We present SVDetect, a program designed to identify genomic structural variations from paired-end and mate-pair next-generation sequencing data produced by the Illumina GA and ABI SOLiD platforms. ...Applying both sliding-window and clustering strategies, we use anomalously mapped read pairs provided by current short read aligners to localize genomic rearrangements and classify them according to their type, e.g. large insertions–deletions, inversions, duplications and balanced or unbalanced inter-chromosomal translocations. SVDetect outputs predicted structural variants in various file formats for appropriate graphical visualization. Availability: Source code and sample data are available at http://svdetect.sourceforge.net/ Contact: svdetect@curie.fr Supplementary information: Supplementary data are available at Bioinformatics online.
Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of ...Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis. High EZH2 expression in tumors results from a tight coupling to proliferation to ensure H3K27me3 homeostasis. However, this process malfunctions in breast cancer. Low EZH2 expression relative to proliferation and mutations in Polycomb genes actually indicate poor prognosis and occur in metastases. We show that while altered EZH2 activity consistently modulates a subset of its target genes, it promotes a wider transcriptional instability. Importantly, transcriptional changes that are consequences of EZH2 loss are predominantly irreversible. Our study provides an unexpected understanding of EZH2's contribution to solid tumors with important therapeutic implications.
Drosophila provides a powerful system for defining the complex genetic programs that drive organogenesis. Under control of the steroid hormone ecdysone, the adult heart in Drosophila forms during ...metamorphosis by a remodelling of the larval cardiac organ. Here, we evaluated the extent to which transcriptional signatures revealed by genomic approaches can provide new insights into the molecular pathways that underlie heart organogenesis. Whole-genome expression profiling at eight successive time-points covering adult heart formation revealed a highly dynamic temporal map of gene expression through 13 transcript clusters with distinct expression kinetics. A functional atlas of the transcriptome profile strikingly points to the genomic transcriptional response of the ecdysone cascade, and a sharp regulation of key components belonging to a few evolutionarily conserved signalling pathways. A reverse genetic analysis provided evidence that these specific signalling pathways are involved in discrete steps of adult heart formation. In particular, the Wnt signalling pathway is shown to participate in inflow tract and cardiomyocyte differentiation, while activation of the PDGF-VEGF pathway is required for cardiac valve formation. Thus, a detailed temporal map of gene expression can reveal signalling pathways responsible for specific developmental programs and provides here substantial grasp into heart formation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Patient-derived xenografts (PDX) have emerged as an important translational research tool for understanding tumor biology and enabling drug efficacy testing. They are established by transfer of ...patient tumor into immune compromised mice with the intent of using them as Avatars; operating under the assumption that they closely resemble patient tumors. In this study, we established 27 PDX from 100 resected gastric cancers and studied their fidelity in histological and molecular subtypes. We show that the established PDX preserved histology and molecular subtypes of parental tumors. However, in depth investigation of the entire cohort revealed that not all histological and molecular subtypes are established. Also, for the established PDX models, genetic changes are selected at early passages and rare subclones can emerge in PDX. This study highlights the importance of considering the molecular and evolutionary characteristics of PDX for a proper use of such models, particularly for Avatar trials.
Abstract
Patient-derived xenograft tumor models (PDX) are of increasing interest for anti-cancer agent testing due to their close resemblance to patient tumors. An accurate molecular characterization ...of the models is essential 1) to select the PDX that best fit the genetic requirements for a successful cancer therapy investigation and 2) to identify potential predictive biomarkers of response. In this study, we evaluated the quality of mutation profiles from whole-exome sequencing (WES) in terms of concordance with previously acquired mutation data in a large collection of PDX. Further, we analyzed the persistence of disclosed mutations at the transcript level with RNA-Seq.
From 339 PDX, DNA was extracted and enriched in exonic regions with Agilent SureSelect kits before Illumina HiSeq 2000 sequencing with a minimum expected average-of-coverage of 100X. Raw paired-end reads were analyzed by a PDX-specific bioinformatics pipeline to identify the human mutation profile. Sequenom Oncocarta and Sanger sequencing data acquired for 29 cancer genes in 272 PDX was used to evaluate the WES mutation profiles. In parallel, 92 PDX were profiled with RNA-Seq (100M sequencing reads required) and we investigated the expressed mutation profiles by comparing with mutations from WES data.
