Melanoma Patient-derived Xenograft Models Research Articles

Prevalence of Homologous Recombination Pathway Gene Mutations in Melanoma: Rationale for a New Targeted Therapeutic Approach

Homologous recombination DNA damage repair (HR-DDR) deficient patients with various solid tumors have been treated with PARP inhibitors. However, the clinical characteristics of patients with melanoma who have HR-DDR gene mutations and the consequences of PARP inhibition are poorly understood. We compared the commercially available next-generation sequencing data from 84 patients with melanomas from our institution with a dataset of 1,986 patients as well as 1,088 patients profiled in cBioportal. In total, 21.4% of patients had ≥1 functional HR-DDR mutation, most commonly involving BRCA1, ARID1A, ATM, ATR, and FANCA. Concurrent NF1, BRAF, and NRAS mutations were found in 39%, 39%, and 22% of cases, respectively. HR-DDR gene mutation was associated with high tumor mutational burden and clinical response to checkpoint blockade. A higher prevalence of HR-DDR mutations was observed in the datasets from Foundation Medicine (Cambridge, CA) and those from the Cancer Genome Atlas. Treatment of HR-DDR‒mutated patient-derived xenograft models of melanoma with PARP inhibitor produced significant antitumor activity invivo and was associated with increased apoptotic activity. RNA sequencing analysis of PARP inhibitor-treated tumors indicated alterations in the pathways involving extracellular matrix remodeling, cell adhesion, and cell-cycle progression. Melanomas with HR-DDR mutations represent a unique subset, which is more likely to benefit from checkpoint blockade and may be targeted with PARP inhibitor.

Drug responses are conserved across patient-derived xenograft models of melanoma leading to identification of novel drug combination therapies

BackgroundPatient-derived xenograft (PDX) mouse tumour models can predict response to therapy in patients. Predictions made from PDX cultures (PDXC) would allow for more rapid and comprehensive evaluation of potential treatment options for patients, including drug combinations.MethodsWe developed a PDX library of BRAF-mutant metastatic melanoma, and a high-throughput drug-screening (HTDS) platform utilising clinically relevant drug exposures. We then evaluated 34 antitumor agents across eight melanoma PDXCs, compared drug response to BRAF and MEK inhibitors alone or in combination with PDXC and the corresponding PDX, and investigated novel drug combinations targeting BRAF inhibitor-resistant melanoma.ResultsThe concordance of cancer-driving mutations across patient, matched PDX and subsequent PDX generations increases as variant allele frequency (VAF) increases. There was a high correlation in the magnitude of response to BRAF and MEK inhibitors between PDXCs and corresponding PDXs. PDXCs and corresponding PDXs from metastatic melanoma patients that progressed on standard-of-care therapy demonstrated similar resistance patterns to BRAF and MEK inhibitor therapy. Importantly, HTDS identified novel drug combinations to target BRAF-resistant melanoma.ConclusionsThe biological consistency observed between PDXCs and PDXs suggests that PDXCs may allow for a rapid and comprehensive identification of treatments for aggressive cancers, including combination therapies.

Establishment and characterization of melanoma patient-derived xenograft models for preclinical evaluation of novel therapeutics.

Patient-derived xenograft (PDX) models mostly retain the histological and genetic features of their donor tumors, which have been used for investigating various types of cancer. However, PDX models for melanoma, especially acral melanoma, are reported occasionally. We aimed to establish a large panel of melanoma PDX models representing the predominant Asian melanomas. Ninety-three fresh melanoma samples were implanted subcutaneously into nonobese diabetic/severe combined immunodeficiency mice. The histological and genetic characteristics were analyzed in both patient tumors and PDX models using immunohistochemistry, PCR amplification, and Sanger sequencing. Furthermore, the sensitivities of PDX models harboring distinct mutation profiles to binimetinib (a MEK inhibitor), vemubrafenib (a BRAF inhibitor), and imatinib (a KIT inhibitor) were also evaluated. Twenty-five PDX models were established successfully [25/93 (26.9%)] and passaged to maintain tumors in vivo. Clinical stage and origin of tumor sample were correlated with successful establishment rates (P=0.008 and <0.001, respectively). The histological (expression of NRAS, P16, and RB) and genetic (mutation status of NRAS, BRAF, and KIT) characteristics were stably maintained from patient tumors to PDX models. Targeted drugs could inhibit the tumor growth of PDX models harboring the corresponding target gene mutations. These PDX models constitute a pharmacological platform, enabling personalized development of therapeutic strategies for Asian melanomas.

