Comparative Oncology Program Laboratory

比较肿瘤学项目实验室

• 批准号: 10262403
• 负责人: Amy Leblanc
• 金额: $ 134.22万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262403
• 关键词: Address; Adolescent; Adult; Adverse event; Age; Agonist; Aliquot; Amputation; Animal Testing; Antineoplastic Agents; Area; Back; Biological; Biological Assay; Biological Markers; Biological Models; Biological Specimen Banks; Biology; Brain Neoplasms; Cancer Biology; Cancer Model; Cancer Patient; Canis familiaris; Carboplatin; Cell Line; Characteristics; Child; Childhood; Childhood Osteosarcoma; Clinical; Clinical Data; Clinical Trials; Collaborations; Collection; Companions; Complex; Conduct Clinical Trials; Custom; DNA; DNA Resequencing; DNA Sequence Alteration; DNA copy number; DNA sequencing; Data; Databases; Decision Making; Development; Disease; Drug Combinations; Drug Targeting; Early treatment; Engraftment; Event; Formalin; Freezing; Future; Gene Expression; Genes; Genome; Genomics; Goals; Growth; Health; Human; Human Cell Line; In Vitro; Infrastructure; Inherited; Investigation; Knowledge; Laboratories; Libraries; Link; Malignant Bone Neoplasm; Malignant Neoplasms; Metastatic Neoplasm to the Lung; Mission; Modeling; Molecular; Multi-Institutional Clinical Trial; Mus; Natural History; Neoplasm Metastasis; Normal tissue morphology; Oncogenes; Outcome; Pathologist; Pathology; Pathway interactions; Patients; Pediatric Neoplasm; Pharmaceutical Preparations; Phase; Physicians; Positioning Attribute; Pre-Clinical Model; Preclinical Drug Development; Preclinical Testing; Primary Neoplasm; Progression-Free Survivals; Proteomics; Puma; RNA; Reagent; Recurrent disease; Regimen; Resource Development; SNP array; Sampling; Single Nucleotide Polymorphism; Specimen; Structure; Survival Rate; Techniques; Testing; Time; Tissue Preservation; Toxicity Tests; Toxicogenomics; Translating; Treatment Failure; Treatment Protocols; Tumor stage; Work; Xenograft procedure; assay development; base; cancer clinical trial; cancer type; central database; chemotherapy; clinical application; cohort; comparative; comparative genomic hybridization; demographics; design; dog genome; drug development; drug efficacy; exome; exome sequencing; experimental study; genome-wide; genomic profiles; improved; improved outcome; in vivo; in vivo Model; inhibitor/antagonist; innovation; laboratory experiment; mouse model; neglect; new therapeutic target; novel therapeutic intervention; novel therapeutics; oncology program; osteosarcoma; pre-clinical; programs; repository; sarcoma; screening; success; transcriptome sequencing; translation to humans; treatment response; tumor

Laboratory activities and projects carried out by the COP laboratory have the specific goal of improving the understanding of the impact of anti-cancer agents on comparative aspects of metastasis biology by virtue of parallel study of murine, canine and human cell lines in a variety of in vitro, ex vivo/in vivo (Pulmonary Metastasis Assay or PuMA) model systems. Data generated in this manner improves understanding of naturally-occurring canine osteosarcoma (OS) models and could be employed to answer unique in vivo questions regarding the anti-metastatic potential of agents, via the COTC clinical trial mechanism. To extend our investigations into the comparative aspects of naturally-occurring OS in dogs to humans, we have recently initiated the DOG2 project: Decoding the Osteosarcoma Genome of the Dog. The DOG2 project fulfills the main mission of the NCI-COP, which is to strategically position the canine cancer patient in studies of cancer biology and drug development, in order to improve outcome for both dogs and humans. The COP has a longstanding focus in osteosarcoma (OS) biology and clinical trials. OS is an aggressive pediatric/AYA malignancy and the most common malignant bone tumor in children and adolescents. OS is also a common naturally-occurring canine cancer with strikingly similar clinical presentation and natural history; preliminary studies suggest a shared molecular landscape. While dogs largely develop OS in adulthood, the similar genomic features and clinical disease characteristics underscore the notion that age does not distinguish canine OS from the disease in children. Both human and canine patients with metastatic or relapsed disease have dismal outcomes, with survival rates less than 20% despite aggressive salvage regimens. We recognize that osteosarcoma is a complex disease and success is unlikely with a single approach. Therefore, to expand this work it is necessary to identify additional targets and drugs. Collectively this work is designed to address the following strategic priorities: 1. Improved knowledge of comparative OS cancer biology to enhance dog to human translation 2. Discovery of new targets and companion biomarkers in support of drug development 3. Assessment and prioritization of new therapeutic strategies in preclinical models 4. Harmonization of comparative canine and human oncology clinical trials to advance new therapeutic concepts Biospecimens. We will utilize an existing high-quality clinically-annotated biospecimen repository of 400 canine OS patient sample