Among 502 point mutations found with classical methods, 95% were retrieved by WES analyses, revealing the very high sensitivity of the PDX-specific bioinformatics pipeline. 5% of mutations were missed because of a low coverage, particularly in the STK11 gene and in the KRAS gene of pancreatic models, possibly due to poor gene enrichment and high mouse stroma content, respectively. Deeper sequencing could potentially overcome this lack of coverage. Additionally, the WES analysis pipeline displays a high specificity, reporting only 1 additional mutation at gene positions covered with the classical methods. Finally, 507 mutations were detected by WES at positions not interrogated by classical methods emphasizing the necessity for next-generation sequencing (NGS) to obtain a comprehensive mutational spectrum. The number of mutations found using RNA-Seq data was on average two times lower and covered 15% of the mutations detected in WES. This was mainly due to the non-expression of genes or isoforms (40%), the mono-allelic expression of genes (30%), and low coverage data (15%). RNA-Seq analysis restricted to expressed genes represents a substantial complement to WES mutation data and enhances understanding of actual gene alterations in cancer cells.
This study demonstrated the high quality of mutation profiles obtained by WES and highlights the importance of integrating expression data to accurately predict the impact of a mutation at the protein level. An accurate molecular characterization of models is crucial for the selection of PDX with a specific genetic background for the evaluation of anticancer agents.
Citation Format: Manuel Landesfeind, Bruno Zeitouni, Anne-Lise Peille, Vincent Vuaroqueaux. Combining whole-exome and RNA-Seq data improves the quality of PDX mutation profiles. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2701.
Abstract
Experimental tumors raised in rodents represent an important preclinical tool to develop innovative anticancer compounds before clinical testing. Amongst others such models include solid ...tumors raised in syngeneic fully immunocompetent hosts and tumors spontaneously growing in genetically engineered mice (GEM) and derivate thereof. These model platforms have gained additional value since the manipulation of the immune system to fight cancer has led to tangible benefits for cancer patients. In the current study, we analyzed somatic mutation profiles from whole-exome sequencing (WES) data in a panel of 14 different mouse models covering 6 major cancer types. 4 models were GEM-derived, all other lines were developed by injection of established cell lines into the corresponding mouse strain. In parallel, these models were evaluated for their sensitivity towards checkpoint inhibitors (α-CTLA-4, α-PD-1 or α-PDL-1) in mono- or combined therapy with cytostatic and/or targeted agents.WES achieved an average-of-coverage of 165X in tumor models and normal DNA. A median mutation rate of 34 somatic mutations (m)/MB was detected, ranging from 7 m/MB (GEM derived NSCLC model KP) to 328 m/MB (syngeneic NSCLC line Lewis Lung) in exons. Mutation rates were markedly lower in GEM-derived models as in syngeneic lines (median of 9 vs 43 m/MB). This reflects very well the different underlying carcinogenic mechanism of these two types of models. The cross-comparison of tissue-transplants vs cell lines from GEM-derived model KP revealed that 75% of the mutations found in the primary KP could also be detected in the corresponding cell lines KP1 and KP4. Of note, the mutation count increased 1.3- (KP4) and 2.9-fold (KP1) during cell line establishment. Every model depicted a distinct profile against modulators of the immune system dividing the panel in responders and non-responders. In our hands no significant correlation could be determined between mutational load and sensitivity towards checkpoint inhibition in vivo. This might be related to the fact that the dataset was not broad enough and the number of models per entity was too small, rendering the subtype analysis within the panel not feasible. However, a strong tendency was observed when investigating the colon lines Colon26, CT26 and MC38 showing best response to the combination of PD-1+CTLA-4 inhibitors and in parallel the highest mutation rates (52, 64 and 59 m/MB, respectively) compared to non-responders B16-F10, CloudmanS91, 4T1 and KP1 (23 m/MB on average). Mouse models of cancer are a relevant tool for preclinical studies specifically for immuno-oncology. The molecular characterization of these models will help to optimize their use in drug discovery. They will support the development of innovative drugs and indentification of biomarkers to classify the patient cohort profiting the most from these new compounds.
Citation Format: Bruno Zeitouni, Cordula Tschuch, Jason M. Davis, Anne-Lise Peille, Yana Raeva, Manuel Landesfeind, Sheri Barnes, Julia B. Schüler. Whole-exome somatic mutation analysis of mouse cancer models and implications for preclinical immunomodulatory drug development abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1840. doi:10.1158/1538-7445.AM2017-1840
Abstract
Despite improvements in treatment, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, with a continuous increase in incidence emphasizing the need for further ...research and therapeutic development. In recent years, we have developed a collection of >40 patient-derived xenograft (PDX) models from PDAC. In order to determine their relevance for anticancer agent testing, we extensively characterized our models for histology features, whole exome mutations (Hiseq 2000), chromosome rearrangements, gene copy number variations (Affymetrix SNP6) and gene expression (Affymetrix U133 Plus2.0).
