1、338 | JUNE 2012 | VOLUME 9 Division of Medical Oncology, Department of Medicine, School of Medicine, University of Colorado Anschutz Medical Campus, 12801 East 17 thAvenue, Aurora, CO 80045, USA (J. J . Tentler , A. C . Tan, C. D . Weekes, A. Jimeno , S. Leong, T. M . Pitts, J. J . Arcaroli , W. A
2、. Messersmith , S. G . Eckhardt). Correspondence to: S. G. Eckhardt gail.eckhardt ucdenver.edu Patient-derived tumour xenografts as models for oncology drug development John J. Tentler, Aik Choon Tan, Colin D. Weekes, Antonio Jimeno, Stephen Leong, Todd M. Pitts, John J. Arcaroli, Wells A. Messersmi
3、th and S. Gail Eckhardt Abstract | Progress in oncology drug development has been hampered by a lack of preclinical models that reliably predict clinical activity of novel compounds in cancer patients. In an effort to address these shortcomings, there has been a recent increase in the use of patient
4、-derived tumour xenografts (PDTX) engrafted into immune-compromised rodents such as athymic nude or NOD/SCID mice for preclinical modelling. Numerous tumour-specific PDTX models have been established and, importantly, they are biologically stable when passaged in mice in terms of global gene-express
5、ion patterns, mutational status, metastatic potential, drug responsiveness and tumour architecture. These characteristics might provide significant improvements over standard cell-line xenograft models. This Review will discuss specific PDTX disease examples illustrating an overview of the opportuni
6、ties and limitations of these models in cancer drug development, and describe concepts regarding predictive biomarker development and future applications. Tentler, J. J. et al. Nat. Rev. Clin. Oncol. 9, 338350 (2012); published online 17 April 2012; doi:10.1038/nrclinonc.2012.61 Introduction One of
7、the most frequently cited reasons for the high failure rate of new agents in oncology is the lack of pre- clinical models that recapitulate the heterogeneity of tumours in patients. 1Although the advent of cancer cell-line culture techniques fuelled an acceleration and expansion of cancer biology di
8、scovery that continues to this day, the harsh reality is that our ability to translate these findings to clinical practice has been hampered by the very models that yielded such valuable insights. Numerous explanations have been suggested for this inconsistency, including the fact that cell lines, e
9、ven when propagated in vivo , are derived from cancer cells that have adapted to growth outside a natural tumour microenvironment, resulting in genetic changes that are distinct from the genetic stress imposed on tumours in patients. 2Likewise, there is strong evidence that a greater genetic diverge
10、nce exists between a primary tumour and the corresponding cell line derived from that tumour, versus a direct xenograft, even after several generations. 2Thus, although the ability to successfully engraft surgically-derived tumours from cancer patients has been established for decades, these preclin
11、ical models are just now being consistently charac terized and applied towards drug development in oncology (Table 1). 39In this Review, we will present the oppor- tunities and challenges of these models in oncology drug development, provide specific disease examples, and describe concepts regarding
12、 predictive biomarker development and future applications. Methodology The methodology of initiation and propagation of patient-derived tumour xenografts (PDTX; Figure 1) has been covered in previous reviews by multiple groups. 711The approach is very straightforward, consisting of obtaining fresh s
13、urgical tissue, sectioning it into 3 mm 3pieces, followed by subcutaneous or orthotopic implant- ation into the flank of an immunodeficient mouse or rat. The generation harbouring the patient-derived mat- erial is termed F 0 , with subsequent generations num- bered consecutively (F 1 , F 2 , F 3and
14、so on), although some groups have named these G 0 , G 1and so on (Figure 1). 10The amount of time required for tumour take is vari- able among tumour types, location of implantation, and recipient strain, but in general this is between 2 months and 4 months, although failure of engraftment should no
15、t be ascertained until at least 6 months. 7In general, the third generation (F 3or G 3 ) can be expanded for drug treatment, and most groups use early passages for such studies. However, the main determinant should be the extent to which the PDTX has diverged from the patient s tumour in terms of ge
16、netics and histology (rather than an arbitrary passage number), two factors that are rarely presented when reporting results of therapeutic studies. Unfortunately, there has not been a comprehensive comparison among recipient strains or hosts, such as athymic nude mice, rats, or NOD/SCID mice, with
17、regards to time-to-engraftment, take rate, genetics, or histology. The majority of investigators report take rates of 75% using athymic nu/nu mice, and NOD/SCID mice are more often used exclusively in F 1or in instances where engraftment is being assessed as a primary end point (such as early stage