sets, unique to the NCI, gathered from ongoing and completed canine OS clinical trials. This repository contains multiple aliquots of both tumor and matched normal tissues, preserved as frozen, RNAlater, and formalin-fixed specimens. Samples have been derived from treatment-naive dogs that all underwent the same treatment protocol (amputation + 4 cycles of carboplatin chemotherapy). We have full clinical data on every patient including demographics, treatment details/adverse events, and event-free and overall survival. We propose beginning with a subset of n = 100 patient samples selected from the ends of a Kaplan-Meier curve that represent early treatment failures (progression-free survival less than 90 days) vs. elite responders (progression-free survival greater than 360 days). We will also query n = 25 matched pairs of tumors with their metastases that developed despite therapy. We have not attempted to perform viability testing for PDX engraftment in NSG mice; rather we use 4 unique canine OS cell lines developed in our lab for dog-in-mouse preclinical testing. All specimens will be subjected to pathology review by an expert board of veterinary and physician sarcoma pathologists to verify adequate sample quality for further genomic study. Genomic profiling. We will employ the following platforms to execute a combination of experiments to understand relationships between genomic complexity and integrity (CGH and WES to identify structural rearrangements, single nucleotide variants, copy number gain/loss), and gene expression (through RNAseq). The specific breed of dog will be verified through use of a germline SNP array recently made available through MARS/Wisdom Health. Samples for genomic profiling will be sent to our collaborator at NCSU (Matthew Breen) for DNA and RNA isolation, library prep, CGH for genome-wide DNA copy number profiling (Agilent CGH array), and for DNA whole exome sequencing (150b paired end reads at 100x for tumors/30x for normal, NovaSeq6000 platform with Roche dog exome kit), methyl-DNA resequencing, as well as RNA sequencing (125b paired end reads, Illumina HiSeq2500 platform). Computational approach. This project is an extension of an existing collaboration with NCSU and the NCATS Division or Preclinical Innovation (DPI) and Therapy for Rare and Neglected Diseases (TRND) program. Through experiments we have already conducted in n = 12 canine OS sample sets similar to those described above as a discovery cohort, the TRND bioinformaticians have developed a strategy that identifies distinct gene co-expression models between and specific to a set of 5 selected canine cancer types, of which OS is one, from which they derive cancer-specific gene panels. A similar analysis is performed on existing RNAseq data publicly available from human tumor samples from the same tumor types to produce cancer-specific human gene panels. These cancer-specific shared gene panels are compared, and genes common to both are retained to facilitate dog-to-human translation of genomic alterations. The RNAseq data, after alignment to reference, is then compared back to CGH and whole-exome DNA sequencing carried out in the same tumor sample to identify genomic changes (CNV, SNV) that are linked to expression changes and that may represent new druggable targets. These oncogenes or other drug targets in given pathway(s) were used to identify inhibitors, suppressors or agonists utilizing Pharos, which interfaces to a central database containing information about the targets collected by the Illuminating the Druggable Genome (IDG) program and the Comparative Toxicogenomic Database (CTD). This combined experimental/computational approach has already been tested using both proteomic and RNAseq data generated from the first n = 12 OS tumor/normal pairs. The data has been analyzed at NCATS to match changes in gene expression to drugs known to modulate the pathways associated with the associated genes. Canine OS cell line screening is already underway through NCATS to determine the in vitro impact of 40 drugs, including 30 synergistic combinations, based on the respective drugs' pathway modulation profile. These data serve as a proving ground for additional work carried out in a larger, outcome-linked, collection of tumor samples proposed herein. Single agents or combinations of drugs identified in Phase I will be tested in our existing panel of human and canine OS cell lines and screened rapidly through available xenograft human-in-mouse and canine-in-mouse models within the COP laboratory. This preclinical animal testing is critical to ranking and advancing appropriate candidates for further study and to understand the comparative features of how drugs behave in our in vivo models. We have expertise in both primary tumor and metastasis modelling to evaluate drug impact at several stages of tumor development and progression. These results will be the basis (go/no go decision making) for canine comparative oncology clinical trials to test the toxicity and efficacy of the drug combinations in vivo. Drug combination candidates that perform well in canine clinical trials would then be considered for human trials.

