Thomas Metz

Correlation of ErbB2 Gene Status, mRNA and Protein Expression in a Panel of >100 Human Tumor Xenografts of Different Origin

Introduction: The receptor tyrosine kinase ErbB2 is an important prognostic marker and therapeutic target in breast cancer. The aim of this study was to investigate the correlation between ErbB2 gene amplification, mRNA and protein expression in a panel of >100 patient-derived nude mouse tumor xenografts of different histological origin. Materials and Methods: Data were obtained using fluorescence in situ hybridization, GenChip® expression analysis, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Tumors included the following types: urinary bladder, breast, colon, stomach, kidney, liver, lung, melanoma, ovary, pancreas, prostate, uterus/cervix uteri and others. Results: All tumors with high-level ErbB2 gene amplification expressed ErbB2 mRNA at a level >10-fold above average and protein at a level >20-fold above average (ELISA). Correlation was found between ErbB2 mRNA and protein expression. Conclusion: Based on expression data, cervical, gastric and adenocarcinomas of the lung emerged as new potential indications for ErbB2-directed cancer therapies.

Abstract A52: PTEN/PTENP1 transcripts expression and alterations in a large panel of human tumor xenograft in nude mice: Implication for resistance to targeted therapies involving EGFR/PI3K/PTEN pathways.

Background: PTEN alterations are major determinants of resistance to several new targeted therapies involving EGFR/PI3K/PTEN pathways. Recent findings also suggested implication of its associated PTENP1 pseudogene. PTEN can be altered at the gene or transcriptional level. PTEN expression is often higher in stroma than in tumor cells complicating readouts of assayed tumor samples. Taking advantage of the stroma of xenografted human tumors in nude mice being produced by the host, we characterized specific PTEN/PTENP1 tumor cell transcript expression levels and mutational status in a large panel of tumor models. Materials and Methods: A total of 192 patient-derived xenografts of 23 different histotypes were investigated. Human and murine PTEN, PTENP1 mRNA levels as well as PTEN gene copy numbers were quantified by quantitative polymerase chain reaction using species specific assays; PTEN transcript was analyzed by sequencing. Results were analyzed by tumor type and compared to sensitivity to cetuximab. Results: PTEN transcript alterations were observed in 32 of the 192 tumor xenografts (17%) and were shown to be due to loss of PTEN gene for 9, to loss of transcript expression for 4 and to frameshift or substitution-missense mutations for 14 and 7 samples, respectively. PTEN expression levels and alteration frequency were both associated with specific histotypes. Melanoma and breast cancer showed most PTEN alterations (6/11 and 6/16 respectively). PTENP1 pseudogene was shown to be well expressed in only 61% of tumor xenografts, whereby expression levels depended on the tumor type. No or low PTENP1 expression was found in tumor models having a PTEN alterations. Finally, in a tumor panel consisting of colon, non-small cell lung, gastric and head and neck cancer we found that none of the PTEN altered tumors analyzed were sensitive to cetuximab in vivo. Conclusion: This study confirmed the multiplicity of PTEN alterations occurring in cancer. PTEN expression levels and alterations were associated with PTENP1 expression levels and depended on tumor types. PTEN alterations were shown to be associated with tumor resistance to cetuximab and their impact on other targeted therapies involving EGFR/PI3K/PTEN pathways should be evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A52.

Abstract 2598: Evaluation of antitumor efficacy of Trabectedin in patient derived tumor xenografts in vitro and in vivo, and determination of a predictive gene signature

