Tissue ProcessingSequencing Facility

组织处理测序设备

• 批准号: 9154313
• 负责人: Cathy D Vocke
• 金额: $ 65.5万
• 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/9154313
• 关键词: Adrenal Gland Neoplasms; Adrenal Mass; Affect; Aliquot; Ascites; Atlases; Basic Science; Biological; Biological Markers; Biopsy; Birt-Hogg-Dube Syndrome; Bladder; Bladder Neoplasm; Blood; Blood specimen; Body Fluids; Cancer Gene Mutation; Cancer Patient; Cancer cell line; Carbon Dioxide; Cell Culture Techniques; Cell Line; Cells; Chest; Clear Cell; Clinical; Clinical Research; Clinical Treatment; Clinical Trials; Collaborations; Collection; Communication; Cyst Fluid; DNA; DNA Sequence; DNA Sequence Analysis; Data; Databases; Deletion Mutation; Detection; Disease; Drug Kinetics; Electron Microscopy; 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; Indium; Inherited; Karyotype determination procedure; Kidney; Kidney Neoplasms; Laboratories; Laboratory Finding; Light; Liquid substance; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Metabolism; Methods; Molecular; Molecular Epidemiology; Molecular Target; Mouse Cell Line; Mus; Mutation; Mutation Analysis; Neoadjuvant Therapy; Neoplasm Metastasis; Nitrogen; Northern Blotting; Nude Mice; Operative Surgical Procedures; Organelles; Other Genetics; Oxygen; Oxygen Consumption; Paper; Papillary; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacodynamics; Pheochromocytoma; Plasma; Polymorphism Analysis; Preclinical Drug Evaluation; Privacy; Procedures; Process; Prostate; Prostate carcinoma; Prostatic Neoplasms; Proteins; Proteomics; Publishing; RNA; Radical Prostatectomy; Reactive Oxygen Species; Reagent; Recording of previous events; Renal Cell Carcinoma; Renal carcinoma; Research; Research Personnel; Resources; Sampling; Scientist; Secure; Security; Single Nucleotide Polymorphism; Site; Specimen; Sterility; Succinate Dehydrogenase; Surgical Pathology; Syndrome; Techniques; Testing; The Cancer Genome Atlas; Therapeutic Agents; Time; 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; base; bench to bedside; bevacizumab; bladder Carcinoma; bladder surgery; cancer genome; comparative genomic hybridization; epidemiology study; exome sequencing; extracellular; gene discovery; kidney surgery; kinase inhibitor; lymph nodes; meetings; metabolomics; next generation sequencing; novel; prospective; protein expression; protein purification; repository; tissue fixing; tissue processing; tumor; tumorigenesis

