Tissue ProcessingSequencing Core

组织处理测序核心

• 批准号: 8554101
• 负责人: Cathy D Vocke
• 金额: $ 57.61万
• 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/8554101
• 关键词: Adrenal Gland Neoplasms; Adrenal Mass; Affect; Aliquot; Ascites; B-Lymphocytes; Basic Science; Biological; Biological Assay; Biological Markers; Biological Preservation; Biopsy; Birt-Hogg-Dube Syndrome; Blood; Blood specimen; Body Fluids; CCR; Cancer Gene Mutation; Cancer Patient; Cancer cell line; Carbon Dioxide; Cell Culture Techniques; Cell Line; Cells; Chest; Chromosome abnormality; Clear Cell; Clinical; Clinical Research; Clinical Treatment; Clinical Trials; Collaborations; Collection; Communication; Cyst Fluid; DNA; DNA Sequence; DNA Sequence Analysis; DNA Sequencing Facility; Data; Databases; Deletion Mutation; Detection; Diagnosis; Disease; Drug Kinetics; Electron Microscopy; Ensure; Enzyme-Linked Immunosorbent Assay; Equipment and supply inventories; Erlotinib; Evaluation; Family; Fluorescent in Situ Hybridization; Formalin; Freezing; Future; Gene Duplication; Gene Expression; Gene Mutation; Genes; Genetic; Genitourinary system; Genome; Glutaral; Goals; Heart; Hereditary Leiomyomatosis and Renal Cell Cancer; Histology; House mice; Human; Immunohistochemistry; Immunoprecipitation; Immunotherapy; Incubators; Inherited; Institutes; Karyotype determination procedure; Kidney Neoplasms; Laboratories; Laboratory Finding; Liquid substance; Location; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Maps; Medical; Metabolism; Methods; Molecular; Molecular Epidemiology; Molecular Target; Mouse Cell Line; Mus; Mutation; Mutation Analysis; 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; Prevalence Study; Privacy; Procedures; Process; Prostate carcinoma; Protein Analysis; Proteins; Protocols documentation; Publishing; RNA; Reagent; Recording of previous events; Renal Cell Carcinoma; Renal carcinoma; Research; Research Personnel; Resources; Sampling; Scientist; Secure; Security; Serum; Severity of illness; Single Nucleotide Polymorphism; Site; Specimen; Sterility; Succinate Dehydrogenase; Surgical Pathology; Syndrome; Techniques; Testing; The Cancer Genome Atlas; Therapeutic Agents; Time; Tissue Procurements; Tissue Sample; Tissues; Tumor Tissue; United States National Institutes of Health; Urine; Urologic Cancer; Urologic Oncology; Uterine Fibroids; Von Hippel-Lindau Syndrome; Western Blotting; Whole Blood; Xenograft Model; Xenograft procedure; ZD-6474; base; bench to bedside; bevacizumab; biobank; bladder Carcinoma; cancer surgery; comparative genomic hybridization; epidemiology study; exome; extracellular; gene discovery; interest; lymph nodes; metabolomics; novel; prospective; protein expression; repository; response; tissue fixing; tissue processing; tumor; tumorigenesis; urologic

The Tissue Procurement and Sequencing Core (TPSC) is essential in providing support and resources for the Urologic Oncology Branch (UOB) and for our collaborators. The TPSC 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 TPSC processes tissue from nearly 100% of UOB surgeries, as well as a subset of biopsies and other procedures. Typically, there are three to five surgeries per week, resulting in surgical samples procured from over 150 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 purification and analysis. In addition, DNA is regularly prepared from blood samples taken from patients with inherited syndromes. Serum or plasma is also collected from select patients for analysis of proteins and other biomarkers. In select cases, whole blood is frozen for future studies, and on occasion, RNA is also prepared from blood. Two to three dozen blood samples may be processed per week. Finally, the core may also procure and process urine, ascites or thoracic fluids, cyst fluids, or other body fluids from surgical or other medical procedures. Frozen samples are stored in liquid nitrogen or a -80 degree centigrade freezer. Specimens are assigned a de-identified lab number and entered into a secure database, Labmatrix. Over 1,400 tissue and blood specimens have been procured within the last year. The entire UOB tissue repository contains in excess of 18,000 tissue samples and DNA from 2,000 blood samples from over 1,000 patients. Most of the samples were collected at the NIH Clinical Center, and full patient histories are incorporated into Labmatrix. Older samples currently in the FreezerWorks biorepository will be integrated into Labmatrix in the near future. 