PDAC from 65 patients were implanted into immuno-compromised mice, resulting in the development of 42 PDX models (success rate 65%). The PDAC from which models were established included moderate and poorly differentiated tumors and were heterogeneous for stroma content. In patient tumors, we showed fibroblast activation but not stroma content predicted poor patient outcome (p<0.0001) and success of PDX establishment (p<0.01). Correlating with those of the parental tumors, the resulting PDAC_PDX also showed heterogeneity for stroma content and for murine fibroblast activation. As seen in patient tumors, the PDAC_PDX were characterized by frequent chromosomal instability, with 38% of the models presenting a moderate hypoploidy while the 62% remaining ones showed a hyperploidy. Two models showed hypermutation due to mismatch repair deficiency while the others had on average a lower mutation load when compared to other histotypes such as colon, lung or melanoma tumors. In total, we identified more than 9000 genes altered by homozygous deletions, high gene amplifications and/or mutations, most of the models showed alterations in KRAS (81%), TP53 (67%), CDKN2A (64%) and TGFBR2/SMAD4 (64%) genes. Next, PDAC_PDX profiles were merged with those of patient tumors to determine PDAC_PDX subtypes and were found to be of the classical (61%), quasi mesenchymal (QM) (34%) and exocrine like subtype (5%) according to the 62 gene expression signature established by Collisson et al. 2011. Preliminary biomarker analysis revealed certain associations between genomic alterations and transcriptome subtypes, such as more frequent alterations in the TGFB pathway in classical PDX (80%) compared to QM (47%). Further detailed biomarkers analyses also addressing questions of molecular determinants of stroma content, of fibroblast activation and of sensitivity toward anticancer agents will be presented.
Extensive characterization of our PDAC_PDX collection revealed similarities with patient tumors with regards of histology features including stroma content and fibroblast activation. At molecular level, the models showed similar genomic and transcriptomic patterns as those reported for patient PDAC, altogether, proving the value of this collection for drug development investigations.
Citation Format: Peter Bronsert, Tim Kees, Bruno Zeitouni, Anne-Lise Peille, Manuel Landesfeind, Heinz-Herbert Fiebig, Simon Kuesters, Vincent Vuaroqueaux. Subtyping of pancreatic cancer patient-derived xenograft tumors and implications for anticancer agent testing. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 639.
Abstract
Non-small cell lung cancer (NSCLC) is a heterogeneous disease comprising different histological subtypes with therapeutic implications. We have established a collection of 76 NSCLC ...patient-derived xenografts (PDX) comprising adenocarcinoma (39), squamous cell carcinoma (24) and large-cell carcinoma (11) models (2 mixed) for use in anti-cancer drug development and pharmacogenomics research. We analyzed the molecular diversity of our PDX collection using whole-exome sequencing, Affymetrix SNP6.0 and RNAseq, assessed their similarity to patient tumors and the impact on drug testing.
Similar to patient lung cancers, genomic analyses revealed that the NSCLC PDX were heavily altered tumors with an overall mutations load ranking from 317 to 1549 per model (mean = 682 mutations). We retrieved signatures of mutational processes known to be associated with ageing, smoking and APOBEC alterations. In addition to the mutations, chromosomal instability characterized by polyploidy including focal gene amplifications (most frequently 2p11.2, 14q32.33) and deletions (e.g. recurrent deletions of 3p14.2, 9p21.3) and gene fusions were detected. While no significant differences in average mutation loads or levels of polyploidy between the histotypes were found, histotype-specific gene alterations and gene expression profiles were identified.
In line with in vivo resistance to anti-EGFR treatment, alterations of the RAS/RAF pathway, PIK3CA and PTEN, as well as the MET amplification occurred more frequently in adenocarcinoma and large cell PDX explaining the resistance of 90% of models to anti-EGFR. Among adenocarcinoma and large cell PDX, we identified three models with EGFR mutations, one of which contained the codon insertion EGFRM766X associated with a strong sensitivity towards cetuximab but not erlotinib.