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Trabectedin is a marine derived antitumoral agent, originally isolated from Ecteinascidia turbinata. It acts by binding to the minor groove of DNA interfering with cell division, transcription, and the DNA repair machinery. Trabectedin was approved by the EMEA as second line therapy for the treatment of advanced soft tissue sarcoma and for ovarian cancer in combination with Doxil. Further Phase II trials with Trabectedin in breast and prostate cancer are underway. We characterized Trabectedin for antitumor efficacy and selectivity in patient-derived tumor xenografts to identify target tumor types for further clinical studies. The compound was tested in 67 tumor xenografts of 15 histo types using an ex vivo clonogenic assay. Pronounced concentration dependent antitumor activity (mean IC70 = 1.3 nM) and selectivity was observed, with sensitive tumor models being on average about 7-fold more sensitive than the average of all tumors tested. Trabectedin was also given to tumor-bearing nude mice at 0.2 and 0.15 mg/kg/d iv once weekly for 3 weeks and showed substantial inhibition of tumor growth at a dose level of 0.2 mg/kg/d in tumors of lung, colon, and breast. The activity data of Trabectedin against 67 tumors in the clonogenic assay were used for further bioinformatic analysis. Subsets of tumors and their corresponding data were randomly split into a training set (n=44) and an independent validation set (n=23). By matching in vitro antitumor efficacy data (IC70) of the tumors with the corresponding gene expression profiles (determined by Affymetrix HG-U133 Plus 2.0 gene chip array), a signature of 19 gene transcripts being specific for the responsiveness towards Trabectedin was determined. The classification border for activity of Trabectedin was IC70 = 0.5 nM. The signature was validated by leave-one-out-cross-validation (LOOCV) on the training set, and prediction of tumors of the independent validation set. In the LOOCV sensitivity or resistance of tumors was predicted correctly in 13/18 (72%) and 26/26 tumors (100%), in the validation set in 4/7 (57%) and 13/16 tumors (81%), respectively. In the validation set, the predicted responders showed a 4.5-fold lower median IC70 compared to the predicted non-responders (p= 0.02). Moreover, the signature was used to predict responsiveness of 173 tumors of the Oncotest xenograft collection with unknown sensitivity to Trabectedin. In this set of tumors, the signature identified sarcoma, leukemia/lymphoma, as well as ovarian, head and neck, small cell lung, mammary and bladder cancer as Trabectedin sensitive tumor types. Experimental testing of the predicted tumors so far confirmed the predictions in 11/13 cases (85%). This study shows the feasibility of combining experimental testing and virtual prediction to identify additional tumor types as candidates for further preclinical and clinical investigations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2598.

Abstract A20: A systematic patient-derived xenograft based solution for pre-clinical biomarker discovery

There is an acute need for biomarkers at every phases of drug development from selecting preclinical models in pharmacogenomic studies to enrollment of patients in clinical trials. However, their identification remains extremely challenging due to the limited availability of clinical samples. In contrast, standard tumor models such as cell lines are available but are genetically relatively far from patient tumors. Use patient-derived xenografts (PDX) for anticancer agent-testing is of increasing interest due to their closer similarity to patient tumors compared to cell lines. Over the last 30 years, we have established a collection of 400 PDX covering more than 30 different cancer types. PDX models have been extensively characterized using the microarray or next-generation sequencing technologies for gene expression, copy number variations and whole-exome mutations. Biomarker research is now possible using these data in combination with drug response data from in vivo or in vitro 2D or 3D assays routinely performed on-site with large panels of 100-200 PDX. We present here a fully integrated bioinformatics pipeline dedicated to biomarker discovery in which the complete molecular profiles of our PDX have been systematically tested for association with drug sensitivity. To identify the biomarkers associated with drug response, several statistical tests have been performed. Drug response data were treated either as continuous variables using the Spearman or Wilcoxon tests, or as categorical variables (with two groups of responders and non-responders) using the LIMMA, t-test or Fisher exact test. Given that high throughput data frequently leads to large biomarker lists, we used specific filters to narrow down the list of candidates by defining thresholds based on corrected p-values, by intersecting results from different tests, or by integrating the tumor type into the statistical tests. Since sensitivity to anticancer agents is often multi-factorial, we also used integrative approaches that combined gene mutations, copy number loss and lack of gene expression for association with drug response. Finally, significant biomarkers were visualized using clustering heatmaps and enrichment GO/pathway approaches to get more insight in their biological function. Using a selection of several datasets of PDX drug responses to chemotherapeutics and targeted therapies (targeting RTK/RAS/RAF and PI3K/MTOR pathways and using specific compounds such as Vemurafenib, Erlotinib or Cetuximab), we demonstrate the efficacy of our approach to retrieve biomarkers of known clinical utility. Using these datasets we also could address the questions of model panel sizes, molecular data type and tumor subtype representation, and show how more accurate biomarkers can be validated using an independent dataset of samples. The development of strategies for testing anticancer agents using PDX in mouse clinical trials, or high throughput in vitro 2D, 3D screening approaches coupled to a more systematic biomarker research should significantly contribute to early biomarker identification and facilitate drug development. Citation Format: Bruno Zeitouni, Anne-Lise Peille, Zakia Amalou, Thomas Metz, Heinz-Herbert Fiebig, Vincent Vuaroqueaux. A systematic patient-derived xenograft based solution for pre-clinical biomarker discovery. [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 A20.

