patients and animal models result in divergent tumor evolution trajectories (Supplementary Note).
Recent genomic analyses have shown that metastases evolve independently from primary tumors, often representing common ancestral subclones that are not detected in individual biopsies of the primary tumor. In contrast to the considerable heterogeneity between primary tumors and metastases, distinct metastatic sites tend to be relatively homogeneous33,61,62. Our findings from PDXs echo those from metastasis. The dominant clones in PDXs often come from minor subclones of the primary tumor, and PDXs that originate from the same primary tumor (the equivalence of multiple metastatic sites) tend to evolve in similar trajectories. It has been suggested that caution is required when inferring the genetic composition of metastatic disease from a primary tumor biopsy, and vice versa33,61,62; similarly, we propose that the genetic composition of a PDX tumor may differ from that of its matched primary tumor, potentially in therapeutically meaningful ways (Supplementary Note). This should be considered when using PDXs as avatar models for personalized medicine or to identify biomarkers of drug sensitivity.
PDX data assembly and processing.
CGH array, SNP array and gene expression microarray data were obtained from the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and EMBL-EBI (http://www.ebi.ac.uk/) repositories. RNA sequencing data were obtained from the NCBI Sequence Read Archive (SRA; https://www.ncbi.nlm.nih.gov/sra/). Accession numbers are provided in Supplementary Table 1. Normalized matrix files were downloaded, and samples were curated manually to identify the cancer tissue type and the PDX passage number. Arrays were analyzed for quality control, and outliers were removed. The final database consisted of 1,110 PDX tumor samples, from 543 unique PDX models across 24 cancer types. The analysis was performed in batches, and normal tissue samples included in each study served as internal controls, whenever available. Data were processed using R statistical software (http://www.r-project.org/). For all platform types, probe sets were organized by their chromosomal location and log2-transformed values were used. Probe sets without an annotated chromosomal location were removed. For gene expression data, all probe sets for each gene were averaged (as well as their chromosomal location), to obtain one intensity value per gene. A threshold expression value was set, and genes with lower expression values were collectively raised to that level: flooring values were 6–7 for the Affymetrix and Illumina platforms and −0.5 for the Agilent platforms. Probe sets not expressed in >20% of the samples within a batch were removed. The 10% of the probe sets with the most variable expression levels were also excluded, to reduce expression noise.
Generation of CNA profiles.
CNA profiles from SNP arrays were generated using the Copy Number Workflow of the Partek Genomics Suite software (http://www.partek.com/pgs), as reported by the original studies. CNA profiles from CGH arrays were generated using CGH-Explorer software (http://heim.ifi.uio.no/bioinf/Projects/CGHExplorer/), using the program's piecewise constant fit (PCF) algorithm with the following set of parameters: least allowed deviation = 0.3; least allowed aberration size = 30; winsorize at quantile = 0.001; penalty = 12; threshold = 0.01. CNA profiles from gene expression data were generated using the protocols developed by Ben-David et al.24 and by Fehrman et al.26. For all gene expression platforms, the e-karyotyping method was applied24: whenever normal tissue samples were available, the median expression value for each gene across the normal samples was subtracted from the expression value for that gene in the tumor samples, to obtain comparative values. These relative gene expression data were then subjected to a CGH-PCF analysis with the following set of parameters: least allowed deviation = 0.25; least allowed aberration size = 30; winsorize at quantile = 0.001; penalty = 12; threshold = 0.01. For Affymetrix gene expression platforms Human Genome U133A and U133Plus2.0, the functional genomic mRNA profiling (FGMP) method26 was also applied: gene expression data were corrected for the first 25 previously identified transcriptional components, and the corrected data were subjected to the same processing steps and CGH-PCF analysis described above with the following set of parameters: least allowed deviation = 0.15; least allowed aberration size = 30; winsorize at quantile = 0.001; penalty = 12; threshold = 0.01. CNA profiles from DNA sequencing were obtained in a processed table form from the publication by Eirew et al.7. CNA profiles from SNP arrays were obtained in a processed form from the publication by Gao et al.3 and compared to CNA profiles from RNA sequencing of the same PDX models. For visualization purposes, moving-average plots were generated using the CGH-Explorer moving average fit tool.
Identification of model-acquired CNAs.
To identify CNAs emerging during the generation and propagation of PDXs, 342 PDX models for which data were available from multiple time points were analyzed. These PDX models were compared either to the primary tumors from which they were derived or to their earliest available passage. For gene expression data, model-acquired CNAs were identified by e-karyotyping. For each probe set, a relative value was obtained by subtracting the early time point value from the late time point value. CGH-PCF analysis was then performed with the same parameters described above. For visualization purposes, moving-average plots were generated using the CGH-Explorer moving average fit tool.