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 TPSC is to support clinical trials within the Branch. We are currently involved in two active clinical trials: Bevacizumab and Erlotinib for patients with metastatic papillary kidney cancer or HLRCC (28 patients accrued to date), and AstraZeneca ZD6474 for patients with VHL syndrome (34 patients accrued to date). Blood samples are processed regularly by the TPSC, for the purpose of investigating pharmacodynamic and phamacokinetic effects of the drugs, as well as other cancer biomarkers. In addition, for the Glaxo GSK1363089 study (now closed to accrual), we obtained and coordinated evaluation of tumor specimens from patients in collaboration with multiple institutes to ascertain tumor histology, gene mutations and chromosomal aberrations, for subsequent correlation with response to the drug. These studies are integral to the GSK1363089 protocol.Many of the tumor samples from kidney cancer surgeries are procured under sterile conditions to establish new cell cultures and mouse xenografts. We have generated over 300 kidney cancer cell lines, 43 of which have been extensively characterized for cancer gene mutations. Lines have been generated from hereditary kidney cancer syndromes (BHD, SDHB, VHL, and 4 HLRCC lines) that provide unique reagents. In the last year, over a dozen kidney 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 (6 this year) that become immortal. In addition, two new bladder cancers were established in the past year. These lines are invaluable for studying both the molecular basis of tumorigenesis and prospective therapies. 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 are being extensively characterized by DNA sequencing of selected genes, array CGH, realtime PCR, Western blotting, oxygen consumption and extracellular acidification rate, and metabolomics. In addition, 6 selected sporadic clear cell lines are being subjected to whole exome sequencing and metabolomic characterization.The Branch has also generated immortal B cell line samples from several hundred UOB patients from families with VHL, BHD, HPRC, HLRCC, or unknown familial kidney cancer syndromes. These lines provide an unlimited resource for germline DNA for characterization of known genetic aberrations, as well as the potential to discover new genes that may involved in inherited kidney cancer in as-yet undiagnosed patients. In addition, these lines provide the opportunity to study genes of interest at the RNA level.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, comparative genome hybridization (CGH) analysis, fluorescent in-situ hybridization (FISH), and/or other genetic studies. Furthermore, during a three year study using array CGH, we have performed fine mapping of germline deletions in 67 VHL families, 8 BHD families, 7 HLRCC families and 2 SDHB families, as well as one novel partial gene duplication in a BHD family. The goal has been twofold: to characterize previously undiagnosed clinically affected patients, and to correlate the sizes and locations of these deletions with the severity of disease and/or response to clinical treatments. 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 TPSC have been characterized extensively in 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 participated in the CCR Multi-Laboratory XMRV Prevalence Study by providing prostate cancer blood and tumor tissue, and are now participating in the Cancer Genome Atlas project by contributing clear cell, papillary and chromophobe kidney cancers. The sizeable biospecimen collection amassed by the UOB over the last 20 years provides an invaluable resource for both basic and clinical research regarding kidney, prostate and bladder cancers.