18、or adjuvant studies). 12,13The development of the NOD/SCID/IL2R nullmice Competing interests The authors declare no competing interests. REVIEWS 2012 Macmillan Publishers Limited. All rights reservedNATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 9 | JUNE 2012 | 339 has allowed for even greater take rates
19、 (approaching 95100%) for tumours that are particularly difficult to engraft, as it further inhibits innate immunity by block- ing the maturation of natural killer (NK) T cells. 14In addition to improved engraftment efficiency, this model has also been reported to maintain human tumour- associated l
20、eukocytes such as effector memory T cells for up to 9 weeks after implantation, providing an improved model of tumourstromal interactions. 15 Multilayered biological assays can be performed on early-passage (F 5 ) PDTX to characterize these models for predictive biomarker development (Figure 1). 16O
21、ne of the main advantages often noted with PDTX models is maintenance of the original tumour architecture and histological characteristics, although there has been con- troversy over how long and to what extent the human- derived microvasculature is maintained. 17,18For example, Gray et al. 18demons
22、trated that prostate cancer tumours implanted in nude mice maintained vessels lined with human endothelial cells and increased mean vessel density over time; however, a similar study with renal cell carcinoma revealed a decrease in human-derived vascu- lature over time. An interesting approach to ci
23、rcumvent this issue used tissue microarrays generated from 150 PDTX samples, which were assessed for VEGF-A, integ- rin 1, cathepsin B, proteinase-activated receptor 1, and MMP1, revealing profiles of an angiogenic phenotype that could be selected for therapies targeting the tumour microenvironment.
24、 19Such approaches, including gene- set enrichment analysis of angiogenic and metastatic pathways, might bypass concerns regarding the ability to completely recapitulate the human microenvironment in PDTX models. 20 Genomic comparisons of PDTX models An important question regarding PDTX model stabil
25、- ity is whether the process of engraftment and expan- sion changes the genetic features of the tumours. Comprehensive genome-wide gene-expression analy- sis studies have demonstrated that PDTX maintain the majority of the key genes and global pathway activity in primary tumours. 2,21For example, in
26、 non-small-cell lung cancer (NSCLC) PDTX models, 21unsupervised hierar- chical clustering of genome-wide gene-expression profiles revealed that nine of the 17 primary tumours clustered directly with the derived PDTX models, with correlation coefficients ranging from 0.78 to 0.95. Importantly, 10 of
27、the 17 primaryPDTX tumour pairs exhibited cor- relation coefficients 0.90 indicating a high degree of similarity between the primary cancer and the corres- ponding PDTX model. 21Similarly, in pancreatic cancer PDTX models, 10 out of 12 primaryPDTX (F 0versus F 3 ) tumour pairs were found to be conco
28、rdant for KRAS mutational and SMAD4 expression status. 11Interestingly, some of the pancreatic cancer PDTX models have been used to enrich the tumour DNA content for the pancre- atic cancer genome sequencing project. 22In a compara- tive study using small-cell lung cancer (SCLC) PDTX models, Daniel
29、et al. 2generated PDTX models from chemotherapy-naive patients with SCLC and compared them to cell lines derived from each PDTX and to subse- quent (after 6 months in culture) cell-line derived PDTX using an Affymetrix platform. 2The direct comparisons among the samples analysed (PDTX and the two ce
30、ll line types) revealed three sets of differentially expressed genes: 395 were significantly different when comparing PDTX to their matched initial cell line, 152 were different when comparing PDTX to their derivative secondary cell line, whereas only 26 genes were differentially expressed when comp
31、aring the initial cell line to those derived from PDTX after 6 months in vitro . These results suggest that global gene expression can change when cell lines are derived in vitro , and that the expression of a significant number of such genes is not restored when the deriva- tive cell line is return
32、ed to growth in vivo , supporting the notion that never in culture PDTX models may more closely recapitulate the genetic characteristics of tumours in patients. 2Figure 2 depicts two comparative examples of matched patientPDTX models using genome-wide gene expression of colorectal cancer (CRC) and p
33、ancre- atic ductal adenocarcinoma (PDA). The F 3CRC PDTX model and the F 5PDA PDTX model demonstrated high correlation of global gene expression with their matched primary tumours (F 0 ). Colorectal cancer In CRC, there is a long history of success in the estab- lishment, maintenance, and study of P