CNA recurrence analysis.
For each tissue type, arm-level CNA recurrence was computed and compared between the PDX data set and the human patient TCGA data set (http://cancergenome.nih.gov/). Chromosome arm-level events in TCGA samples were called using a new approach to be described in a separate publication (Taylor, J.S. and G.H. et al., unpublished data). Briefly, segments of CNAs identified by ABSOLUTE63 were determined as loss, neutral or gain relative to each sample's predicted tumor ploidy. Consecutive segments were iteratively joined such that the combined segment was no less than 80% altered in a given direction (gain or loss, but not both). For each combination of arm/chromosome and direction of alteration within each TCGA tumor type, the start coordinates, end coordinates and proportion of the chromosome arm altered (based on the longest joined segment) were clustered across samples using a 3D Gaussian mixture model. The optimal clustering solution was chosen on the basis of the Bayesian information criterion. Clusters whose mean fraction altered in either specific direction was ≥80% were considered 'aneuploid', and those whose mean fraction altered (in both directions) was ≤20% were considered 'non-aneuploid'. Chromosome arm-level events in PDX samples were determined using the CNA status of the largest overlapping segment from the e-karyotyping analysis. Comparisons of absolute CNA landscapes were performed using the FGMP-derived CNA profiles, and comparisons of model-acquired CNAs were performed using the e-karyotyping-derived CNA profiles. Comparisons between early- and late-passage PDXs were performed using FGMP-derived CNA profiles: samples from P1 or earlier were defined as early passage, and those from P3 or later were defined as late passage. Heat maps were generated using the pheatmap R package, and clustering was performed using Euclidean distance and complete linkage.
DGI comparison across passages and tissue types.
The DGI of each sample was determined in three ways: (i) the fraction of the genome affected by model-acquired CNAs per passage, (ii) the number of discrete events per passage, and (iii) the fraction of altered genes per passage. For each cancer type, the median number of model-acquired arm-level CNAs across all PDX samples was determined and compared to several TCGA statistics: the percentage of samples with WGD taken from the publication by Zack et al.27, the median number of arm-level CNAs per sample and the median number of clones per sample taken from the publication by Andor et al.30.
PDX samples derived from the same primary tumors but propagated in different mice starting from their initial transplantation (transplanted into different P0 mice) were defined as 'siblings'. PDX samples derived from the same primary tumors and propagated in the same mice at some point during PDX propagation were excluded from the analysis. PDX samples from distinct primary tumors were defined as 'non-siblings'. Similarity scores were calculated for each pair of samples on the basis of the arm-level events that occurred during their in vivo passaging (model-acquired CNAs), using a modified Jaccard similarity coefficient. This similarity coefficient was inversely weighted to account for the observed prevalence of each CNA in each PDX tissue type. Therefore, the similarity score was calculated using the following equation
and freq(k) is the frequency of event k in that tumor type.
Allelic copy number data were obtained from the publication by Eirew et al.7. Using 10-Mb windows along the genome, we identified the following scenarios: (i) the minor allele was 0 (LOH) in a primary tumor but >0 (presence of both alleles) in the tumor-derived PDX model and (ii) the minor allele was 0 (LOH) in an early-passage PDX but >0 in a later passage of the same PDX model. These instances of apparent reversion of LOH were visualized using the Integrative Genomics Viewer (IGV; https://www.broadinstitute.org/igv/) and replotted in Figure 2f.
Gene expression signature scores.
The apoptosis and proliferation gene sets were derived from the Molecular Signature Database (http://software.broadinstitute.org/gsea/msigdb) using the Hallmark_Apoptosis64 and Benporath_Proliferation65 gene sets, respectively. The CIN70 gene set was derived from the publication by Carter et al.13. Signature scores were generated for all PDX models reported by Gao et al.3. For each gene set, genes not expressed at all in the PDX data set were removed and the remaining gene expression values were log2 transformed and scaled by subtracting the gene expression means. The signature score was defined as the sum of these scale-normalized gene expression values.
Advanced disease data assembly, processing and CNA profiling.
CGH array and SNP array data were obtained from the GEO and EMBL-EBI repositories. CNA profiles from DNA sequencing were obtained in a processed table form from the authors of the respective publications. Accession numbers are provided in Supplementary Table 4. The final database consisted of 306 tumor samples across five cancer types. Chromosome arm-level events in advanced-disease samples were determined as described above. Progression-acquired CNAs were determined as events that were identified in the primary tumor but not in its advanced-disease sample (metastasis, recurrence or progressed sample), or vice versa.