Analysis of NRAS gain in 657 patients with melanoma and evaluation of its sensitivity to a MEK inhibitor

BackgroundNeuroblastoma rat-sarcoma (NRAS) mutations have been described in Chinese patients with melanoma. However, the status and the clinical significance of NRAS gain have not been investigated on a large scale. MethodsA total of 657 melanoma samples were included in the study. NRAS copy number was examined using the QuantiGene Plex DNA assay. The sensitivities of cell lines and patient-derived xenograft (PDX) models containing NRAS gain to a MAP/ERK kinase (MEK) inhibitor (binimetinib) were also evaluated. ResultsThe overall incidence of NRAS gain was 14.0% (92 of 657). Incidence of NRAS gain in acral, mucosal, chronic sun-induced damage (CSD) and non-CSD melanomas was 12.2%, 15.8%, 9.5% and 19.4%, respectively. NRAS gain was mutually exclusive to NRAS mutations (P = 0.036). The median survival time for melanoma patients with NRAS gain was significantly shorter than that for patients with normal NRAS copy number (P = 0.006). For patients containing NRAS gain, the median survival time for higher copy number (>4 copies) was significantly shorter than those with lower copy number (2–4 copies; P = 0.002). The MEK inhibitor (binimetinib) inhibited the proliferation of melanoma cells and the tumour growth of PDX models with NRAS gain. ConclusionsNRAS gain is frequent in patients with melanoma and may predict a poor prognosis of melanoma. The melanoma cells and PDX models containing NRAS gain are sensitive to MEK inhibitor (binimetinib), indicating that NRAS gain might be a new therapeutic target for melanoma.

Identification of coexistence of BRAF V600E mutation and EZH2 gain specifically in melanoma as a promising target for combination therapy

BackgroundCoexistence of enhancer of zeste homolog 2 (EZH2) and BRAF gene aberrations has been described in many cancer types. In this study, we aim to explore the coexistence status of BRAF V600E mutation and the copy number variation of EZH2 and explore the potential of this combination as a therapeutic target.MethodsA total of 138 cases of melanoma samples harboring BRAF V600E mutation were included, and EZH2 copy numbers were examined by QuantiGenePlex DNA Assays. Clinical pathological distinction between patient groups with or without EZH2 amplification (hereafter referred to as EZH2 gain) was statistically analyzed. The sensitivity of melanoma cell lines and patient-derived xenograft (PDX) models containing BRAF V600E mutation with or without EZH2 gain to vemurafenib (BRAF inhibitor), GSK2816126 (EZH2 inhibitor) and a combination of both agents was evaluated.ResultsIn our cohort, the coexistence rate of BRAF V600E mutation and EZH2 gain was up to 29.0%, and significant differences in overall survival and disease-free survival were found between no EZH2 copy number gain and gain groups (P = 0.038, P = 0.030), gain and high EZH2 copy number gain groups (P = 0.006, P = 0.010). Combination with BRAF and EZH2 inhibition showed better inhibitory efficacy in melanoma prevention compared with vemurafenib monotherapy. More importantly, this improved therapeutic effect was observed especially in melanoma cell lines and PDX models containing concurrently BRAF V600E mutation and EZH2 gain.ConclusionsCoexistence of BRAF V600E mutation and EZH2 gain is rather prevalent in melanoma. Our findings provided evidence for the feasibility of combination therapy with EZH2 and BRAF inhibitors in melanoma with concurrent BRAF V600E mutation and EZH2 gain.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 1191: Translational Proof-of-Concept (TransPoC), a not-for-profit research organization enabling access to large-scale translational oncology platforms: The Patient-Derived Xenograft network

