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) 对于为泌尿肿瘤科 (UOB) 和我们的合作者提供支持和资源至关重要。 TPSC 处理 UOB 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，以实现阐明与肾癌、前列腺癌和膀胱癌相关的生物学途径的最终目标。TPSC 处理来自近 100% UOB 手术的组织， 以及活检和其他程序的子集。通常，每周进行三到五次手术，每年从 150 多名患者那里采集手术样本，包括肾癌、前列腺癌和膀胱癌、肾上腺肿瘤、子宫肌瘤、淋巴结转移以及与散发性和家族性泌尿系统癌症综合征相关的其他样本。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。组织被快速冷冻，保存在福尔马林或戊二醛中，或进行生物分子纯化和分析处理。此外，定期从遗传综合征患者的血液样本中制备 DNA。还从选定的患者身上收集血清或血浆用于蛋白质和其他生物标志物的分析。在某些情况下，全血会被冷冻以供将来的研究使用，有时还会从血液中制备 RNA。每周可处理两到三打血液样本。 最后，核心还可以获取和处理来自外科手术或其他医疗程序的尿液、腹水或胸液、囊液或其他体液。 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，我们采购了 1,400 多个组织和血液样本。整个大华银行组织储存库包含超过 18,000 个组织样本以及来自 1,000 多名患者的 2,000 份血液样本的 DNA。大部分样本是在 NIH 临床中心采集的，完整的患者病史均已纳入 Labmatrix。目前 FreezerWorks 生物存储库中的较旧样本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。TPSC 的一个关键功能是支持分支机构内的临床试验。 我们目前正在进行两项临床试验：针对转移性乳头状肾癌或 HLRCC 患者的贝伐单抗和厄洛替尼（迄今为止已累计 28 名患者），以及针对 VHL 综合征患者的阿斯利康 ZD6474（迄今为止已累计 34 名患者）。 TPSC 定期处理血液样本，以研究药物以及其他癌症生物标志物的药效学和药代动力学效应。此外，对于葛兰素GSK1363089研究（现已关闭应计），我们与多个机构合作获得了患者的肿瘤标本并进行了协调评估，以确定肿瘤组织学、基因突变和染色体畸变，以便随后与药物反应相关。这些研究是 GSK1363089 方案的组成部分。肾癌手术中的许多肿瘤样本都是在无菌条件下获取的，用于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 多种肾癌细胞系，其中 43 种已被广泛表征为癌症基因突变。遗传性肾癌综合征的细胞系（BHD、SDHB、VHL 和 4 个 HLRCC 细胞系）产生，可提供独特的试剂。去年，十多个肾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可存活，少数（今年有 6 个）可以永生。此外，去年还发现了两种新的膀胱癌。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。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。 通过选定基因的 DNA 测序、阵列 CGH、实时 PCR、蛋白质印迹、耗氧量和细胞外酸化率以及代谢组学，对遗传系进行了广泛的表征。 此外，正在对 6 个选定的零星透明细胞系进行全外显子组测序和代谢组学表征。该分支机构还从来自 VHL、BHD、HPRC、HLRCC 或未知家族性肾癌综合征家族的数百名 UOB 患者中生成了永生 B 细胞系样本。这些细胞系为种系 DNA 提供了无限的资源，用于表征已知的遗传畸变，并有可能在尚未确诊的患者中发现可能与遗传性肾癌有关的新基因。此外，这些细胞系还提供了在 RNA 水平上研究感兴趣基因的机会。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、比较基因组杂交 (CGH) 分析、荧光原位杂交 (FISH) 和/或其他遗传学研究来分析几十个血液样本。此外，在使用阵列 CGH 的一项为期三年的研究中，我们对 67 个 VHL 家族、8 个 BHD 家族、7 个 HLRCC 家族和 2 个 SDHB 家族中的种系缺失以及 BHD 家族中一个新的部分基因重复进行了精细定位。 目标有两个：描述先前未诊断的临床受影响患者的特征，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、缺失和扩增。 TPSC 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 中通过免疫组织化学、定量 PCR、表达微阵列、Northern 和 Western 印迹、免疫沉淀和 DNA 测序进行了广泛的表征。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。该分院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研究以及前列腺癌的分子流行病学研究。我们通过提供前列腺癌血液和肿瘤组织参与了 CCR 多实验室 XMRV 患病率研究，现在通过提供透明细胞癌、乳头状癌和嫌色肾癌参与癌症基因组图谱项目。大华银行在过去 20 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。