Clinical Tissue Procurement and Sequencing Core

临床组织采购和测序核心

• 批准号: 7970014
• 负责人: Cathy D Vocke
• 金额: $ 35.37万
• 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/7970014
• 关键词: Adrenal Gland Neoplasms; Adrenal Mass; Aliquot; B-Lymphocytes; Basic Science; Biological; Biological Markers; Biopsy; Birt-Hogg-Dube Syndrome; Bladder; Blood; Blood specimen; Cancer Gene Mutation; Cancer Patient; Cancer cell line; Carbon Dioxide; Cell Culture Techniques; Cell Line; Chromosome abnormality; Chromosomes; Clear Cell; Clinical; Clinical Research; Clinical Treatment; Clinical Trials; Collaborations; Collection; DNA; DNA Sequence; DNA Sequence Analysis; DNA Sequencing Facility; DNA analysis; Data; Databases; Deletion Mutation; Detection; Development; Diagnosis; Disease; Drug Kinetics; Electron Microscopy; Ensure; Equipment and supply inventories; Family; Fluorescent in Situ Hybridization; Formalin; Freezing; Future; Gene Mutation; Genes; Genetic; Genetic Transcription; Genitourinary system; Glutaral; Goals; Heart; Hereditary Renal Cell Carcinoma; Histology; House mice; Human; Immunohistochemistry; Immunoprecipitation; Immunotherapy; Incubators; Indium; Inherited; Kidney; Kidney Neoplasms; Laboratories; Laboratory Finding; Leiomyomatosis; Liquid substance; Location; Malignant Neoplasms; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Maps; Medical; Molecular; Molecular Epidemiology; Molecular Target; Mouse Cell Line; Mus; Mutation; Neoplasm Metastasis; Nitrogen; Nucleotides; Nude Mice; Oncogenes; Operative Surgical Procedures; Organelles; Other Genetics; Oxygen; Papillary; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacodynamics; Pheochromocytoma; Plasma; Privacy; Procedures; Process; Prostate; Protein Analysis; Proteins; Protocols documentation; RNA; Reagent; Recording of previous events; Renal Cell Carcinoma; Renal carcinoma; Research; Research Personnel; Resources; Sampling; Scientist; Secure; Security; Serum; Severity of illness; Site; Specimen; Sterility; Succinate Dehydrogenase; Surgical Pathology; Syndrome; System; TSC1/2 gene; Techniques; Testing; Therapeutic Agents; Tissue Procurements; Tissue Sample; Tissues; Tuberous Sclerosis; Tumor Suppressor Genes; Tumor Tissue; Tumor-Associated Process; United States National Institutes of Health; Urine; Urologic Cancer; Urologic Oncology; Uterine Fibroids; Western Blotting; Xenograft Model; Xenograft procedure; ZD-6474; base; bench to bedside; biobank; bladder Carcinoma; cancer stem cell; cancer surgery; comparative genomic hybridization; deletion analysis; epidemiology study; gene discovery; interest; lymph nodes; pancreatic neoplasm; prospective; repository; response; tissue fixing; tissue processing; tumor; tumorigenesis

The Clinical Tissue Procurement and Sequencing Core (CTPSC) is essential in providing support and resources for the Urologic Oncology Branch (UOB) and for our collaborators. It is the CTPSC's responsibility to handle every biospecimen that is generated within the UOB, to process each specimen in order to preserve biomolecules, to keep an accurate inventory of each procurement, and to assist in the scientific analysis of select specimens, for the ultimate goal of elucidating biological pathways relating to kidney, prostate, and bladder cancers. The CTPSC processes tissue from nearly 100% of UOB surgeries, as well as a subset of biopsies and other medical procedures. Typically, there are between two and five surgeries per week, resulting in surgical samples from about 100 patients procured per year, including kidney, prostate, and bladder carcinomas, adrenal tumors, uterine leiomyomas, lymph node metastases, pancreatic tumors, 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, blood samples are regularly taken from patients with inherited kidney cancer syndromes, in order to prepare DNA for analysis. Serum or plasma is also collected from select patients for analysis of proteins and other biomarkers. Ten to twenty blood samples may be processed per week. Frozen samples are stored in liquid nitrogen or a -80 degree centigrade freezer. All specimens are assigned a laboratory number and entered into a secure database, Labmatrix. In all, over 700 tissue and blood specimens have been procured within the last year. The entire UOB tissue repository contains in excess of 16,000 tissue samples and DNA from 1,200 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 are also being entered promptly into Labmatrix, with the aim of completing the incorporation into Labmatrix within the next fiscal year. 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 CTPSC is to support clinical trials in progress within the Branch. We are currently involved in two clinical trials: Glaxo GSK1363089 for patients with type 1 papillary kidney cancer, and AstraZeneca ZD6474 for patients with VHL or sporadic clear cell kidney cancer. Blood samples are processed weekly by the CTPSC, for the purpose of investigating pharmacodynamic (PD) and phamacokinetic (PK) effects of the drugs, as well as the study of other cancer biomarkers. In addition, in a collaborative effort with other laboratories to ascertain tumor histology, gene mutations, and chromosomal aberrations, we obtain and process tumor specimens from these patients. These studies are integral to the Glaxo GSK1363089 drug 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. These include four lines generated from familial kidney cancer syndromes (BHD, SDHB, and 2 HLRCC lines) that provide unique reagents for studying the activity of these respective tumor suppressor genes. In the last year, approximately two 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 successfully established as immortal lines (six within the past year). These lines are invaluable for studying both the molecular basis of tumor development 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 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 in order to characterize genetic aberrations of known genes of interest, as well as the potential to discover new genes that may involved in inherited kidney cancer in patients that have not yet been diagnosed. In addition, these lines provide the opportunity to study genes of interest at the RNA expression 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. Approximately 30 blood samples per year are analyzed by DNA sequencing, comparative genome hybridization (CGH) analysis, fluorescent in-situ hybridization (FISH), and other genetic studies. Furthermore, by using array CGH, we have performed fine mapping of 3 distinct germline deletions from HLRCC families and 61 distinct germline deletions from VHL families, with the goal of correlating the sizes and locations of these deletions with the severity of disease and/or response to clinical treatments. By combining array CGH with DNA sequencing on an ABI Prism 310 Gene Analyzer, we have mapped 47 VHL family deletions to the exact nucleotide (33 of these within the past year), for ultimate accuracy of characterization of their germline aberrations. 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 CTPSC have been used extensively by the UOB for characterization using techniques such as 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 stem cell markers, protein and RNA studies of adrenal masses (pheochromocytomas), and molecular epidemiology studies of prostate cancers. Serum and plasma from select kidney cancer and prostate cancer patients are being collected for molecular studies by other laboratories as well as ours. 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.