Abstract 2890: Single mouse trials, a concept using patient-derived tumor xenografts for large scale in vivo screens

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Patient-derived xenografts (PDX) passaged in immunocompromised mice are a well-established system for preclinical efficacy testing of anti-cancer agents. Typically, PDXs are pre-screened either in vitro or via molecular analysis, and selected models are then tested in groups of 8 to 12 mice to confirm or evaluate the efficacy of a treatment relative to a vehicle control group. This approach results in highly reliable and reproducible efficacy data. However, it is often desirable to test large panels of tumor models in vivo in order to properly take genetic diversity into account. Such experiments can be cost-prohibitive in conventional study layouts, which results in fewer models being tested. The Single Mouse Trial (SMT) format addresses the need for compound testing in larger, more diverse tumor populations. This format employs a single mouse per PDX model and treatment arm, thereby enabling the investigation of efficacy in substantially larger panels of PDX models. In order to better mirror inter-patient response diversity observed in the clinic, less emphasis is put on the statistical robustness of response data for individual models. In the study presented here, six SoC drugs were tested in the SMT format in colorectal (cetuximab, oxaliplatin, irinotecan, 5-FU) and non-small-cell lung cancer (cetuximab, paclitaxel) PDXs, and results were compared to those obtained from standard format experiments with 5 - 10 mice per group. Dosing and schedules were adapted to clinical standards. Data for 19 CXF and 16 NSCLC models showed that in 77% of the cases, results obtained from the single mouse trial format were in line with results from standard efficacy tests. Furthermore, in 10% of the cases, efficacy was similar by trend between the two formats. Notable discrepancies were seen mainly in tumors exhibiting intermediate sensitivity. Our findings indicate that for the drugs tested here, the risk of misjudging the efficacy in a given PDX model based on SMT is low. This risk could be further lowered by increasing group sizes to 3 mice. For the identification of biomarkers which requires accurate efficacy data, such an intermediate format between the SMT and the standard format might be advantageous. Citation Format: Christina Gredy, Julia B. Schuler, Nina Zanella, Heinz-Herbert Fiebig, Thomas Metz. Single mouse trials, a concept using patient-derived tumor xenografts for large scale in vivo screens. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2015-2890

Abstract C30: A KRAS pathway activation index predicting response to MEK inhibitors in patient-derived tumor xenografts.

Introduction: MEK 1/2 inhibitors (MEKi) are promising compounds for the treatment of cancer due to frequent activation of the RAS/MAPK/ERK oncogenic pathway. CI-1040 and PD0325901 are newly developed MEKi that are currently being tested in clinical trials. In the present study, we investigated MEKi response in different tumor types and we determined whether an index of KRAS pathway activation (K-PAI) could predict response to MEKi. Material and Methods: CI-1040 and PD0325901 were tested using an ex vivo 3D Tumor Clonogenic Assay (TCA) in a panel of 63 patient-derived tumor xenografts (PDX) covering 15 tumor histotypes. The K-PAI was determined by identifying gene expression patterns (Affymetrix HGU133 plus 2.0 arrays) associated with activation of the pathway and KRAS mutational status (determined by Sanger sequencing). Results: The absolute activities (IC50) of CI-1040 and PD0325901 correlated in most of the tumor models tested (r=0.87). Most of the melanomas were sensitive to both MEKi tested, whereas variable response profiles were observed in colon cancers and non-small cell lung cancers (NSCLC). Ovarian and pancreatic cancer xenografts displayed in most instances weak responses. The KRAS and BRAF statuses were significantly associated with MEKi IC50 (p=0.0001 and p=0.0002, respectively). The melanomas which frequently displayed BRAF mutations (13/21), were highly sensitive to MEKi treatment, whereas ovarian and pancreatic tumors, which frequently harbored KRAS mutations (1/3 and 2/2), were resistant. Moreover, we found that the K-PAI correlated significantly with MEKi IC50 (r>0.5, p<0.0001 for CI-1040 and PD0325901).= Melanomas with low K-PAI values were highly sensitive to MEKi treatment whereas ovarian and pancreatic tumors with high K-PAI values were resistant. Interestingly, the K-PAI was also predictive of response to MEKi treatment for tumors expressing wild-type BRAF and KRAS.Conclusion: This large ex vivo PDX study showed that tumor sensitivity to MEKi is related to histology and to RAS pathway activation. KRAS and BRAF mutations were predictive of MEKi response (resistance and sensitivity, respectively). These results are consistent with published data on cell lines and on small patient cohorts and demonstrate that PDX are adequate models to test targeted drugs such as MEKi. The correlation of tumor sensitivity to MEKi and the RAS pathway activation level (K-PAI) should be further evaluated in ex vivo 3D assays utilizing PDXs or with an in vivo study, to be able to better stratify patients when testing the predictive potential of K-PAI in clinical trials. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C30. Citation Format: Anne-Lise Peille, Armin Maier, Frederic Foucault, Rebekka Krumbach, Tim Kees, Torsten Giesemann, Thomas Metz, Thomas Metcalfe, Heinz-Herbert Fiebig, Vincent Vuaroqueaux. A KRAS pathway activation index predicting response to MEK inhibitors in patient-derived tumor xenografts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C30.