Abstract TransPoC is a not-for-profit research organization that will deliver open-access, large-scale translational oncology platforms to enable greater clinical proof-of-concept success for new cancer therapies. TransPoC will comprise three platforms: 1. CPN - Cancer Cell “PoC” Network for screening compounds against 1000+ genomically-characterized cell lines; 2. MPN- Mouse “PoC” Network - a multi-site platform for mouse preclinical trials using genomically-characterized Patient-Derived Xenograft (PDX) models; 3. BioIT - analysis and integration of genomic information and pharmacological profiling data. Here we present an overview of the Mouse “PoC” Network, define a path to implementation of multi-center pre-clinical trials in mice and describe a pilot study to demonstrate the feasibility of implementing such a network. PDX models are increasingly used in pre-clinical studies as they capture and retain the histological, molecular, and genetic heterogeneity of the original tumor compared to cell line derived xenografts and are therefore a closer representation of a patient's tumor in situ. To enable transformative preclinical studies, models need to be characterized in a manner similar to tumor samples in The Cancer Genome Atlas and the International Cancer Genome Consortium, and must be assembled in sufficient quantity to capture clinically relevant major cancer (sub)types. To achieve this, TransPoC is building a global network of mouse PDX “hospitals” with genomic and metabolomic profiles characterized in a consistent manner. In addition, each mouse hospital will utilize common SOPs to generate comparable pharmacology data sets across sites that will include testing standard of care agents. BioIT will enable deep interrogation of data sets and provide pipelines for pharmacogenomics correlates of response to both standard and novel agents. To date, the network has collated over 2,000 PDX models and will enable sponsors to execute multi-center pre-clinical trials in a manner similar to those used in multi-institutional cooperative clinical trials. To demonstrate the viability of MPN, a pilot study has been initiated at 6 sites located in Canada, Italy, China and USA to evaluate the activity of MEK and RAF inhibitors against a panel of BRAF/KRAS mutant melanoma and colorectal cancer PDX models. An update on the initial tolerability, PK/PD/efficacy studies and molecular characterization of PDX models in the network will be presented. TransPoC continues to recruit new sites and characterize their PDX models for incorporation into MPN for use by TransPoC sponsors. Through this effort TransPoC enables rapid assessment of standard and novel investigational therapies to determine their therapeutic potential for translation to clinical trials with a mission to improve the chance of observing clinical proof-of-concept. Citation Format: Peter G. Smith, David Sutton, Andrea Bertotti, Livio Trusolino, Susan Airhart, Ming S. Tsao, Bradly G. Wouters, S. Gail Eckhardt, Lai Wang, Tim Heffernan, David Verbel, Andrea Gerken, Peter Fekkes, Lihua Yu, Lihua Yu, Markus Warmuth. Translational Proof-of-Concept (TransPoC), a not-for-profit research organization enabling access to large-scale translational oncology platforms: The Patient-Derived Xenograft network. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1191. doi:10.1158/1538-7445.AM2014-1191

Abstract 3699: Molecular profiling of BRAFi-resistance in melanoma cancer models using high-throughput sequencing in patient-derived xenografts

