Tissue Processing-Sequencing Facility

组织处理测序设备

• 批准号: 10703039
• 负责人: Cathy D Vocke
• 金额: $ 91.73万
• 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/10703039
• 关键词: Adrenal Gland Neoplasms; Adrenal Mass; Affect; Aliquot; Ascites; Basic Science; Biological; Biopsy; Bladder; Bladder Neoplasm; Blood; Blood specimen; Body Fluids; Cancer Gene Mutation; Cancer Patient; Cancer cell line; Carbon Dioxide; Cell Culture Techniques; Cell Line; Cellular Metabolic Process; Chest; Clear Cell; Clinical; Clinical Research; Clinical Treatment; Clinical Trials; Collaborations; Collection; Communication; Cyst Fluid; DNA; DNA analysis; DNA sequencing; Data; Databases; Detection; Disease; Drug Screening; Electron Microscopy; Elements; Ensure; Equipment and supply inventories; Erlotinib; Fluorescent in Situ Hybridization; Formalin; Freezing; Future; Gene Expression; Genes; Genetic study; Genitourinary system; Genome; Glutaral; Goals; Heart; Hereditary Leiomyomatosis and Renal Cell Cancer; House mice; Human; Immunohistochemistry; Immunoprecipitation; Immunotherapy; Incubators; Inherited; Karyotype; Karyotype determination procedure; Kidney; Kidney Neoplasms; Laboratories; Laboratory Finding; Light; Liquid substance; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Metastatic Neoplasm to Lymph Nodes; Methods; Methylation; Mitochondrial DNA; Molecular; Molecular Epidemiology; Molecular Target; Mus; Mutation; Mutation Analysis; Nitrogen; Northern Blotting; Nuclear; Nude Mice; Operative Surgical Procedures; Organelles; Other Genetics; Oxygen; Oxygen Consumption; Paper; Papillary; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacodynamics; Pheochromocytoma; Plasma; Polymorphism Analysis; Procedures; Process; Prostate; Prostate carcinoma; Prostatic Neoplasms; Proteins; Proteomics; Publishing; RNA; Reactive Oxygen Species; Reagent; Recording of previous events; Renal Cell Carcinoma; Renal carcinoma; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Saliva; Sampling; Scientist; Secure; Serum; Single Nucleotide Polymorphism; Site; Specimen; Sterility; Succinate Dehydrogenase; Surgical Pathology; Syndrome; Techniques; Testing; The Cancer Genome Atlas; Therapeutic Agents; Time; Tissue Banks; Tissue Sample; Tissues; Tuberous Sclerosis; Tumor Tissue; United States National Institutes of Health; Urine; Urogenital Cancer; Urologic Cancer; Urologic Oncology; Uterine Fibroids; Western Blotting; Whole Blood; Xenograft Model; Xenograft procedure; accurate diagnosis; bench to bedside; bevacizumab; bladder Carcinoma; bladder surgery; cancer biomarkers; cancer type; central database; clinical center; comparative genomic hybridization; epidemiology study; exome; exome sequencing; extracellular; gene discovery; inhibitor; kidney surgery; kinase inhibitor; metabolomics; molecular targeted therapies; new therapeutic target; next generation; pharmacokinetics and pharmacodynamics; preservation; prospective; protein expression; protein metabolite; tissue processing; tumor; tumorigenesis; whole genome

The Tissue Processing and Sequencing Facility (TPSF) is essential in providing support and resources for the Urologic Oncology Branch (UOB) and for our collaborators. The TPSF handles every biospecimen that is generated within the UOB, processes each specimen in order to preserve biomolecules, keeps an accurate inventory of each procurement, and assists in the scientific analysis of select specimens, for the ultimate goal of elucidating biological pathways relating to kidney, prostate, and bladder cancers. The TPSF processes tissue from nearly 100% of UOB surgeries, as well as a subset of biopsies and other procedures. Typically, there are 3 to 8 surgeries and 10 to 20 biopsies per week, resulting in tissue samples procured from over 400 patients per year, including kidney, prostate, and bladder carcinomas, adrenal tumors, uterine leiomyomas, lymph node metastases, and other specimens relating to sporadic and familial urologic cancer syndromes. Tissue is always procured in cooperation with Surgical Pathology, to ensure proper handling and accurate diagnosis. Tissue is snap frozen, preserved in formalin or glutaraldehyde, or processed for biomolecule (DNA, RNA, protein, metabolite) purification and analysis. In addition, DNA, serum, and plasma are regularly prepared from blood samples taken from patients with inherited syndromes. Whole blood or RNA may also be procured and stored from select patients. Over two dozen blood samples may be processed per week. Finally, the core also procures and processes urine, ascites or thoracic fluids, cyst fluids, saliva, and other body fluids. Frozen samples are stored in liquid nitrogen or in a -80 degree centigrade freezer. Specimens