Tissue Processing-Sequencing Facility

组织处理测序设备

基本信息

批准号：
10926613
负责人：
Cathy D Vocke
金额：
$ 97.35万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10926613
关键词：
Adrenal Gland Neoplasms Adrenal Mass Affect Aliquot Ascites Basic Science Biological Biopsy Bladder Bladder Neoplasm Blood Blood specimen Body Fluids Cancer Gene Mutation Cancer Patient Cancer cell line Carbon Dioxide Caring Cell Culture Techniques Cell Line Cellular Metabolic Process Chest Chromosome inversion Clear Cell Clinical Clinical Research Clinical Treatment Clinical Trials Collaborations Collection Communication Cyst Fluid DNA DNA Sequence Rearrangement DNA analysis DNA sequencing Data Databases Detection Disease Dose Drug Screening Electron Microscopy Elements Ensure Equipment and supply inventories Erlotinib Family Fluorescent in Situ Hybridization Formalin Freezing Future Gene Expression Genes Genetic study Genitourinary system Genome Genomics Glutaral Goals Heart Hereditary Leiomyomatosis and Renal Cell Cancer House mice Human Immunohistochemistry Immunoprecipitation Immunotherapy Incubators Inherited Karyotype Karyotype determination procedure Kidney Kidney Neoplasms Laboratories Laboratory Finding Light Liquid substance Malignant Neoplasms Malignant neoplasm of prostate Malignant neoplasm of urinary bladder Metastatic Neoplasm to Lymph Nodes Methods Methylation Mitochondrial DNA Molecular Molecular Epidemiology Molecular Target Mus Mutation Mutation Analysis Nitrogen Northern Blotting Nuclear Operative Surgical Procedures Organelles Other Genetics Oxygen Oxygen Consumption PSA level Paper Papillary Pathway interactions Patient Selection Patients Pharmaceutical Preparations Pharmacodynamics Pheochromocytoma Plasma Polymorphism Analysis Privacy Procedures Process Prostate Prostate carcinoma Prostatic Neoplasms Proteins Proteomics Publishing RNA Reactive Oxygen Species Reagent Recording of previous events Renal Cell Carcinoma Renal carcinoma Research Research Personnel Resources Saliva Sampling Scientist Secure Security Serum Single Nucleotide Polymorphism Site Specimen Sterility Succinate Dehydrogenase Surgical Pathology Syndrome Techniques Testing The Cancer Genome Atlas Therapeutic Agents Time Tissue Banks Tissue Sample Tissues Tuberous Sclerosis Tumor Suppressor Genes 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 bench to bedside bevacizumab bladder Carcinoma bladder surgery cancer biomarkers cancer type cell immortalization central database clinical center comparative genomic hybridization epidemiology study exome exome sequencing extracellular gene discovery inhibitor kidney surgery metabolomics molecular targeted therapies new therapeutic target next generation pharmacokinetics and pharmacodynamics preservation prospective protein expression tissue fixing tissue processing transcriptome sequencing tumor tumorigenesis whole genome

