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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）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。TPSF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。TPSF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8次手术和10至20次活检，每年从400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，代谢物）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、血清和血浆。全血或RNA也可以从选定的病人身上获取和储存。每周可能要处理超过24份血液样本。最后，核心还获取和处理尿液、腹水或胸腔液体、囊肿液、唾液和其他体液。冷冻样品储存在液氮中或-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，已经采购和处理了大约2000个组织和800个血液标本。整个大华银行组织库包含超过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。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。TPSF的一项关键职能是支持该科的临床试验。在过去的一年里，我们处理了几个临床试验中的患者样本，这些临床试验是开放的：用于转移性乳头状肾癌或HLRCC患者的贝伐单抗、厄洛替尼和阿特唑单抗，用于VHL患者的HIF2抑制剂，低剂量阿帕鲁胺对前列腺特异性抗原（PSA）水平的影响，前列腺癌患者的护理和前列腺癌组织的前瞻性采购。一些新的临床试验目前也正在进行中。定期处理血液和/或尿液样本，以调查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。许多来自肾脏和膀胱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92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、SDHD、VHL、BAP1、HPRC和HLRCC）和罕见的肾癌类型（憎色、TFE-3 RCC和髓质RCC）中产生的细胞系提供了独特的试剂。去年，大约有12个肾肿瘤和一些前列腺和膀胱肿瘤被放置在细胞培养中，其中一部分在短期内可以存活，而一小部分（今年有两个）可以永生。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。在最近的一项研究中，我们的一部分生产线接受了药物筛选。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。一些品系已经通过核和线粒体DNA测序和选定基因的拷贝数、阵列CGH、核型、甲基化、RNAseq、实时PCR、Western blotting、耗氧量和细胞外酸化率以及代谢组学进行了广泛的鉴定。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测序进行分析，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。在过去的一年里，我们发现了一个新的肿瘤抑制基因，PRDM10，在我们的一个家族中负责遗传性肾癌，以及一个独特的染色体反转，在我们的一个临床VHL家族中负责VHL功能的丧失。通过免疫组织化学、定量PCR、表达微阵列、Northern和Western blotting、免疫沉淀、核和线粒体DNA测序，TPSF处理的肾脏、前列腺癌和膀胱癌的冷冻和福尔马林固定组织都在UOB中得到了广泛的表征。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。大华银行还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份。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。我们参与了多个癌症基因组图谱（TCGA）项目，贡献了透明细胞瘤、憎色瘤和乳头状肾肿瘤以及前列腺肿瘤。大华银行在过去的35年里积累了大量的生物标本，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。

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）

UOK 262 cell line, fumarate hydratase deficient (FH-/FH-) hereditary leiomyomatosis renal cell carcinoma: in vitro and in vivo model of an aberrant energy metabolic pathway in human cancer.

DOI：
10.1016/j.cancergencyto.2009.08.018
发表时间：
2010-01-01
期刊：
Cancer genetics and cytogenetics
影响因子：
0
作者：
Yang Y;Valera VA;Padilla-Nash HM;Sourbier C;Vocke CD;Vira MA;Abu-Asab MS;Bratslavsky G;Tsokos M;Merino MJ;Pinto PA;Srinivasan R;Ried T;Neckers L;Linehan WM
通讯作者：
Linehan WM

Clinical and Molecular Characterization of Microphthalmia-associated Transcription Factor (MITF)-related Renal Cell Carcinoma.

DOI：
10.1016/j.urology.2020.11.025
发表时间：
2021-03
期刊：
Urology
影响因子：
2.1
作者：
Lang M;Vocke CD;Ricketts CJ;Metwalli AR;Ball MW;Schmidt LS;Linehan WM
通讯作者：
Linehan WM

BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports.

BHD突变，Birt-Hogg-Dubé综合征的临床和分子遗传研究：一个新的50个家庭系列和已发表报告的评论。

DOI：
10.1136/jmg.2007.054304
发表时间：
2008-06
期刊：
JOURNAL OF MEDICAL GENETICS
影响因子：
4
作者：
Toro, J. R.;Wei, M-H;Glenn, G. M.;Weinreich, M.;Toure, O.;Vocke, C.;Turner, M.;Choyke, P.;Merino, M. J.;Pinto, P. A.;Steinberg, S. M.;Schmidt, L. S.;Linehan, W. M.
通讯作者：
Linehan, W. M.

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

DOI：
10.1038/nrurol.2015.206
发表时间：
2015-10
期刊：
Nature reviews. Urology
影响因子：
0
作者：
通讯作者：

Hereditary kidney cancer: unique opportunity for disease-based therapy.

DOI：
10.1002/cncr.24230
发表时间：
2009-05-15
期刊：
Cancer
影响因子：
6.2
作者：
Linehan WM;Pinto PA;Bratslavsky G;Pfaffenroth E;Merino M;Vocke CD;Toro JR;Bottaro D;Neckers L;Schmidt LS;Srinivasan R
通讯作者：
Srinivasan R