Establishing and characterizing patient-derived xenografts using pre-chemotherapy percutaneous biopsy and post-chemotherapy surgical samples from a prospective neoadjuvant breast cancer study.
ABSTRACT: Patient-derived xenografts (PDXs) are increasingly used in cancer research as a tool to inform cancer biology and drug response. Most available breast cancer PDXs have been generated in the metastatic setting. However, in the setting of operable breast cancer, PDX models both sensitive and resistant to chemotherapy are needed for drug development and prospective data are lacking regarding the clinical and molecular characteristics associated with PDX take rate in this setting.The Breast Cancer Genome Guided Therapy Study (BEAUTY) is a prospective neoadjuvant chemotherapy (NAC) trial of stage I-III breast cancer patients treated with neoadjuvant weekly taxane+/-trastuzumab followed by anthracycline-based chemotherapy. Using percutaneous tumor biopsies (PTB), we established and characterized PDXs from both primary (untreated) and residual (treated) tumors. Tumor take rate was defined as percent of patients with the development of at least one stably transplantable (passed at least for four generations) xenograft that was pathologically confirmed as breast cancer.Baseline PTB samples from 113 women were implanted with an overall take rate of 27.4% (31/113). By clinical subtype, the take rate was 51.3% (20/39) in triple negative (TN) breast cancer, 26.5% (9/34) in HER2+, 5.0% (2/40) in luminal B and 0% (0/3) in luminal A. The take rate for those with pCR did not differ from those with residual disease in TN (p?=?0.999) and HER2+ (p?=?0.2401) tumors. The xenografts from 28 of these 31 patients were such that at least one of the xenografts generated had the same molecular subtype as the patient. Among the 35 patients with residual tumor after NAC adequate for implantation, the take rate was 17.1%. PDX response to paclitaxel mirrored the patients' clinical response in all eight PDX tested.The generation of PDX models both sensitive and resistant to standard NAC is feasible and these models exhibit similar biological and drug response characteristics as the patients' primary tumors. Taken together, these models may be useful for biomarker discovery and future drug development.
Project description:Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer.Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively.The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-? secretion by tumour cells.Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.
Project description:Using patient-derived xenografts (PDXs) for preclinical cancer research demands proper storage of tumour material to facilitate logistics and to reduce the number of animals needed. We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%. Corresponding cells from mouse replaced human tumour stromal and endothelial cells in second generation PDXs as demonstrated with mouse-specific vimentin and CD31 immunohistochemical staining. For biobanking purposes two cryopreservation methods, a fetal calf serum (FCS)-based (95%v/v) "FCS/DMSO" protocol and a low serum-based (10%v/v) "vitrification" protocol were tested. After primary cryopreservation, tumour take rates were 38% and 67% using either the vitrification or FCS/DMSO-based cryopreservation protocol, respectively. Cryopreserved tumour tissue of established PDXs achieved take rates of 67% and 94%, respectively compared to 91% using fresh PDX tumour tissue. Genotyping analysis showed that no changes in copy number alterations were introduced by any of the biobanking methods. Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues. However, FCS/DMSO-based cryopreservation is more successful. Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.
Project description:Patient derived xenografts (PDXs) are increasingly appreciated models in cancer research, particularly for preclinical testing, as they reflect the patient's tumor biology more accurately than cancer cell lines. We have established a collection of 20 breast PDXs and characterized their biological and clinical features, as well as their genetic stability. While most PDXs originated from triple negative breast cancers (70%), our collection comprised five ER + cases (25%). Remarkably, the tumors that produced PDXs derived from a subset of aggressive breast cancers with a high proportion of grade 3 tumors and reduced recurrence-free survival. Consistent with this, we found significant differences between the transcriptomic signatures of tumors that produced a PDX (Take) and those that did not (No Take). The PDXs faithfully recapitulate the histological features of their primary tumors, and retain an excellent conservation of molecular classification assignment and Copy Number Change (CNC). Furthermore, the CNC profiles of different PDXs established from the same tumor overlap significantly. However, a small fraction of CNCs in the primary tumor that correspond to oligoclonal events were gradually lost during sequential passaging, suggesting that the PDXs' genetic structure eventually stabilizes around a dominant clone present in the tumor of origin. Finally, de novo occurring genetic events covering up to 9% of the genome were found in only a minority of the PDXs, showing that PDXs have limited genetic instability. These data show that breast cancer PDXs represent a subset of aggressive tumors prone to relapse, and that despite of an excellent conservation of original features, they remain genetically dynamic elements.