Abstract 3835: Ex vivo 3D assay: rapid and reliable replication of the in vivo anti-tumor efficacy of c-Met inhibitors.

For compounds directed against molecular targets expressed in tumor cells, the ex vivo 3D tumor clonogenic assay (TCA) is a rapid and reliable ex vivo assay with a high predictive value for in vivo tumor sensitivity. Single cell suspensions prepared from patient-derived tumor xenografts (PDX) growing subcutaneously in nude mice or from cultured human tumor cell lines are seeded in semisolid medium and tumor colony formation is monitored in the presence or absence of test compounds over a period of one to three weeks. Based on experiments with up to 70 PDX, we have demonstrated that the TCA accurately replicates the in vivo sensitivity of PDX towards cMet inhibitors across all major tumor histologies. More specifically, all three NSCLC PDX that regressed in response to cMet inhibition in in vivo efficacy tests were sensitive to several cMet inhibitors in the ex vivo TCA. By contrast, data obtained with a 2D cell proliferation and survival assay did not correlate with the 3D and in vivo situations, suggesting that cMet function does not affect cell survival and proliferation on plastic but confers the capacity for anchorage-independent growth. The correlation of 3D but not 2D data with in vivo sensitivity was confirmed using anti-cMet siRNAs in selected PDX-derived non-small cell lung cancer cell lines. Preliminary immunohistochemical analysis revealed that PDX sensitive to cMet inhibitors expressed high cMet levels while not all PDX expressing high cMet levels were sensitive to cMet inhibitors. In conclusion, for cMet inhibitors the TCA replicates in vivo sensitivities of PDX to a high degree. Due to its short duration the TCA is an excellent tool for the screening of large numbers of cMet inhibitors and PDX. As all of the PDX qualified for use in the TCA (>200) have been extensively molecularly characterised (gene expression, gene copy number variation and mutation analysis) this assay is also an excellent tool for generating high quality biomarker hypotheses during the preclinical profiling of molecules intended for oncology indications. Citation Format: Sabine Gorynia, Jianing Guo, Andreas Ackermann, Armin Maier, Rebekka Krumbach, Gerhard Kelter, Vincent Vuaroqueaux, Thomas Metz, Thomas Metcalfe, Heiner H. Fiebig. Ex vivo 3D assay: rapid and reliable replication of the in vivo anti-tumor efficacy of c-Met inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3835. doi:10.1158/1538-7445.AM2013-3835

Abstract 5572: Breastin a natural product from Nerium Oleander exhibits high activity in a panel of human tumor cell lines.

Background: More than half of all registered anticancer agents were originally derived from natural sources. We isolated and screened >2.000 pure compounds, and purified extracts from microorganisms and plants in a panel of human tumor cell lines (Kelter et al. ENA 2008). Here we report on the activity of Breastin, a defined extract isolated from the plant Nerium Oleander in 63 human cell lines. The mode of action was elucidated by comparing the activity profile with that of known anticancer agents with defined mechanism. Methods: Breastin was isolated as cold extract from the leaves of the plant Nerium Oleander. It contains defined amounts of glycosides, flavonoides and polysaccharides. In-vitro activity was determined in 63 human tumor cell lines in a monolayer assay. 4 to 10,000 cells were seeded in 96 well plates; one day later Breastin was added at 5 concentrations and incubated with the cells for 4 days. The read-out was propidium iodide-based fluorescence. Based on IC50 and IC70 values tumor selectivity was analyzed. In the Compare Analysis the IC50 and IC70 pattern of Breastin was compared with the corresponding patterns of 180 known substances using Spearman Correlations. Results: Breastin showed a dose-dependent antitumor activity, the mean IC50 and IC70 were 1.1 and 2.7 μg/ml, respectively. It was the most potent and selective preparation out of a series of 36 extracts that were obtained with different extraction procedures. 31 / 63 cell lines investigated showed IC50 The Compare Analyses showed no similarity with 180 known anticancer agents, the coefficients of correlation were In former studies in-vivo activity was determined in murine models: B16 melanoma and Lewis-Lung-carcinoma were sensitive, leukemias L1210 and P388 were resistant. Conclusion: The evaluation of 36 extracts from Nerium Oleander identified the cold extract called Breastin as most promising anticancer agent with selective in-vitro activity found in bladder, CNS, colon and NSC lung cancer cell lines as well as in pancreas and prostate models. Moreover, in combination studies Breastin showed synergism with severall cytotoxic drugs. Citation Format: Heinz H. Fiebig, Gerhard Kelter, Armin Maier, Thomas Metz, Luay J. Rashan. Breastin a natural product from Nerium Oleander exhibits high activity in a panel of human tumor cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5572. doi:10.1158/1538-7445.AM2013-5572

