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.

组织加工和测序设施（TPSF）对于为泌尿外科肿瘤分支（UOB）和我们的合作者提供支持和资源至关重要。 TPSF处理在UOB中生成的每种生物异常，处理每个标本以保留生物分子，保留每种采购的准确库存，并有助于对选定标本进行科学分析，以阐明与肾脏，前列腺和蓝晶犬有关的生物学途径的最终目标。 TPSF从近100％的UOB手术以及活检和其他手术的子集中处理组织。通常，每周有3至8次手术和10至20次活检，导致每年400多名患者采购的组织样本，包括肾脏，前列腺和膀胱癌，肾上腺肿瘤，子宫平滑肌瘤，淋巴结转移酶以及其他与孢子虫蛋白尿液学癌症癌症有关。始终与手术病理合作采购组织，以确保正确处理和准确的诊断。组织被冻结，保存在福尔马林或戊二醛中，或用于生物分子（DNA，RNA，蛋白，蛋白，代谢物）纯化和分析的加工。另外，从遗传综合征患者采集的血液样本中定期制备DNA，血清和血浆。也可以从某些患者中采购并存储全血或RNA。每周可能会处理超过二十个血液样本。最后，核心还采购和处理尿液，腹水或胸腔液，囊肿液，唾液和其他体液。冷冻样品存储在液氮或-80度摄氏冰箱中。标本被分配一个去识别的实验室号，并输入到安全数据库LabMatrix中。在过去的一年中，已经采购和处理了大约2,000个组织和800个血样。整个UOB组织存储库中包含超过25,000个组织样品，以及来自5,000多个血液样本和800个肿瘤的DNA。大多数样品是在NIH临床中心收集的，并将完整的患者历史纳入Labmatrix。未来的目标是将所有临床和实验室发现都纳入LabMatrix，以为我们从长凳到床边的所有研究提供无障碍资源。 TPSF的关键功能是支持分支机构内的临床试验。 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高级膀胱癌患者的Vicinium。目前还建立了一些新的临床试验。定期处理血液和/或尿液样品，目的是研究药物的药效学和phamacokinetic效应以及其他癌症生物标志物。肾脏和膀胱手术中的许多肿瘤样品都是在无菌条件下采购的，以建立新的细胞培养物和小鼠异种移植物。我们已经产生了300多种肾脏癌细胞系，其中92种针对癌症基因突变进行了广泛的特征。系列是由遗传性肾癌综合征（BHD，SDHB，SDHD，VHL，BAP1，HPRC和HLRCC）以及稀有肾癌类型（Chromophobe，TFE-3 RCC和Medullary RCC）产生的。去年，在细胞培养和/或SCID/Beig或裸鼠中，大约有十几个肾脏肿瘤以及一些前列腺肿瘤和膀胱肿瘤被放置在短期中，其中一部分在短期内生长出来，而少量（今年两个）变得不朽。这些线对于研究肿瘤发生的分子基础和前瞻性疗法是无价的。在最近的一项研究中，我们的一部分线进行了药物筛查。将细胞系保持在标准二氧化碳或低氧气孵化器中或储存在液氮中，并将小鼠放置在适当的现场设施中。一些线的特征是核和线粒体DNA测序以及选定基因的拷贝数，阵列CGH，核型，甲基化，实时PCR，Western斑点，氧气消耗，细胞外酸化速率以及代谢组学。 DNA样品用于检测和表征种系疾病突变的收集是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，整个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/或其他遗传研究。我们继续使用这些技术来发现新的突变，缺失，易位和放大。由TPSF处理的肾脏，前列腺和膀胱癌的冷冻和福尔马林固定的组织都通过免疫组织化学，定量PCR，表达微阵列，北部和蛋白质斑点，北部和蛋白质斑点，免疫沉淀，以及核和核和线粒体的dna sequecencing在UOB中广泛特征。 为了表征亚细胞细胞器，已将戊二醛固定组织用于电子显微镜。正确处理我们的手术标本一直是确保最佳质量实验室结果的重要因素。 UOB还参与了其许多采购的肿瘤组织和血液样本的等分试样，以进行合作实验室。该分支机构具有长期的合作，我们将组织分配给其他实验室，以进行细胞培养和肾脏癌患者的免疫疗法，分析肾脏癌细胞标记，癌症基因突变分析，肾上腺肿块的蛋白质和RNA研究（pheochrosocytomas）以及前列腺癌的分子流行病学研究。我们通过贡献透明细胞，铬虫和乳头状肾肿瘤以及前列腺肿瘤参加了几个癌症基因组图集（TCGA）项目。在过去的35年中，UOB积累的大量生物循环收集为肾脏，前列腺和膀胱癌的基本和临床研究提供了宝贵的资源。