COP实验室开展的实验室活动和项目的具体目标是通过在各种体外、离体/体内（肺转移试验或PuMA）模型系统中对小鼠、犬和人类细胞系进行平行研究，提高对抗癌药物对转移生物学比较方面的影响的理解。以这种方式产生的数据提高了对自然发生的犬骨肉瘤（OS）模型的理解，并且可以通过COTC临床试验机制来回答有关药物抗转移潜力的独特体内问题。为了将我们的研究扩展到狗与人类自然发生的骨肉瘤的比较方面，我们最近启动了DOG2项目：解码狗的骨肉瘤基因组。DOG2项目完成了NCI-COP的主要任务，即在癌症生物学和药物开发研究中战略性地定位犬癌症患者，以改善犬和人类的预后。COP长期专注于骨肉瘤（OS）生物学和临床试验。OS是一种侵袭性儿科/AYA恶性肿瘤，是儿童和青少年中最常见的恶性骨肿瘤。OS也是一种常见的自然发生的犬癌，具有惊人相似的临床表现和自然病史；初步研究表明存在共同的分子景观。虽然狗大部分在成年后发展为OS，但相似的基因组特征和临床疾病特征强调了年龄不能区分犬OS和儿童OS的概念。患有转移性或复发性疾病的人类和犬患者的预后都很糟糕，尽管采用了积极的挽救方案，但生存率仍低于20%。我们认识到骨肉瘤是一种复杂的疾病，单一的治疗方法是不可能成功的。因此，为了扩大这项工作，有必要确定更多的靶点和药物。总的来说，这项工作旨在解决以下战略重点：提高了比较OS癌症生物学的知识，增强了狗对人类的翻译。发现新的靶点和伴生生物标志物，支持药物开发临床前模型中新治疗策略的评估和优先排序协调比较犬和人类肿瘤临床试验，以推进新的治疗概念。我们将利用现有的高质量临床注释生物标本库，其中包含400个犬OS患者样本集，这些样本集是NCI独有的，来自正在进行和已完成的犬OS临床试验。该存储库包含肿瘤和匹配的正常组织的多个等份，保存为冷冻、RNAlater和福尔马林固定标本。样本来自未接受治疗的狗，它们都接受了相同的治疗方案（截肢+ 4个周期的卡铂化疗）。我们有每位患者的完整临床数据，包括人口统计数据、治疗细节/不良事件、无事件生存期和总生存期。我们建议从代表早期治疗失败（无进展生存期小于90天）和精英应答者（无进展生存期大于360天）的Kaplan-Meier曲线末端选择n = 100例患者样本的子集开始。我们还将查询n = 25对匹配的肿瘤及其在治疗后发生的转移。我们尚未尝试在NSG小鼠中进行PDX植入的活力测试；相反，我们使用我们实验室开发的4种独特的犬OS细胞系进行犬鼠临床前测试。所有标本将由兽医和内科肉瘤病理学家组成的专家委员会进行病理检查，以验证样本质量是否足够，以便进一步进行基因组研究。基因组分析。我们将采用以下平台进行组合实验，以了解基因组复杂性和完整性（CGH和WES鉴定结构重排、单核苷酸变异、拷贝数增加/减少）和基因表达（通过RNAseq）之间的关系。具体品种的狗将通过使用最近由MARS/Wisdom Health提供的种系SNP阵列进行验证。基因组分析的样本将发送给我们在NCSU的合作者（Matthew Breen）进行DNA和RNA分离、文库准备、全基因组DNA拷贝数分析的CGH （Agilent CGH阵列）、DNA全外显子组测序（150b对末端读取100倍的肿瘤/30倍的正常，NovaSeq6000平台与罗氏狗外显子组试剂盒）、甲基DNA重测序以及RNA测序（125b对末端读取，Illumina HiSeq2500平台）。计算的方法。该项目是与NCSU和NCATS的临床前创新（DPI）和罕见和被忽视疾病治疗（TRND）项目现有合作的延伸。通过实验，我们已经在n = 12个犬OS样本集中进行了类似于上述发现队列的实验，TRND生物信息学家已经制定了一种策略，确定了5种选定犬癌症类型之间和特异性的不同基因共表达模型，其中OS是其中一种，他们从中得出癌症特异性基因面板。对来自相同肿瘤类型的人类肿瘤样本的现有公开的RNAseq数据进行类似的分析，以产生癌症特异性的人类基因面板。这些癌症特异性共享基因组进行比较，并保留两者共同的基因，以促进狗对人类基因组变化的翻译。将RNAseq数据与参考文献比对后，与同一肿瘤样本中进行的CGH和全外显子组DNA测序进行比较，以鉴定与表达变化相关的基因组变化（CNV， SNV），这些变化可能代表新的药物靶点。这些癌基因或其他药物靶点在给定的途径中被用来识别抑制剂、抑制剂或激动剂，使用Pharos，它连接到一个中央数据库，该数据库包含由照亮可药物基因组（IDG）计划和比较毒物基因组数据库（CTD）收集的靶点信息。这种实验/计算相结合的方法已经通过前n = 12对OS肿瘤/正常对产生的蛋白质组学和RNAseq数据进行了测试。NCATS对这些数据进行了分析，以将基因表达的变化与已知调节与相关基因相关的途径的药物相匹配。犬OS细胞系筛选已经通过NCATS进行，以确定40种药物的体外影响，包括30种协同组合，基于各自药物的通路调节特征。这些数据可以作为进一步工作的试验场，在这里提出了一个更大的、与结果相关的肿瘤样本收集。在第一阶段确定的单一药物或药物组合将在我们现有的人类和犬类OS细胞系中进行测试，并通过COP实验室中可用的人鼠和犬鼠异种移植模型进行快速筛选。这种临床前动物试验对于为进一步研究排序和推进合适的候选药物以及了解药物在体内模型中的行为的比较特征至关重要。我们在原发肿瘤和转移模型方面拥有专业知识，以评估药物在肿瘤发展和进展的几个阶段的影响。这些结果将成为犬比较肿瘤学临床试验的基础（进行/不进行决策），以测试药物组合在体内的毒性和功效。在犬类临床试验中表现良好的候选药物组合将被考虑用于人体试验。