Tissue Processing-Sequencing Facility

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

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 15 biopsies per week, resulting in tissue samples procured from over 400 patients per year, including kidney, prostate, bladder and other genitourinary carcinomas, adrenal tumors, uterine leiomyomas, lymph node metastases, and other specimens relating to sporadic and familial urologic cancer syndromes. Tissue is 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 visiting clinic or having surgery. Whole blood or RNA may also be procured and stored from select patients. Over a 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 1,500 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 1,000 tumors. Most of the samples were collected at the NIH Clinical Center, and full patient histories are incorporated into Labmatrix. 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, and Palbociclib and Sasanlimab for advanced renal cell carcinomas, Enzalutamide Implants (Enolen) in Patients With Prostate Cancer. Several new clinical trials are also currently being established. 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 immortal kidney cancer cell lines, 92 of which have been extensively characterized for cancer gene mutations. Some lines have been extensively characterized by nuclear and mitochondrial DNA sequencing and copy number of selected genes, karyotype, methylation, RNAseq, real-time PCR, Western blotting, oxygen consumption and extracellular acidification rate, and metabolomics. Lines have been generated from hereditary kidney cancer syndromes (BHD, SDHB, VHL, BAP1, HPRC, and HLRCC) and from rare kidney cancer types (chromophobe, TFE-3 RCC, and medullary RCC) that provide unique reagents. We continue to attempt to grow renal tumors in culture. 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 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, and other genetic studies. We continue to use these techniques to discover new mutations, deletions, genomic rearrangements, and amplifications. This past year, we have identified the first multigenerational family who possess a mutation in the ELOC gene, which is responsible for this family's hereditary kidney cancer (a previous outside report described a single patient with a germline ELOC mutation and clinical features of disease, but with no family history). We have also recently identified two rare germline variants in FLCN: a genomic inversion and a deep intronic variant that causes splicing errors. Both frozen and formalin-fixed tissues from genitorurinary 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. 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.