Project description:Patient derived xenografts (PDXs) represent an essential tool in oncologic research, and we sought to further expand our repertoire of head and neck squamous cell carcinoma (HNSCC) while determining potential boundaries for this system.We consented new patients for PDX development and determined if a 24-h time delay from tumor excision to xenograft implantation affected PDX establishment. We developed a tissue microarray (TMA) from formalin fixed, paraffin embedded PDXs and their subsequent passages and carried out quantitative immunohistochemistry for EGFR, pEGFR, pAkt, pERK and ERCC1. First and last passaged PDXs were compared via a paired t-test to examine for the stability of protein expression across passages. We performed a similar comparison of the mutational profile of the patient tumor and resulting xenografts using a targeted sequencing approach.No patient/tumor characteristics influenced PDX take rate and the 24-h time delay from tumor excision to xenograft implantation did not affect PDX establishment, growth or histology. There was no significant difference in biomarker expression between the first and last passaged PDXs for EGFR, pEGFR, pAkt, and ERCC1. For pERK there was a significant difference (p=0.002), but further analysis demonstrated this only arose in three of 15 PDXs. Targeted sequencing revealed striking stability of passenger and likely driver mutations from patient to xenograft.The stability of protein expression across PDX passages will hopefully allow greater investigation of predictive biomarkers in order to identify ones for further pre-clinical and clinical investigation.
Project description:Patient-derived xenografts (PDXs) are widely recognised as a more physiologically relevant preclinical model than standard cell lines, but are expensive and low throughput, have low engraftment rate and take a long time to develop. Our newly developed conditional reprogramming (CR) technology addresses many PDX drawbacks, but lacks many in vivo factors. Here we determined whether PDXs and CRCs of the same cancer origin maintain the biological fidelity and complement each for translational research and drug development. Four CRC lines were generated from bladder cancer PDXs. Short tandem repeat (STR) analyses revealed that CRCs and their corresponding parental PDXs shared the same STRs, suggesting common cancer origins. CRCs and their corresponding parental PDXs contained the same genetic alterations. Importantly, CRCs retained the same drug sensitivity with the corresponding downstream signalling activity as their corresponding parental PDXs. This suggests that CRCs and PDXs can complement each other, and that CRCs can be used for in vitro fast, high throughput and low cost screening while PDXs can be used for in vivo validation and study of the in vivo factors during translational research and drug development.
Project description:Advanced Epithelial Ovarian Cancer (EOC) patients frequently relapse by 24 months and develop resistant disease. Research on EOC therapies relies on cancer cell lines established decades ago making Patient Derived Xenografts (PDX) attractive models, because they are faithful representations of the original tumor. We established 35 ovarian cancer PDXs resulting from the original graft of 77 EOC samples onto immuno-compromised mice. PDXs covered the diversity of EOC histotypes and graft take was correlated with early patient death. Fourteen PDXs were characterized at the genetic and histological levels. PDXs reproduced phenotypic features of the ovarian tumors of origin and conserved the principal characteristics of the original copy number change (CNC) profiles over several passages. However, CNC fluctuations in specific subregions comparing the original tumor and the PDXs indicated the oligoclonal nature of the original tumors. Detailed analysis by CGH, FISH and exome sequencing of one case, for which several tumor nodules were sampled and grafted, revealed that PDXs globally maintained an oligoclonal structure. No overgrowth of a particular subclone present in the original tumor was observed in the PDXs. This suggested that xenotransplantation of ovarian tumors and growth as PDX preserved at least in part the clonal diversity of the original tumor. We believe our data reinforce the potential of PDX as exquisite tools in pre-clinical assays.
