Tissue ProcessingSequencing Core

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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.