In addition, we identified adenocarcinoma PDX models in which KIT was amplified and large cell PDX with deletions in CDKN2A/B, alterations in the RAS/RAF pathway (73%) and epigenetic regulation pathways (64%). Squamous cell PDX were less frequently altered in the RAS/RAF pathway, but frequently had altered TP53 (90%), PIK3CA and PTEN (30%) and high expression of TP63 and FGFR3. Furthermore, a highly expressed FGFR3-TACC3 fusion gene was identified in one squamous cell PDX, which is under evaluation for sensitivity towards anti-FGFR3. Regarding PDX response to standard-of-care agents, squamous cell PDX were the most sensitive to cisplatin whereas adenocarcinoma PDX were the most resistant (p = 0.003). In contrast, both groups were more sensitive to paclitaxel than large cell PDX (p = 0.03). These findings suggest distinct molecular profiles influence the response to cytotoxic agents.
Molecular profiling of our NSCLC PDX collection confirmed its similarity to patient tumors and highlighted the distinct molecular specificities of each subtype, suggesting different therapeutic approaches. This extensive characterization will help improve the selection of PDX models in future drug development.
Citation Format: Anne-Lise Peille, Bruno Zeitouni, Vanina Fiebig, Heinz-Herbert Fiebig, Vincent Vuaroqueaux. Molecular profiling of a non-small cell lung PDX collection by whole exome sequencing and RNAseq revealed subtype specificities with therapeutic implications. abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A102.
Abstract
Small Cell Lung Cancers (SCLC) are fast growing tumors with frequent neuroendocrine differentiation. They represent 10-15% of lung cancers and arise in heavy smokers. Most SCLC cancer ...patients respond well to chemotherapy initially but rapidly develop resistance and die within a few months. Thus, the identification of alternative therapeutic approaches is urgently needed to improve patient's prognosis. Since SCLC is rarely treated by surgery, the number of SCLC models developed for pharmacogenomic research is very limited, however we have established 6 patient-derived xenograft (PDX) models from primary and metastasis SCLC. Complementary to histology, growth characteristic and response to SOCs, here we present comprehensive genomic analyses of these models, revealing similarity with patient tumors and druggable genes and pathways.
First we confirmed that the SCLC PDX models retained the morphology characteristics of SCLC tumors and all were heterogeneous in stroma and vasculature content. The median volume doubling time of SCLC-PDX was 7.4 days, which was faster than adenoma (9.6 days) or squamous (9.5 days) NSCLC and slower than large cell NSCLC-PDX (5.7 days). In vivo analyses demonstrated that the SCLC-PDX responded to the SOCs irinotecan and cyclophosphamide but were relatively resistant to cisplatin.
Whole exome sequencing, RNAseq and SNP6.0 microarray data revealed mutations in 1542 genes (bi-allelic mutations occurring in 328 of these) as well as larger chromosomal alterations including high copy number gains in 33 genes (of the 33 gene copy gains found, 28 occurred in a model with amplification of chromosome 1), 3 homozygous gene deletions and >20 gene fusions. Genes involved in cell cycle/apoptosis, ECM and focal adhesion via PI3K/AKT were frequently altered, as identified using KEGG database resources and as in patient tumors, recurrent mutations were seen in TP53, RB1 and SPDYE5. Furthermore, similar to SCLC data reported by the TCGA, we saw alterations of the NOTCH signaling pathway in some models, with mutations in NOTCH 1, 2 or 4 genes as well as downstream signaling molecules (CTBP2, KAT2A, DTX2). A search for drug targetable gene alterations identified mutations in BRAF, ERBB2, ALK, mTOR and JAK3 genes while EGFR, KRAS, PI3KCA and PTEN genes were not affected in these models. We also discovered gene fusions such as IP6K1-TRAIP or TM9SF4 fused with the oncogene SRC. At the gene expression level (RNAseq and Affymetrix U133 2Plus), 5 out of 6 SCLC-PDX expressed high levels of neuroendocrine markers characteristic of SCLC such as ASCL1, DLK1, GRP or CHGA, and had gene signatures characteristic of high proliferation and expression of stem cell markers such as LGR5. Analysis of the model with amplification of chromosome 1 confirmed that gene copy number gains were frequently associated with increased expression. This included the MYC family member MCL1, suggesting MYC-driven tumors and sensitivity toward compounds such as aurora kinase inhibitors.
In conclusion, we show here SCLC-PDX closely resemble to patient tumors for histology and genomic profiles resulting in fast growing models of various sensitivity toward chemotherapeutics. Numerous drug targetable alterations have been revealed by comprehensive genomic profile analysis. These models therefore represent useful tools for pharmacogenomics investigations.
Citation Format: Vincent Vuaroqueaux, Anne-Lise Peille, Bruno Zeitouni, Vanina Fiebig, Heinz-Herbert Fiebig. Comprehensive genomic profile analyses of small cell lung cancer patient-derived xenografts for pharmacogenomics. abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A101.
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