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 6 surgeries and 10 to 16 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) 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, 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. Over 1,500 tissue and over 700 blood specimens have been procured and processed within the past year. The entire UOB tissue repository contains in excess of 20,000 tissue samples, and DNA from over 3,500 blood samples and 500 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 four clinical trials that are open to accrual: Bevacizumab and Erlotinib for patients with metastatic papillary kidney cancer or HLRCC (44 patients to date), a Met kinase inhibitor INC280 for patients with papillary kidney cancer (7 patients to date), PANVAC-BCG vs. BCG for patients with high grade bladder cancer (11 patients to date), and Neoadjuvant rFowlpox-PSA for patients undergoing radical prostatectomy (14 patients to date). 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, 85 of which have been extensively characterized for cancer gene mutations. Lines have been generated from hereditary kidney cancer syndromes (BHD, SDHB, VHL, BAP1, HPRC, and HLRCC) 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 (2 this year) that become immortal. These lines are invaluable for studying both the molecular basis of tumorigenesis and prospective therapies. In a current study, 15 of our lines are being subjected to drug screening in a collaboration with NCATS. 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. The hereditary lines have been extensively characterized by DNA sequencing of selected genes, array CGH, realtime 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-gen sequencing techniques, 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, 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 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 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 have 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 25 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）在为泌尿外科肿瘤科（UOB）和我们的合作者提供支持和资源方面至关重要。TPSF处理在UOB内产生的每个生物标本，处理每个标本以保存生物分子，保持每次采购的准确库存，并协助选定标本的科学分析，以阐明与肾癌，前列腺癌和膀胱癌相关的生物途径的最终目标。TPSF处理几乎100%大华银行手术的组织，以及活检和其他手术的一部分。通常，每周有3至6次手术和10至16次活检，每年从400多名患者获得组织样本，包括肾癌、前列腺癌和膀胱癌、肾上腺肿瘤、子宫平滑肌瘤、淋巴结转移瘤以及其他与散发性和家族性泌尿系统癌症综合征相关的标本。组织总是与外科病理学合作，以确保正确的处理和准确的诊断。组织被快速冷冻，保存在福尔马林或戊二醛中，或处理用于生物分子（DNA， RNA，蛋白质）纯化和分析。此外，从遗传综合征患者的血液样本中定期制备DNA、血清和血浆。全血或RNA也可以从选定的病人身上获取和储存。每周可能要处理超过24份血液样本。最后，核心还获取和处理尿液、腹水或胸腔液体、囊肿液体和其他体液。冷冻样品储存在液氮中或-80摄氏度的冷冻室中。标本被分配一个去识别的实验室编号，并进入一个安全的数据库，Labmatrix。在过去的一年里，已经采购和处理了1500多个组织和700多个血液样本。整个大华银行组织库包含超过20,000个组织样本，以及来自3,500多个血液样本和500个肿瘤的DNA。大多数样本是在美国国立卫生研究院临床中心收集的，完整的患者病史被纳入Labmatrix。未来的目标是将所有临床和实验室结果纳入Labmatrix，为我们从实验室到床边的所有研究提供可访问的资源。TPSF的一项关键职能是支持该科的临床试验。在过去的一年里，我们处理了来自4个临床试验的患者样本，这些临床试验是开放的：Bevacizumab和Erlotinib用于转移性乳头状肾癌或HLRCC患者（迄今为止44例），Met激酶抑制剂INC280用于乳头状肾癌患者（迄今为止7例），PANVAC-BCG与BCG用于高级别膀胱癌患者（迄今为止11例），以及新辅助rFowlpox-PSA用于根治性前列腺切除术患者（迄今为止14例）。定期处理血液和/或尿液样本，以调查药物的药效学和药代动力学效应，以及其他癌症生物标志物。许多来自肾脏和膀胱手术的肿瘤样本是在无菌条件下获得的，用于建立新的细胞培养和小鼠异种移植。我们已经产生了300多个肾癌细胞系，其中85个已被广泛表征为癌症基因突变。从遗传性肾癌综合征（BHD、SDHB、VHL、BAP1、HPRC和HLRCC）中产生的细胞系提供了独特的试剂。去年，大约有12个肾肿瘤和一些前列腺和膀胱肿瘤被放置在细胞培养和/或SCID/BEIG或裸鼠中，其中一部分在短期内生长存活，少数（今年2个）成为不朽的。这些线对于研究肿瘤发生的分子基础和前瞻性治疗都是无价的。在目前的一项研究中，我们的15个品系正在与NCATS合作进行药物筛选。细胞系保存在标准的二氧化碳或低氧孵化器中，或储存在液氮中，小鼠被安置在适当的现场设施中。这些遗传系已通过选定基因的DNA测序、阵列CGH、实时PCR、Western blotting、耗氧量和细胞外酸化率以及代谢组学进行了广泛的表征。收集DNA样本以检测和表征种系疾病突变一直是大华银行基因发现过程的核心。每年有几十份血液样本通过DNA测序进行分析，包括下一代测序技术、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、缺失和扩增。通过免疫组织化学、定量PCR、表达微阵列、Northern和Western blotting、免疫沉淀和DNA测序，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都在UOB中得到了广泛的表征。戊二醛固定组织已用于电子显微镜，以表征亚细胞细胞器。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。大华银行参与向合作实验室提供其获得的许多肿瘤组织和血液样本的等量。该科有长期的合作，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，肾癌细胞标记分析，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，以及前列腺癌的分子流行病学研究。我们参与了多个癌症基因组图谱（TCGA）项目，贡献了透明细胞瘤、憎色瘤和乳头状肾肿瘤以及前列腺肿瘤。在过去的25年里，大华银行收集了大量的生物标本，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。