are assigned a de-identified lab number and entered into a secure database, Labmatrix. Approximately 2,000 tissue and 800 blood specimens have been procured and processed within the past year. The entire UOB tissue repository contains in excess of 25,000 tissue samples, and DNA from over 5,000 blood samples and 800 tumors. Most of the samples were collected at the NIH Clinical Center, and full patient histories are incorporated into Labmatrix. A future goal is for all clinical and laboratory findings to be incorporated into Labmatrix to provide an accessible resource for all of our studies from bench to bedside. A key function of the TPSF is to support clinical trials within the Branch. This past year we handled samples from patients in several clinical trials that are open to accrual: Bevacizumab and Erlotinib for patients with metastatic papillary kidney cancer or HLRCC, a Met kinase inhibitor INC280 for patients with papillary kidney cancer, a HIF2 inhibitor for patients with VHL, PANVAC-BCG vs. BCG for patients with high grade bladder cancer, and Durvalumab and Vicinium for patients with high grade bladder cancer. Several new clinical trials are also currently being established. Blood and/or urine samples are processed at regular intervals, for the purpose of investigating pharmacodynamic and phamacokinetic effects of the drugs, as well as other cancer biomarkers. Many of the tumor samples from kidney and bladder surgeries are procured under sterile conditions to establish new cell cultures and mouse xenografts. We have generated over 300 kidney cancer cell lines, 92 of which have been extensively characterized for cancer gene mutations. Lines have been generated from hereditary kidney cancer syndromes (BHD, SDHB, SDHD, VHL, BAP1, HPRC, and HLRCC) and from rare kidney cancer types (chromophobe, TFE-3 RCC, and medullary RCC) that provide unique reagents. In the last year, about a dozen kidney tumors and a few prostate and bladder tumors have been placed in cell culture and/or in SCID/BEIG or nude mice, with a subset of these growing viably in the short-term and a small number (two this year) that become immortal. These lines are invaluable for studying both the molecular basis of tumorigenesis and prospective therapies. In a recent study, a subset of our lines were subjected to drug screening. Cell lines are kept in standard carbon dioxide or low oxygen incubators or stored in liquid nitrogen, and mice are housed in an appropriate on-site facility. Some lines have been extensively characterized by nuclear and mitochondrial DNA sequencing and copy number of selected genes, array CGH, karyotype, methylation, real-time PCR, Western blotting, oxygen consumption and extracellular acidification rate, and metabolomics. The collection of DNA samples for the detection and characterization of germline disease mutations has been at the heart of the gene discovery process in the UOB. Several dozen blood samples per year are analyzed by DNA sequencing including next-generation whole exome, whole genome, and targeted gene sequencing, comparative genome hybridization (CGH) analysis, fluorescent in-situ hybridization (FISH), and/or other genetic studies. We continue to use these techniques to discover new mutations, deletions, translocations, and amplifications. Both frozen and formalin-fixed tissues from kidney, prostate, and bladder cancers that have been processed by the TPSF have been characterized extensively within the UOB by immunohistochemistry, quantitative PCR, expression microarrays, Northern and Western blotting, immunoprecipitation, and nuclear and mitochondrial DNA sequencing. Glutaraldehyde-fixed tissues have been used for electron microscopy, in order to characterize subcellular organelles. Proper handling of our surgical specimens has been an essential factor in assuring the best quality laboratory results. The UOB is also involved in providing aliquots of many of its procured tumor tissues and blood samples to collaborating laboratories. The Branch has long-standing collaborations in which we distribute tissue to other laboratories for cell culture and immunotherapy for kidney cancer patients, analysis of kidney cancer cellular markers, cancer gene mutation analysis, protein and RNA studies of adrenal masses (pheochromocytomas), and molecular epidemiology studies of prostate cancers. We participated in several Cancer Genome Atlas (TCGA) projects, by contributing clear cell, chromophobe and papillary renal tumors as well as prostate tumors. The sizeable biospecimen collection amassed by the UOB over the last 35 years provides an invaluable resource for both basic and clinical research regarding kidney, prostate and bladder cancers.