Project description:Patient-derived xenografts (PDXs) are resected human tumors engrafted into mice for preclinical studies and therapeutic testing. It has been proposed that the mouse host affects tumor evolution during PDX engraftment and propagation, affecting the accuracy of PDX modeling of human cancer. Here, we exhaustively analyze copy number alterations (CNAs) in 1,451 PDX and matched patient tumor (PT) samples from 509 PDX models. CNA inferences based on DNA sequencing and microarray data displayed substantially higher resolution and dynamic range than gene expression-based inferences, and they also showed strong CNA conservation from PTs through late-passage PDXs. CNA recurrence analysis of 130 colorectal and breast PT/PDX-early/PDX-late trios confirmed high-resolution CNA retention. We observed no significant enrichment of cancer-related genes in PDX-specific CNAs across models. Moreover, CNA differences between patient and PDX tumors were comparable to variations in multiregion samples within patients. Our study demonstrates the lack of systematic copy number evolution driven by the PDX mouse host.
Project description:Patient-derived xenografts (PDXs) are powerful tools for translational cancer research. Here, we established PDX models from different molecular subtypes of breast cancer for in vivo drug tests and compared the histopathologic features of PDX model tumors with those of patient tumors. Predictive biomarkers were identified by gene expression analysis of PDX samples using Nanostring nCount cancer panels. Validation of predictive biomarkers for treatment response was conducted in established PDX models by in vivo drug testing. Twenty breast cancer PDX models were generated from different molecular subtypes (overall success rate, 17.5%; 3.6% for HR<sup>+</sup>/HER2<sup>-</sup>, 21.4% for HR<sup>+</sup>/HER2<sup>+</sup>, 21.9% for HR<sup>-</sup>/HER2<sup>+</sup> and 22.5% for triple-negative breast cancer (TNBC)). The histopathologic features of original tumors were retained in the PDX models. We detected upregulated HIF1A, RAF1, AKT2 and VEGFA in TNBC cases and demonstrated the efficacy of combined treatment with sorafenib and everolimus or docetaxel and bevacizumab in each TNBC model. Additionally, we identified upregulated HIF1A in two cases of trastuzumab-exposed HR<sup>-</sup>/HER2<sup>+</sup> PDX models and validated the efficacy of the HIF1A inhibitor, PX-478, alone or in combination with neratinib. Our results demonstrate that PDX models can be used as effective tools for predicting therapeutic markers and evaluating personalized treatment strategies in breast cancer patients with resistance to standard chemotherapy regimens.
Project description:The research was to appraise the utility of the patient-derived tumor xenografts (PDXs) as models of estrogen receptor positive (ER+HER2- and ER+HER2+) breast cancers. We compared protein expression profiles by Reverse Phase Protein Array (RPPA) in tumors that resulted in PDXs compared to those that did not. Our overall PDX intake rate for ER+ breast cancer was 9% (9/97). The intake rate for ER+HER2+ tumors (3/16, 19%) was higher than for ER+HER2- tumors (6/81, 7%). Heat map analyses of RPPA data showed that ER+HER2- tumors were divided into 2 groups by luminal A/B signature [protein expression of ER, AR, Bcl-2, Bim (BCL2L11), GATA3 and INPP4b], and this expression signature was also associated with the rate of PDX intake. Cell survival pathways such as the PI3K/AKT signaling and RAS/ERK pathways were more activated in the specimens that could be established as PDX in both classes. Expression of the ER protein itself may have a bearing on the potential success of an ER+ PDX model. In addition, HER2 and its downstream protein expressions were up-regulated in the ER+HER2+ patient tumors that were successfully established as PDX models. Moreover, the comparison of RPPA data between original and PDX tumors suggested that the selection/adaptation process required to grow the tumors in mice is unavoidable for generation of ER+ PDX models, and we identified differences between patient tumor samples and paired PDX tumors. A better understanding of the biological characteristics of ER+PDX would be the key to using PDX models in assessing treatment strategies in a preclinical setting.