34、DTX models. 2326CRC PDTX models are quite easy to establish with take rates of over 75%, they can be successfully cryopreserved prior to implantation, and closely recapitulate the genetic alterations and histology of the fresh tumour. 2326Indeed, even in early stage CRC tumours exhibiting chromo- so
35、mal instability, establishment and propagation as PDTX retains the intratumoural clonal heterogeneity, chromosomal instability, and histology of the parent tumour for up to 14 passages, providing a unique oppor- tunity to study new agents for adjuvant therapy targeted towards specific molecular subt
36、ypes. 25 An important component in the validation of disease- specific PDTX is determining the response to standard agents and correlating responses of the xenograft to the Key points Many preclinical animal models fail to accurately predict the clinical efficacy of novel anticancer agents, largely
37、due to their inability to reflect the complexity and heterogeneity of human tumours Patient-derived tumour xenograft models (PDTX), where surgically resected tumour samples are engrafted directly into immune-compromised mice, offer several advantages over standard cell-line xenograft models PDTX tum
38、ours maintain the molecular, genetic and histological heterogeneity typical of tumours of origin through serial passaging in mice The tumour histology of PDTX models provides an excellent in vivo preclinical platform to study cancer stem-cell biology and stromaltumour interactions; novel cancer ther
39、apeutics can also be assessed Well-characterized PDTX models represent an information-rich preclinical resource for analysis of drug activity, including novelnovel drug combinations, as well as predictive biomarker discovery The PDTX approach to modelling of specific cancer types could potentially r
40、educe non-informative animal studies while providing a more-relevant system to test clinically directed hypotheses REVIEWS 2012 Macmillan Publishers Limited. All rights reserved340 | JUNE 2012 | VOLUME 9 response of the patient. In one study, 15 CRC PDTX models were established and treated with 5-f
41、luorouracil, oxaliplatin, or irinotecan with reasonable concord- ance between known response rates to these drugs and between the patient and corresponding xenograft. 26For example, five out of 15 xenografts were treated with cytostatic chemotherapeutics and all five of these exhib- ited similar res
42、ponses to their corresponding patients. 26In addition, these models retained the histological features of the parent tumour, although there was an increase in epithelial (EpCAM) and tumour markers (CEA) with passage. 26 With the advent of biological agents for the treatment of CRC, there has been a
43、recent focus on the use of PDTX models to further refine the mechanisms of resistance to these agents and develop rational combination strat- egies. In one such study, 23 CRC PDTX models were treated with the EGFR inhibitor cetuximab, profiled for KRAS, NRAS, BRAF, and assessed for epi regulin, amph
44、iregulin, as well as total and activated EGFR, MET , AKT , and HER3, to derive a cetuximab response score . 27The cetuximab response score comprised positive points associated with high levels of EGFR, epi regulin and/or amphiregulin, and negative points for KRAS, NRAS and/or BRAF mutations or high
45、levels of activated MET , HER3, AKT , or undetectable EGFR. Although not indepen dently validated in another set of PDTX models, the cetuximab response score was 90% accurate in pre- dicting the responsiveness of the CRC explants, indica- ting the utility of these models in generating clinically rel
46、evant hypotheses that can be subsequently tested in other PDTX and, ultimately, in patients. 27 PDTX have been used to functionally cross validate predictive biomarkers obtained retrospectively; KRAS, NRAS and BRAF mutations predicted non-responsiveness to cetuximab in both patients and the correspo
47、nding PDTX, which led to the identification of HER2 ampli- fication in cetuximab-resistant tumours that were wild type for KRAS, NRAS, BRAF and PI3K. 28This study illus- trates the ability to conduct xenopatient trials where the rational combination of cetuximab with pertuzumab or lapatinib was asse
48、ssed in the subset of tumours with resistance to cetuximab and HER2 amplification. 28These studies, and others, emphasize the potential impact of combining genomics data and the PDTX models with a closely associated clinical translation to accelerate drug development and predictive biomarker identif
49、ication and validation in CRC and other diseases. 16,29,30 Often overlooked, owing to the greater emphasis placed on in vivo models, the establishment of new patient-derived cell lines is also important to update the current library of CRC cell lines in which initial drug screening and functional studies can be performed. PDTX models can also be used for establishing new cell lines; however, available data suggest, not surprisingly, that there is greater genetic divergence from the paren- tal tumour when e