组织处理和测序设施 (TPSF) 对于为泌尿肿瘤科 (UOB) 和我们的合作者提供支持和资源至关重要。 TPSF 处理大华银行内生成的每个生物样本，处理每个样本以保存生物分子，保留每次采购的准确库存，并协助对选定样本进行科学分析，以实现阐明与肾脏相关的生物学途径的最终目标、前列腺癌和膀胱癌。 TPSF 处理来自近 100% UOB 手术的组织，以及一部分活检和其他手术。通常，每周进行 3 至 8 次手术和 10 至 20 次活检，每年从 400 多名患者采集组织样本，包括肾癌、前列腺癌和膀胱癌、肾上腺肿瘤、子宫肌瘤、淋巴结转移瘤和其他样本与散发性和家族性泌尿系统癌症综合征有关。组织始终与外科病理学合作采购，以确保正确处理和准确诊断。组织被速冻、保存在福尔马林或戊二醛中，或进行处理以进行生物分子（DNA、RNA、蛋白质、代谢物）纯化和分析。此外，定期从遗传综合征患者的血液样本中制备 DNA、血清和血浆。还可以从特定患者处获取并储存全血或 RNA。每周可能会处理超过两打血液样本。最后，核心还获取并处理尿液、腹水或胸液、囊肿液、唾液和其他体液。冷冻样品储存在液氮或-80℃冰箱中。样本被分配一个去识别化的实验室编号，并输入安全数据库 Labmatrix。去年，我们采购并处理了大约 2,000 份组织样本和 800 份血液样本。整个 UOB 组织存储库包含超过 25,000 个组织样本以及来自 5,000 多个血液样本和 800 个肿瘤的 DNA。大部分样本是在 NIH 临床中心收集的，完整的患者病史已纳入 Labmatrix。未来的目标是将所有临床和实验室发现纳入 Labmatrix，为我们从实验室到临床的所有研究提供可访问的资源。 TPSF 的一项关键职能是支持分部内的临床试验。去年，我们处理了几项开放的临床试验中的患者样本：用于治疗转移性乳头状肾癌或 HLRCC 患者的贝伐珠单抗和厄洛替尼、用于乳头状肾癌患者的 Met 激酶抑制剂 INC280、用于 VHL 患者的 HIF2 抑制剂、PANVAC-BCG 与 BCG 对比治疗高级别膀胱癌患者，以及 Durvalumab 和 Vicinium 治疗高级别膀胱癌患者。目前还正在建立一些新的临床试验。定期处理血液和/或尿液样本，以研究药物以及其他癌症生物标志物的药效和药代动力学效应。许多来自肾脏和膀胱手术的肿瘤样本都是在无菌条件下获得的，以建立新的细胞培养物和小鼠异种移植物。我们已经生成了 300 多种肾癌细胞系，其中 92 种已被广泛表征为癌症基因突变。细胞系是从遗传性肾癌综合征（BHD、SDHB、SDHD、VHL、BAP1、HPRC 和 HLRCC）和罕见肾癌类型（嫌色细胞、TFE-3 RCC 和髓质 RCC）中产生的，可提供独特的试剂。去年，大约有十几个肾肿瘤和一些前列腺和膀胱肿瘤被置于细胞培养物和/或 SCID/BEIG 或裸鼠中，其中一部分在短期内存活且数量很少。 （今年二）即成仙。这些细胞系对于研究肿瘤发生的分子基础和前瞻性治疗具有无价的价值。在最近的一项研究中，我们对部分品系进行了药物筛选。细胞系保存在标准二氧化碳或低氧培养箱中或储存在液氮中，小鼠则饲养在适当的现场设施中。一些品系已通过核和线粒体 DNA 测序和所选基因的拷贝数、阵列 CGH、核型、甲基化、实时 PCR、蛋白质印迹、耗氧量和细胞外酸化率以及代谢组学进行了广泛的表征。用于检测和表征种系疾病突变的 DNA 样本收集一直是 UOB 基因发现过程的核心。每年通过 DNA 测序分析几十个血液样本，包括下一代全外显子组、全基因组和靶向基因测序、比较基因组杂交 (CGH) 分析、荧光原位杂交 (FISH) 和/或其他遗传学研究。我们继续使用这些技术来发现新的突变、缺失、易位和扩增。 TPSF 处理的肾癌、前列腺癌和膀胱癌的冷冻和福尔马林固定组织已在 UOB 内通过免疫组织化学、定量 PCR、表达微阵列、Northern 和 Western 印迹、免疫沉淀以及核 DNA 和线粒体 DNA 进行了广泛的表征测序。 戊二醛固定的组织已用于电子显微镜，以表征亚细胞器。正确处理我们的手术标本是确保最佳质量实验室结果的重要因素。大华银行还参与向合作实验室提供其采购的许多肿瘤组织和血液样本的等分试样。该分部与其他实验室有着长期的合作关系，我们将组织分发给其他实验室，用于肾癌患者的细胞培养和免疫治疗、肾癌细胞标志物分析、癌症基因突变分析、肾上腺肿块（嗜铬细胞瘤）的蛋白质和RNA研究以及分子生物学研究。前列腺癌的流行病学研究。我们参与了多个癌症基因组图谱 (TCGA) 项目，贡献了透明细胞、嫌色细胞和乳头状肾肿瘤以及前列腺肿瘤。大华银行在过去 35 年中收集的大量生物样本为肾癌、前列腺癌和膀胱癌的基础和临床研究提供了宝贵的资源。