CLDX data assembly, processing and CNA profiling.
Gene expression microarray data from the NCI MicroXeno project were downloaded from the GEO repository under accession GSE48433 (ref. 45). Data were processed as described above. CNA profiles were generated using the FGMP method, and model-acquired CNAs were identified by e-karyotyping, as described above. The gene expression values for in vitro–cultured (P0) cell lines were used as the reference in the e-karyotyping analysis. The numerical karyotypic complexity categorization of the cell lines was obtained from the publication by Roschke et al.66.
Cell line data assembly, processing and CNA profiling.
Whole-exome sequencing data from nine newly derived cell lines were obtained from Tseng et al. (Y.-Y.T., unpublished data). CNA profiles were generated from these data using the ReCapSeg program (http://gatkforums.broadinstitute.org/gatk/categories/recapseg-documentation), from the ratio of tumor read depth to the expected read depth (as determined from a panel of normal samples). Gene expression microarray data were obtained from the GEO repository. Accession numbers are provided in Supplementary Table 3. Data were processed as described above. CNA profiles were generated using the FGMP method, and model-acquired CNAs were identified by e-karyotyping, as described above. Renal cancer CNA data were obtained directly from Cifola et al.44, and model-acquired CNAs were identified as described above. For comparison of model-acquired CNA rates across passages, samples were compared to the earliest available passage (P0 or P1). Samples from P7 or earlier were defined as early passage, samples from P10 were defined as medium passage and samples from P19 or later were defined as late passage.
PDX drug response association analyses.
PDX drug response data were obtained from the publication by Gao et al.3. For analysis of the association between chemotherapy response and absolute levels of aneuploidy, the CNA fraction was determined according to the FGMP-derived CNA profiles of the sample from the latest passage available for each model. Low CNA levels were determined as CNA fraction < 0.3; intermediate CNA levels were determined as 0.3 < CNA fraction < 0.7; and high CNA levels were determined as CNA fraction > 0.7. For analysis of the association between chemotherapy response and DGI, the DGI level of each model was determined as the number of discrete model-acquired CNAs per passage, using the latest passage available for each model: low DGI was determined as DGI = 0; intermediate DGI was determined as 0 < DGI < 4; and high DGI was determined as DGI > 4. BestAvgResponse values were used to make response calls. Association tests were conducted in each available tissue type independently, yielding a total of six drug–tissue association tests (representing five chemotherapies in five tissue types). For analysis of the association between targeted therapy response and the existence of specific arm-level events, the arm-level copy number status of each model was set according to the FGMP-derived CNA profiles of the sample from the latest passage available for that model. BestAvgResponse values were used to make response calls. For each of the 12 recurrent TCGA events that tended to disappear throughout PDX passaging, the association of the event with PDX drug response was evaluated in the relevant cancer type. All targeted drugs that were used as single agents and yielded at least partial response in at least one mouse were evaluated. Drug combinations were also evaluated if one or both of the drugs in the combination was not tested as a single agent. A total of 54 association tests were performed (representing 15 single-agent drugs and 5 drug combinations in three tissue types).
Cell line gene expression, genetic dependency and drug response association analyses.
Chromosome arm-level events in cell line samples were called using the same approach used to call arm-level events in TCGA samples (described above). Gene expression was measured for CCLE cell lines using RNA-seq (n = 936 cell lines with arm-level CNA calls) and was normalized to RPKM values for each gene60. Gene essentiality profiles for each cell line were derived from genome-wide RNAi screens across 503 cell lines (n = 446 with arm-level CNA calls), using the DEMETER algorithm to isolate the effects of gene knockdown from off-target effects60. Drug sensitivity measurements were taken from the Cancer Therapeutics Response Portal (CTRP v2) data set (downloaded from https://ocg.cancer.gov/programs/ctd2/data-portal/). These data represented dose–response curve AUC measures for 887 cell lines (n = 804 with arm-level CNA calls) across 545 compounds. Comparisons of gene expression, RNAi-based gene essentiality and compound sensitivity for cell lines with and without particular arm-level CNAs were made using standard linear modeling tools developed for differential expression analysis: the R package limma was used to derive P values from empirical Bayes moderated t statistics67. In all cases, we tested the effect of arm-level CNAs while controlling for between-lineage differences by including lineage as a covariate in the model67. Gene set testing was performed using a parametric approximation to permutation-based testing, implemented in the R package npGSEA68. Between-lineage differences were controlled for by regressing lineage out of both the arm-level CNA calls and the variable of interest68.