Abstract Patients with metastatic V600E mutant melanomas treated with BRAF inhibitors (BRAFi) frequently develop resistant tumors with aggressive phenotypes. Furthermore, approximately 20-40% of V600E mutant tumors are intrinsically resistant to BRAFi. Suitable models for studying mechanisms of acquired and intrinsic resistance to BRAFi are therefore necessary. In the present study, we applied deep sequencing techniques to identify possible mechanisms of intrinsic and acquired resistance in our collection of melanoma patient-derived xenograft models (MEXFs) treated with BRAFi. Mutational and expression profiles in MEXFs were characterized by whole-exome sequencing (WES) and HG-U133 Plus 2.0 Affymetrix chips and correlated with BRAFi efficacy data from 3D Tumor Clonogenic Assays (TCA). In addition, four resistant cell lines were created by continuously treating 2D monolayer cultures of tumor cells with Vemurafenib, all of which were initially sensitive to BRAFi and carry the BRAF V600E mutation. Expression profiles and mutations of these cell lines were analyzed from RNA-seq data. Potential gene candidates responsible for conferring resistance were further investigated by Q-PCR and Western-blot experiments. WES data revealed that the number of mutations per model was highly variable. Some MEXFs showed a hyper-mutated profile (&amp;gt;2000 mutations) and were characterized by specific mutational signatures in agreement with those found in melanoma (Alexandrov et al, Nature, 2013). Mutation frequencies of genes typically mutated in melanoma are very similar to those found in The Cancer Genome Atlas. We observed a high correlation of mutations between our MEXFs and their respective cell lines. Among the models with a V600E mutation, only one (MEXF 462) showed resistance to BRAFi-treatment as identified by 3D TCA analyses. In this model, we identified gene point mutations and a high over-expression of EGFR, MEK1, PDGFRA and NF1 in contrast to the other models, suggesting a specific regulation of the RTK signaling pathways. In 2D assays, synergistic interaction of Vemurafenib and Erlotinib was shown. RNA-seq analysis of the cell line established from MEXF 276, in which resistance to Vemurafenib was induced, revealed around 20% of genes being differentially expressed (FC&amp;gt;2) between sensitive and resistant cell lines. An up-regulation of EGFR was also found in this resistant cell line and was confirmed by Western-blot. In addition, an overexpression of PLAU, a biomarker of invasiveness, was identified. We currently perform expression profiling of three other models resistant to BRAFi. Preliminary results suggest different patterns of gene regulation involved in acquisition of resistance. A precise map of transcriptomic and mutational profiles of the four cell lines will be generated and we are investigating if invasiveness is increased upon acquisition of resistance. Citation Format: Bruno Zeitouni, Gerhard Kelter, Armin Maier, Florian Kiefer, Frederic Foucault, Anne-Lise Peille, Tim Kees, Torsten Giesemann, Vincent Vuaroqueaux, Thomas Metcalfe, Heinz-Herbert Fiebig. Molecular profiling of BRAFi-resistance in melanoma cancer models using high-throughput sequencing in patient-derived xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3699. doi:10.1158/1538-7445.AM2014-3699

Abstract A13: Establishment and characterization of a panel of melanoma patient-derived xenograft (PDX) models.

Abstract Background: A majority of metastatic melanomas are driven by BRAF and NRAS mutations in the MAP/ERK kinase pathway. Recently approved therapies targeting B-Raf and Erk kinases have improved patient outcome; however, anticancer effects of these agents are transient, indicating additional pathways and mutations may play a role in disease progression. To better understand these mechanisms we have established a panel of melanoma patient-derived xenograft (PDX) models and characterized them by mutation, receptor and ligand density and drug sensitivity. Methods: PDX melanoma models were established in athymic nude mice from primary or metastatic patient tissue and once established were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. For each model, DNA was extracted and subjected to exon sequencing of 207 known oncogenes; growth factor receptor and ligand densities were interrogated using immunohistochemistry and quantitative RNA in situ hybridization. Drug sensitivity studies were performed evaluating sensitivity of models towards chemotherapy and targeted agents including oral vemurafenib and trametinib. Vemurafenib was administered twice daily at 50 mg/kg and trametinib once daily at 1-3 mg/kg for at least twenty-eight days; oral temozolomide was tested at 100 mg/kg on a qdx5 schedule. Study endpoints included tumor volume and time from treatment initiation; with tumor growth inhibition, delay and regression reported at study completion. Results: BRAF mutations were identified in approximately 50% of melanoma models and 8% reported NRAS mutation; all identified mutations were concordant with clinical results reported from patients linked to the models. EGFR expression was reported in 13% of models (1+ to 3+) and HER3 expression (1+ to 3+) found in 55% of models with associated heregulin expression; HER2 was not found expressed in any evaluated melanoma model. Temozolomide tested at its maximum tolerated dose was ineffective in most models while vemurafenib was active in all evaluated B-RafV600E models and inactive in models with wildtype or alternate mutations. Trametinib was active in most B-RafV600E models and in some N-Ras-mutated models. Interestingly the ST054 model harboring B-RafL584 and N-RasQ61L mutations was sensitive to trametinib but not vemurafenib. Conclusion: We have established a panel of melanoma PDX models and characterized mutation status, receptor and ligand density and drug sensitivity. We found HER 3 expressed in a majority of models and benchmarked vemurafenib and trametinib activity in these models. This panel can be utilized as a valuable screening tool in early and late-stage oncology drug development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A13. Citation Format: Lizette Gamez, Michael J. Wick, Teresa L. Vaught, Monica Farley, Anthony W. Tolcher, Drew Rasco, Amita Patnaik, Alex Miller, Ron Drengler, Kyriakos P. Papadopoulos. Establishment and characterization of a panel of melanoma patient-derived xenograft (PDX) models. [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 A13.