临床组织采购和测序核心 (CTPSC) 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。 CTPSC 的责任是处理 UOB 内生成的每个生物样本，处理每个样本以保存生物分子，保持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。 CTPSC 处理来自近 100% 的 UOB 手术的组织，以及活检和其他医疗程序的子集。通常，每周进行两到五次手术，每年从约 100 名患者身上获取手术样本，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、胰腺肿瘤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，我们还定期从患有遗传性肾癌综合征的患者身上采集血液样本，以制备 DNA 进行分析。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。每周可处理十到二十个血液样本。 冷冻样品储存在液氮或-80℃冰箱中。所有样本均分配有实验室编号并输入安全数据库 Labmatrix。去年总共采购了 700 多个组织和血液样本。整个 UOB 组织储存库包含超过 16,000 个组织样本以及来自 1,000 多名患者的 1,200 个血液样本的 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也将立即输入 Labmatrix，目标是在下一财年完成并入 Labmatrix。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。 CTPSC 的一项关键职能是支持该分会内正在进行的临床试验。我们目前正在进行两项临床试验：针对1型乳头状肾癌患者的葛兰素GSK1363089，以及针对VHL或散发性透明细胞肾癌患者的阿斯利康ZD6474。 CTPSC 每周处理一次血液样本，目的是研究药物的药效 (PD) 和药代动力学 (PK) 作用，以及其他癌症生物标志物的研究。此外，在与其他实验室合作确定肿瘤组织学、基因突变和染色体畸变的过程中，我们获取并处理了这些患者的肿瘤标本。这些研究是葛兰素史克 GSK1363089 药物方案的组成部分。许多来自肾癌手术的肿瘤样本都是在无菌条件下获得的，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 多种肾癌细胞系，其中 43 种已被广泛表征为癌症基因突变。其中包括从家族性肾癌综合征产生的 4 个品系（BHD、SDHB 和 2 个 HLRCC 品系），它们为研究这些各自的肿瘤抑制基因的活性提供了独特的试剂。去年，大约两打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存活，少数成功建立永生系（去年有 6 个）。这些细胞系对于研究肿瘤发展的分子基础和前瞻性治疗非常有价值。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。该分支机构还从数百名来自 VHL、BHD、HPRC、HLRCC 或未知家族性肾癌综合征家族的 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，以便表征已知感兴趣基因的遗传畸变，并有可能在尚未诊断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 表达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) 和其他遗传学研究对大约 30 个血液样本进行分析。此外，通过使用阵列 CGH，我们对 HLRCC 家族中的 3 个不同种系缺失和 VHL 家族中的 61 个不同种系缺失进行了精细定位，目的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相关联。通过在 ABI Prism 310 基因分析仪上将阵列 CGH 与 DNA 测序相结合，我们已将 47 个 VHL 家族缺失映射到精确的核苷酸（其中 33 个是在过去一年中发生的），以最终准确地表征其种系畸变。我们继续使用这些技术来发现新的突变、缺失和扩增。 UOB 广泛使用 CTPSC 处理过的肾癌、前列腺癌和膀胱癌的冷冻组织和福尔马林固定组织，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 测序等技术进行表征。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。该分部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、肾癌干细胞标记物分析、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。 其他实验室以及我们的实验室正在收集精选的肾癌和前列腺癌患者的血清和血浆进行分子研究。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。