Tissue ProcessingSequencing Core

组织采购和测序核心（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的职责是处理在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，并协助对选定标本进行科学分析，以阐明与肾癌，前列腺癌和膀胱癌相关的生物学途径。TPSC处理几乎100%的大华银行手术组织，以及活检和其他医疗程序的一部分。通常，每周有2到5次手术，每年从100多名患者中获取手术样本，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相关的样本。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纯化和分析。此外，还定期从遗传综合征患者身上采集血液样本，以便为分析准备DNA。还从选定的患者中收集血清或血浆，用于分析蛋白质和其他生物标志物。在某些情况下，全血被冷冻以备将来的研究，在极少数情况下，也可以从血液中制备RNA。每周可能会处理两到三十份血液样本。最后，核心还处理额外的体液，如尿液、腹水或胸腔液体以及囊肿液体，这些体液可能来自外科手术或其他医疗程序。冷冻样品储存在液氮或零下80摄氏度的冷冻室中。所有的标本都被分配了一个去识别的实验室编号，并进入一个安全的数据库，Labmatrix。在过去的一年里，总共获得了一千多个组织和血液样本。整个大华银行组织库包含超过16,000个组织样本和来自1000多名患者的1200个血液样本的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。目前在FreezerWorks生物储存库中的旧样品也被迅速输入Labmatrix，目的是在不久的将来完成Labmatrix的合并。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正在进行的临床试验。我们目前参与了两项临床试验：贝伐单抗和厄洛替尼用于1型乳头状肾癌或HLRCC患者，阿斯利康ZD6474用于VHL或散发性透明细胞肾癌患者。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，以及研究其他癌症生物标志物。此外，在gsk GSK1363089研究中，我们与其他实验室合作获取并协调对患者肿瘤标本的处理和评估，以确定肿瘤组织学、基因突变和染色体畸变，从而与药物反应进行后续关联。这些研究是GSK1363089药物方案的组成部分。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。其中包括从家族性肾癌综合征（BHD、SDHB、VHL和2个HLRCC系）产生的细胞系，这些细胞系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，大约有24个肾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生长存活，少数成功建立为不朽系（去年有4个）。这些线对于研究肿瘤发展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。BHD， HLRCC， SDHB和VHL系通过选定基因的DNA测序，阵列CGH，实时PCR， Western blotting，氧消耗和细胞外酸化率以及代谢组学进行了广泛的表征。此外，选择6个散发性透明细胞系进行全外显子组测序和代谢组学表征。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。这些细胞系为种系DNA提供了无限的资源，以表征已知基因的遗传畸变，以及发现可能涉及尚未确诊的遗传性肾癌患者的新基因的潜力。此外，这些细胞系提供了在RNA表达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66个VHL家族、6个BHD家族、3个HLRCC家族和1个SDHB家族的种系缺失进行了精细定位，并在一个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已被UOB广泛用于免疫组织化学、定量PCR、表达微阵列、Northern和Western blotting、免疫沉淀和DNA测序等技术的表征。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，肾癌干细胞标记物的分析，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。我们现在通过提供透明细胞癌和乳头状肾癌参与癌症基因组图谱项目，并通过提供前列腺癌血液和肿瘤组织参与CCR多实验室XMRV患病率研究。在过去的20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。

The Tissue Procurement and Sequencing Core (TPSC) is essential in providing support and resources for the Urologic Oncology Branch (UOB) and for our collaborators. It is the TPSC'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 TPSC 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 over 100 patients procured 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, blood samples are regularly taken from patients with inherited syndromes, in order to prepare DNA for analysis. 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 rare occasions, RNA is also prepared from blood. Two to three dozen blood samples may be processed per week. Finally, the core also processes additional body fluids, such as urine, ascites or thoracic fluids, and cyst fluids, that may be taken from surgical or other medical procedures. Frozen samples are stored in liquid nitrogen or a -80 degree centigrade freezer. All specimens are assigned a de-identified lab number and entered into a secure database, Labmatrix. In all, over a thousand 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 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 in progress within the Branch. We are currently involved in two clinical trials: Bevacizumab and Erlotinib for patients with type 1 papillary kidney cancer or HLRCC, and AstraZeneca ZD6474 for patients with VHL or sporadic clear cell kidney cancer. Blood samples are processed regularly by the TPSC, for the purpose of investigating pharmacodynamic and phamacokinetic effects of the drugs, as well as the study of other cancer biomarkers. In addition, for the Glaxo GSK1363089 study, we obtained and coordinated processing and evaluation of tumor specimens from patients in collaborative effort with other laboratories to ascertain tumor histology, gene mutations and chromosomal aberrations, for subsequent correlation with response to the drug. These studies are integral to the 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 lines generated from familial kidney cancer syndromes (BHD, SDHB, VHL, 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 (four 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 BHD, HLRCC, SDHB, and VHL 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 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. 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 66 VHL families, 6 BHD families, 3 HLRCC families and one SDHB family, 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 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. We are now participating in both the Cancer Genome Atlas project by contributing clear cell and papillary kidney cancers, and the CCR Multi-Laboratory XMRV Prevalence Study by providing prostate cancer blood and tumor tissue. 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.