Abstract 2774: The molecular determinants of sensitivity to HER2 targeted therapy in Patient Derived Xenograft gastric tumor models from Caucasian and Eastern Asian patients.

Acknowledgements Gastric tumors were xenografted into nude mice and characterized for gene copy number variation (Affymetrix SNP V6.0 array and qPCR), for mutations (Sanger sequencing and Sequenom MassARRAY OncoCarta panels 1, 2 and 3), for mRNA expression (Affymetrix HGU133 plus 2.0) and for protein expression (immunohistochemistry, IHC). Response to HER2targeted therapy was assessed in vivo by treating PDX with Trastuzumab at 10 mg/kg/day on days 0, 7, 14, and 21.  A total of 26 gastric cancer PDX were established (6 PDXs from patients of Caucasian and 20 from patients of Eastern Asian origin) (see poster R. Krumbach, abs 2789).  Based on transcriptomic profiles, gastric PDXs clustered in distinct groups. Caucasian PDXs clustered in a separate group.  5 Gastric PDXs highly expressed HER2 mRNA with all except one clustering together.  Amphiregulin was the predominant growth factor in most of the HER2amp gastric PDXs.  HER2 and HER3 were predominantly expressed (not EGFR or HER4).  4 out of 5 HER2amp gastric PDXs expressed Met mRNA.

Abstract 4170: Mutation and chemosensitivity profiling of 18 human melanoma cell lines

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Malignant melanomas account for a substantial proportion of cancers worldwide and are largely resistant to conventional therapy. The heterogeneity and the invasiveness of melanomas present notable challenges for anti-cancer treatment. Recently, several mutations in RAF, RAS, and PTEN were identified that allow for prognosis of cancer development and progression. Therefore, inhibitors of the RAF-MEK-ERK pathway form attractive candidates for molecular-based targeted treatment of melanoma cancer. Here, we present the molecular characterization and chemosensitivity profiling of 18 human melanoma cell lines. Eight of these were established by Oncotest from human tumor xenografts originally derived from primary patient material. The molecular profiling included mutational analysis for BRAF, PIK3CA, KRAS, NRAS, TP53, and PTEN by sequencing. The chemosensitivity profiles were determined in vitro using a fluorescence-based cytotoxicity assay and included commonly used chemotherapeutic agents for treatment of melanoma, targeted agents (sorafenib and bortezomib) and various BRAF (PLX-4032, PLX-4720, GDC-0879) and MEK (CI-1040, PD0325901) inhibitors. The clinically relevant BRAF V600E substitution was the most prevalent mutation and was found in more than 50% of the melanoma cell lines. The chemosensitivity profiling revealed diverse patterns of selectivity and potency with mean IC50 values between 0.4 nM (vinblastine) and 8.3 µM (carboplatin). Each of the BRAF inhibitors tested was clearly more active in melanoma cell lines carrying a BRAF mutation. On average, IC50 values of BRAF inhibitors were more than 20-fold lower for the BRAF V600E mutated cell lines compared to the wild type cell lines. However, the BRAF mutated cell line MEXF 462NL was resistant towards BRAF inhibitors and it is warranted to compare its gene expression profile and mutational status with BRAF mutated cell lines responding to BRAF inhibitors. By using an ex vivo clonogenic assay, similar activity patterns for BRAF inhibitors were found, with mutated BRAF tumor xenografts showing markedly higher sensitivity. Potential synergistic activity of BRAF inhibitors with other inhibitors of the RAF-MEK-ERK pathway or with standard chemotherapeutic agents will be presented. The mutational status of selected cancer related genes, as well as responsiveness towards many cytotoxic and targeted agents was analyzed for 18 melanoma cell lines. This extensive genotypic and phenotypic characterization makes this melanoma cell line panel a valuable tool for in vitro profiling of novel compounds, in particular inhibitors of the RAF-MEK-ERK pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4170. doi:1538-7445.AM2012-4170