Mutation allelic fraction analysis.
The allelic fraction (AF) and predicted effects of point mutations were obtained from the publication by Eirew et al.7. Shifts in allelic fraction were determined as |AF(primary) − AF(PDX)| > 0.2. Missense and nonsense coding mutations were considered separately from the rest of the mutations, and their shifts in allelic fraction are plotted in Supplementary Figure 4.
Comparison of CNA-based and mutation-based phylogenetic trees.
Copy number and mutation data were obtained from Gibson et al.33 and Bi et al.69. To construct single-nucleotide variant (SNV)-based trees, SNVs present at low allelic fractions and SNVs from regions of low sequencing coverage were first excluded. To construct arm-level CNA–based trees, copy number data from the tumors were converted into arm-level calls. Phylogenetic trees were then generated separately. In each case, branch points in the tree were assigned on the basis of the overall similarity in shared events and branch lengths were set to be proportional to the number of shared events. Patients for whom trees could not be generated owing to insufficient information (for example, no branch points identified) were excluded from the analysis. CNA-based trees were then compared to SNV-based trees to determine their sensitivity (the proportion of SNV-based branch points identified in CNA-based trees) and specificity (the proportion of CNA-based branch points identified in SNV-based trees).
The significance of differences in prevalence and rates of absolute CNAs and model-acquired CNAs between PDX passages, between primary and metastatic PDXs, between PDXs with wild-type and mutated or deleted p53, between PDXs from the most stable (upper quartile) and least stable (lower quartile) tissue types, between cell line passages, between CLDX passages and between CLDXs from cell lines of distinct numerical karyotypic complexity was determined using the two-tailed Wilcoxon rank-sum test. The significance of differences in similarity scores between siblings and non-siblings, the significance of differences in CNA rates between PDXs and cell lines, and the significance of differences between PDX-acquired and progression-acquired CNA fractions were determined using a stratified bootstrap test, permuting the data 100,000 times within each tissue type. The significance of the gene expression signature trends observed throughout PDX passaging was determined by the Kruskal–Wallis rank-sum test. The significance of correlations between PDX and TCGA data was determined using a Spearman's correlation test. To evaluate the tendency to acquire or lose recurrent TCGA CNAs during PDX propagation and during disease progression in patients, recurrent CNAs were defined for each tissue type as those that recurred in over 40% of the samples and the number of events that involved these CNAs were computed in the lineage-matched PDX cohorts; the significance of differences between the emergence frequency and the loss frequency in PDXs and in advanced disease was determined using McNemar's test, and the significance of differences between the two groups was determined using a chi-squared test for equality of proportions (using the proportion observed in humans as the expected proportion). The significance of the differences in CNA prevalence between early- and late-passage PDX samples was evaluated using the one-tailed Fisher's exact test. The significance of the association between chromosome arms and drug response was determined using the Wilcoxon rank-sum test, with false discovery rate (FDR) multiple-test correction performed for each tissue type independently. Box plots show the median, 25th and 75th percentiles, lower whiskers show data within the 25th percentile − 1.5 times the IQR, upper whiskers show data within the 75th percentile + 1.5 times the IQR, and circles show the actual data points. Violin plots show the combination of a box plot and a kernel density plot, in which the width is proportional to the relative frequency of the measurements. All of the statistical tests were performed using R statistical software, and the box plots and violin plots were generated using the boxplot and vioplot R packages, respectively. A Life Sciences Reporting Summary is available.
The codes used to generate and/or analyze the data during the current study are publically available or are available from the authors upon request.
The data sets generated during and/or analyzed during the current study are available within the article and its supplementary information files or are available from the authors upon request.
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Gene Expression Omnibus
We thank L. Franke for assistance with functional genomic mRNA profiling; A. Bass, K. Ligon, A.J. Aguirre and J. Lorch for providing the clinical samples for cell line derivation; A. Tubelli for assistance with figure preparation; M. Meyerson, A.J. Cherniack, A. Taylor, A. Pearson and Z. Tothova for helpful discussions; and W.J. Gibson for copy number data. U.B.-D. is supported by a Human Frontiers Science Program postdoctoral fellowship, R.B. received support from the US National Institutes of Health (R01 CA188228) and the Gray Matters Brain Cancer Foundation, and T.R.G. received support from the Howard Hughes Medical Institute.
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Journal of Hematology & Oncology (2018)