组织采购和测序核心（TPSC）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。 TPSC处理UOB中生成的每种生物测量，处理每个样品以保留生物分子，保持每种采购的准确库存，并有助于对精选标本进行科学分析，以阐明与肾脏，前列腺和bladcecer.tpsce的最终目标，以阐明生物学途径。以及活检和其他程序的子集。通常，每周有三到五次手术，导致每年有150多名患者采购的手术样本，包括肾脏，前列腺和膀胱癌，肾上腺肿瘤，子宫平滑肌瘤，淋巴结淋巴结转移酶以及其他与孢子虫和家族性和家族性的尿毒学癌症综合征有关。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，或加工以进行生物分子纯化和分析。另外，从遗传综合征患者采集的血液样本中定期制备DNA。还从特定患者中收集血清或血浆，以分析蛋白质和其他生物标志物。在某些情况下，全血被冷冻以未来的研究，有时还会从血液中制备RNA。每周可以处理两到三打血液样本。 最后，核心还可以从外科手术或其他医疗程序中采购和处理尿液，腹水或胸腔液，囊肿流体或其他体液。 冷冻样品存储在液氮或-80摄氏度的冰箱中。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。去年，已经购买了超过1,400张组织和血液标本。整个UOB组织存储库中包含超过18,000个组织样品和来自1,000多名患者的2,000个血液样本的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。目前在冰箱生物座设备中的较旧样品将在不久的将来集成到Labmatrix中。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可访问的资源。TPSC的关键功能是支持分支机构内的临床试验。 目前，我们参与了两项活跃的临床试验：用于转移性乳头状肾癌或HLRCC患者的贝伐单抗和厄洛替尼（迄今为止累积的28例患者），VHL综合征患者的阿斯利康ZD6474（迄今为止，34例患者）。血样定期通过TPSC处理，目的是研究药物的药效和phamacokinetic效应以及其他癌症生物标志物。此外，对于葛兰素GSK1363089的研究（现已关闭为应计），我们与多家机构合作地对患者的肿瘤标本进行了协调评估，以确定肿瘤组织学，基因突变和染色体类似，以与对药物的反应相关。这些研究是GSK1363089方案不可或缺的。肾癌手术的肿瘤样品在无菌条件下采购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43种针对癌症基因突变进行了广泛的特征。线条是由遗传性肾脏癌综合征（BHD，SDHB，VHL和4 HLRCC系）产生的，这些疾病提供了独特的试剂。去年，已将十几个肾脏肿瘤放在细胞培养和/或SCID/BEIG或裸鼠中，其中这些肿瘤的子集在短期内生长出来，而少量（今年6）变得不朽。此外，在过去的一年中建立了两种新的膀胱癌。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。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。 遗传线的广泛特征是选定基因，阵列CGH，实时PCR，蛋白质印迹，消耗氧和细胞外酸化速率和代谢组学的DNA测序。 此外，该分支从患有VHL，BHD，HPRC，HLRCC或不知名的家族肾癌综合症的几百名UOB患者中产生了几百名UOB患者的不朽B细胞系样品。这些线为生殖线DNA提供了无限的资源，用于表征已知的遗传畸变，并有可能发现可能涉及在尚未诊断的患者中可能涉及遗传性肾癌的新基因。此外，这些线条提供了研究RNA水平上感兴趣的基因的机会。用于检测和表征种系疾病突变的DNA样品是UOB中基因发现过程的核心。每年通过DNA测序，比较基因组杂交（CGH）分析，荧光原位杂交（FISH）和/或其他遗传研究来分析几十个血样。此外，在使用Array CGH的三年研究中，我们在67个VHL家族，8个BHD家族，7个HLRCC家庭和2个SDHB系列中进行了细菌缺失，并在BHD家族中进行了一个新颖的部分基因重复。 目标是双重的：要表征先前未诊断的临床影响患者，并将这些缺失的大小和位置与疾病的严重程度和/或对临床治疗的反应相关联。我们继续使用这些技术来发现新的突变，缺失和放大。由TPSC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，免疫沉淀和DNA序列在UOB中广泛表征。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参与了其许多采购的肿瘤组织和血液样本的等分试样，以合作实验室。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。我们通过提供前列腺癌的血液和肿瘤组织参加了CCR多实验室XMRV患病率研究，现在通过贡献清晰细胞，乳头状和成以下的肾脏肾脏来参与癌症基因组计划。在过去的20年中，UOB积累的大量生物循环收集为有关肾脏，前列腺和膀胱癌的基本和临床研究提供了宝贵的资源。