Tissue ProcessingSequencing Core

组织处理测序核心

• 批准号: 8763752
• 负责人: Cathy D Vocke
• 金额: $ 56.54万
• 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/8763752
• 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; 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; DNA Sequencing Facility; Data; Databases; Deletion Mutation; Detection; Diagnosis; Disease; Drug Kinetics; Electron Microscopy; Ensure; Enzyme-Linked Immunosorbent Assay; Equipment and supply inventories; Erlotinib; Family; Fluorescent in Situ Hybridization; Formalin; Freezing; Future; Gene Duplication; Gene Expression; Genes; Genetic; Genitourinary system; Genome; Glutaral; Goals; Heart; Hereditary Leiomyomatosis and Renal Cell Cancer; House mice; Human; Immunohistochemistry; Immunoprecipitation; Immunotherapy; Incubators; Inherited; 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; Privacy; Procedures; Process; Prostate; Prostate carcinoma; Prostatic Neoplasms; Proteins; 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 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 sequencing; extracellular; falls; gene discovery; interest; kinase inhibitor; lymph nodes; meetings; metabolomics; novel; prospective; protein expression; protein purification; repository; response; tissue fixing; tissue processing; tumor; tumorigenesis; urologic

The Tissue Processing/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 3 to 5 surgeries and 2 to 6 biopsies per week, resulting in tissue samples procured from over 300 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 is regularly prepared from blood samples taken from patients with inherited syndromes. Serum or plasma, whole blood, or RNA may also be procured and stored from select patients. 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 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 2,000 tissue and blood specimens have been procured within the last year. The entire UOB tissue repository contains in excess of 20,000 tissue samples and DNA from 2,500 blood samples from over 1,200 patients. Most of the samples were collected at the NIH Clinical Center, and full patient histories are incorporated into Labmatrix. Older samples are currently in the FreezerWorks biorepository. 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 (36 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. A new clinical trial using a Met kinase inhibitor for papillary kidney cancer patients is expected to begin this year. 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 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. 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. 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 are now participating in the Cancer Genome Atlas (TCGA) project by contributing clear cell, papillary and chromophobe kidney cancers, and are currently collecting prostate specimens to be sent to the TCGA this fall. 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/测序核心（TPSC）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。TPSC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。TPSC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5次手术和2至6次活检，每年从300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和家族性泌尿系统癌症综合征有关的标本。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。血清或血浆、全血或RNA也可从选定的患者中获取和储存。每周可能会处理两到三十份血液样本。最后，核心还可以从医疗程序中获取和处理尿液、腹水或胸腔液体、囊肿液体或其他体液。冷冻样品储存在液氮或零下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。在过去一年中，已经采购了2000多个组织和血液样本。整个大华银行组织库包含超过20,000个组织样本和来自1,200多名患者的2,500份血液样本的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。较旧的样本目前在FreezerWorks生物库中。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。TPSC的一项关键职能是支持该科的临床试验。我们目前参与了两项活跃的临床试验：贝伐单抗和厄洛替尼用于转移性乳头状肾癌或HLRCC患者（迄今累计36例），阿斯利康ZD6474用于VHL综合征患者（迄今累计34例）。血液样本由TPSC定期处理，目的是研究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。一项使用Met激酶抑制剂治疗乳头状肾癌患者的新临床试验预计将于今年开始。许多来自肾癌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43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和4个HLRCC系）中产生的细胞系提供了独特的试剂。去年，十多个肾脏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存活，少数（今年2例）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。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。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，对这些遗传系进行了广泛的表征。该科还从数百名患有VHL、BHD、HPRC、HLRCC或未知家族性肾癌综合征的大华银行患者中获得了不朽的B细胞系样本。这些细胞系为已知遗传畸变的鉴定提供了无限的生殖细胞DNA资源，也有可能在尚未确诊的患者中发现可能涉及遗传性肾癌的新基因。此外，这些细胞系提供了在RNA水平上研究感兴趣基因的机会。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、比较基因组杂交（CGH）分析、荧光原位杂交（FISH）和/或其他遗传研究进行分析。此外，在使用阵列CGH进行的为期三年的研究中，我们对67个VHL家族、8个BHD家族、7个HLRCC家族和2个SDHB家族的种系缺失进行了精细定位，并在BHD家族中发现了一个新的部分基因重复。目的有两个：一是确定以前未确诊的临床受影响患者的特征，二是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联系起来。我们继续使用这些技术来发现新的突变、缺失和扩增。经TPSC处理的冷冻和福尔马林固定的肾癌、前列腺癌和膀胱癌组织在UOB中通过免疫组织化学、定量PCR、表达微阵列、Northern和Western blotting、免疫沉淀和DNA测序进行了广泛的表征。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。我们现在正在参与癌症基因组图谱（TCGA）项目，贡献透明细胞癌、乳头状癌和憎色肾癌，目前正在收集前列腺标本，将于今年秋天送到TCGA。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。