项目摘要

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 8 surgeries and 10 to 20 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, metabolite) 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, saliva, 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. Approximately 2,000 tissue and 800 blood specimens have been procured and processed within the past year. The entire UOB tissue repository contains in excess of 25,000 tissue samples, and DNA from over 5,000 blood samples and 800 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 several clinical trials that are open to accrual: Bevacizumab, Erlotinib, and Atezolizumab for patients with metastatic papillary kidney cancer or HLRCC, a HIF2 inhibitor for patients with VHL, Effects of Low Dose Apalutamide on Prostate-Specific Antigen (PSA) Levels, and Care of the Prostate Cancer Patient and Prospective Procurement of Prostate Cancer Tissue. Several new clinical trials are also currently being established. 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, 92 of which have been extensively characterized for cancer gene mutations. Lines have been generated from hereditary kidney cancer syndromes (BHD, SDHB, SDHD, VHL, BAP1, HPRC, and HLRCC) and from rare kidney cancer types (chromophobe, TFE-3 RCC, and medullary RCC) 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, with a subset of these growing viably in the short-term and a small number (two this year) that become immortal. These lines are invaluable for studying both the molecular basis of tumorigenesis and prospective therapies. In a recent study, a subset of our lines were subjected to drug screening. 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. Some lines have been extensively characterized by nuclear and mitochondrial DNA sequencing and copy number of selected genes, array CGH, karyotype, methylation, RNAseq, real-time 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-generation whole exome, whole genome, and targeted gene sequencing, genomic copy number analysis, fluorescent in-situ hybridization (FISH), and/or other genetic studies. We continue to use these techniques to discover new mutations, deletions, genomic rearrangements, and amplifications. This past year we discovered a new tumor suppressor gene, PRDM10, responsible for the hereditary kidney cancer in one of our families, as well as a unique chromosomal inversion, responsible for loss of VHL function in one of our families with clinical VHL. 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 nuclear and mitochondrial 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 also 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 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 35 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) 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。 TPSF 处理大华银行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，以实现阐明与肾脏相关的生物学途径的最终目标、前列腺癌和膀胱癌。 TPSF 处理来自近 100% 的 UOB 手术的组织，以及一部分活检和其他手术。通常，每周进行 3 至 8 次手术和 10 至 20 次活检，每年从 400 多名患者采集组织样本，包括肾癌、前列腺癌和膀胱癌、肾上腺肿瘤、子宫肌瘤、淋巴结转移瘤和其他样本与散发性和家族性泌尿系统癌症综合征有关。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，或进行处理以进行生物分子（DNA、RNA、蛋白质、代谢物）纯化和分析。此外，定期从遗传综合征患者的血液样本中制备 DNA、血清和血浆。还可以从选定的患者处获取并储存全血或 RNA。每周可能会处理超过两打血液样本。最后，核心还获取并处理尿液、腹水或胸液、囊肿液、唾液和其他体液。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，我们采购并处理了大约 2,000 份组织样本和 800 份血液样本。整个 UOB 组织存储库包含超过 25,000 个组织样本以及来自 5,000 多个血液样本和 800 个肿瘤的 DNA。大部分样本是在 NIH 临床中心收集的，完整的患者病史均已纳入 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。 TPSF 的一项关键职能是支持分部内的临床试验。去年，我们处理了几项开放的临床试验中的患者样本：用于治疗转移性乳头状肾癌或 HLRCC 患者的贝伐珠单抗、厄洛替尼和 Atezolizumab，一种用于治疗 VHL 患者的 HIF2 抑制剂，低剂量阿帕鲁胺对前列腺的影响特异性抗原 (PSA) 水平、前列腺癌患者的护理以及前列腺癌组织的预期采购。目前还正在建立一些新的临床试验。定期处理血液和/或尿液样本，以研究药物以及其他癌症生物标志物的药效和药代动力学效应。许多来自肾脏和膀胱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。我们已经生成了 300 多种肾癌细胞系，其中 92 种已被广泛表征为癌症基因突变。细胞系是从遗传性肾癌综合征（BHD、SDHB、SDHD、VHL、BAP1、HPRC 和 HLRCC）和罕见肾癌类型（嫌色细胞、TFE-3 RCC 和髓样 RCC）中产生的，可提供独特的试剂。去年，大约有十几个肾肿瘤以及一些前列腺和膀胱肿瘤被置于细胞培养中，其中一部分在短期内可以存活，少数（今年有两个）可以永生。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。在最近的一项研究中，我们对部分品系进行了药物筛选。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。一些品系已通过核和线粒体 DNA 测序和所选基因的拷贝数、阵列 CGH、核型、甲基化、RNAseq、实时 PCR、蛋白质印迹、耗氧量和细胞外酸化率以及代谢组学进行了广泛的表征。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 测序分析几十个血液样本，包括下一代全外显子组、全基因组和靶向基因测序、基因组拷贝数分析、荧光原位杂交 (FISH) 和/或其他遗传学研究。我们继续使用这些技术来发现新的突变、缺失、基因组重排和扩增。去年，我们发现了一种新的肿瘤抑制基因 PRDM10，它导致了我们一个家族中的遗传性肾癌，以及一种独特的染色体倒位，导致了我们一个患有临床 VHL 家族的 VHL 功能丧失。 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已在 UOB 内通过免疫组织化学、定量 PCR、表达微阵列、Northern 和 Western 印迹、免疫沉淀以及核 DNA 和线粒体 DNA 进行了广泛的表征测序。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。大华银行还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研究以及分子生物学研究。前列腺癌的流行病学研究。我们参与了多个癌症基因组图谱 (TCGA) 项目，贡献了透明细胞、嫌色细胞和乳头状肾肿瘤以及前列腺肿瘤。大华银行在过去 35 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。

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Protein kinase D inhibitor CRT0066101 suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M.

蛋白激酶 D 抑制剂 CRT0066101 通过阻断 G2/M 细胞周期来抑制体外膀胱癌和异种移植物的生长。

DOI：
发表时间：
2018
期刊：
影响因子：
0
作者：
Li, Qingdi Quentin;Hsu, Iawen;Sanford, Thomas;Railkar, Reema;Balaji, Navin;Sourbier, Carole;Vocke, Cathy;Balaji, K C;Agarwal, Piyush K
通讯作者：
Agarwal, Piyush K

Comprehensive characterization of Alu-mediated breakpoints in germline VHL gene deletions and rearrangements in patients from 71 VHL families.

71 个 VHL 家族患者种系 VHL 基因缺失和重排中 Alu 介导的断点的综合表征。

DOI：
发表时间：
2021
期刊：
影响因子：
3.9
作者：
Vocke, Cathy D;Ricketts, Christopher J;Schmidt, Laura S;Ball, Mark W;Middelton, Lindsay A;Zbar, Berton;Linehan, W Marston
通讯作者：
Linehan, W Marston

Novel renal medullary carcinoma cell lines, UOK353 and UOK360, provide preclinical tools to identify new therapeutic treatments.

新型肾髓样癌细胞系 UOK353 和 UOK360 为识别新的治疗方法提供了临床前工具。

DOI：
发表时间：
2020
期刊：
影响因子：
0
作者：
Wei, Darmood;Yang, Youfeng;Ricketts, Christopher J;Vocke, Cathy D;Ball, Mark W;Sourbier, Carole;Wangsa, Darawalee;Wangsa, Danny;Guha, Rajarshi;Zhang, Xiaohu;Wilson, Kelli;Chen, Lu;Meltzer, Paul S;Ried, Thomas;Thomas, Craig J;Merino, Maria J
通讯作者：
Merino, Maria J

Identification of intragenic deletions and duplication in the FLCN gene in Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé 综合征中 FLCN 基因的基因内缺失和重复的鉴定。

DOI：
发表时间：
2011-06
期刊：
影响因子：
0
作者：
Benhammou, Jihane N;Vocke, Cathy D;Santani, Avni;Schmidt, Laura S;Baba, Masaya;Seyama, Kuniaki;Wu, Xiaolin;Korolevich, Susana;Nathanson, Katherine L;Stolle, Catherine A;Linehan, W Marston
通讯作者：
Linehan, W Marston

Molecular genetics and clinical features of Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé 综合征的分子遗传学和临床特征。