Abstract B12: Overcoming acquired resistance to vemurafenib using clinically relevant PDX models of melanoma.

Abstract Development of resistance is a significant clinical problem for virtually all targeted cancer therapies. We have generated a reproducible, patient derived xenograft (PDX) model of acquired vemurafenib resistance to address these challenges. Continuous treatment of V600E melanoma tumors, caused synchronous tumor stasis for approximately 7 weeks, following which, all tumors displayed simultaneous resistance marked by rapid tumor growth. Additionally, this model maintains the resistance phenotype upon serial transplantation, providing a platform for testing rational drug selection. The fidelity of the PDX models was further confirmed using a BRAF V600V tumor which did not respond to vemurafenib. Onset of vemurafenib resistance is accompanied by increased phosphor-ERK signifying re-engagement of the MAPK signaling pathway and supporting MEK as a potential target. MEK inhibition in vemurafenib resistant tumors using PD0325901, resulted in rapid tumor shrinkage and dramatically reduced phosphor-ERK levels. Treatment of resistant tumors with PD0325901 alone, whilst leading to rapid tumor shrinkage, showed significant host toxicity and onset of acquired MEKi resistance. Interestingly, combination of vemurafenib + PD0325901 was non-toxic, and showed dramatic and sustained tumor suppression. Upon cessation of PD0325901 at 70 days the tumors remained undetectable for the duration of the study (&amp;gt;100 days). These data support the use of MEK inhibitors post-development of vemurafenib resistance and demonstrate that combination therapy mitigates systemic MEKi toxicity and results in persistent tumor inhibition/eradication. PDX models of acquired resistance provide a unique opportunity to bridge the gap between patients and the basic in vitro biology. Additionally, this PDX system allows the interrogation of the kinetics involved in the development of resistance by longitudinal tumor tissue sampling. Numerous mechanisms have been identified as potential causes of the resistance phenotype. Many have been identified in vitro but not all have been confirmed in patients. We detected no evidence of increased BRAF copy number or expression, although alternative BRAF splicing was identified in resistant tumors. Using differential gene expression accompanied by pathway and network analysis we identified distinct differences in the PDX tumors at various time points during the development of resistance. In particular, a potential role for interferon signaling in resistant tumors was observed. Furthermore, changes in the metabolic profiles were identified with untreated and resistant tumors favoring glycolytic pathways, whereas growth arrested tumors exhibited a preference for oxidative phosphorylation. In conclusion, these results demonstrate the value of PDX models for contributing to clinical cancer management through the decryption of complex drug resistance mechanisms and accelerating the identification of rationally selected drug combinations for bench to bedside applications. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B12. Citation Format: Noel R. Monks, David J. Monsma, David M. Cherba, Emily Eugster, Dawna Dylewski, Mary E. Winn, Andrew S. Borgman, Paula J. Davidson, Chelsea A. Peterson, Jose M. Pimiento, Alexander E. Ivliev, Yuri Nikolsky, Marina Bessarabova, Valerie S. Calvert, Mariaelena Pierobon, Emanuel F. Petricoin, Craig P. Webb, Brian J. Nickoloff. Overcoming acquired resistance to vemurafenib using clinically relevant PDX models of melanoma. [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 B12.