Clinical Ligand Assay Society (CLAS)
31st International Meeting
Molecular Basis of Health and Disease
Tools, Technologies & Applications
General Chair: James L. Wittliff, Ph.D., M.D. hc
Clinical Ligand Assay Society (CLAS)
31st International Meeting
Molecular Basis of Health and Disease
Tools, Technologies & Applications
General Chair: James L. Wittliff, Ph.D., M.D. hc
AWARDS & RECOGNITION:
D. Alan Kerr II:
Grafton D. Chase Education Award – in honor of Grafton D. Chase, Ph.D., a founding member, Past-President and good friend of CLAS, the Executive Board established the “Grafton D. Chase Memorial Endowment Fund” in 1994. The purpose of this Fund is to provide an annual award recognizing a student or post-doctoral fellow for excellence in research and academic achievement related to ligand assays. This year's Grafton Chase Award is presented to D. Alan Kerr II , who received a Bachelor's Degree in Chemistry from the Johns Hopkins University prior to being appointed a Research Scholar in the Hormone Receptor Laboratory at the University of Louisville . After completing training in molecular endocrinology, he and his co-workers received the Best Abstract Award at CLAS 2003 for research focusing on the application of Pressure Cycling Technology in clinical chemistry. Alan, who is also associated with the J. Graham Brown Cancer Center, was awarded a Graduate Fellowship from the Integrated Programs in Biomedical Sciences to pursue the Ph.D. degree in Biochemistry & Molecular Biology. The subject of his Grafton Chase Award presentation represents the topic of his doctoral dissertation studies with his mentor, Dr. Wittliff.
Integrity & Yields of RNA Isolated from LCM-Procured Cells to Discover Molecular Signatures and Drug Targets
D. Alan Kerr II, Sarah A. Andres, Irina A. Smolenkova and James L. Wittliff, Department of Biochemistry & Molecular Biology, Brown Cancer Center and Institute for Molecular Diversity & Drug Design, University of Louisville, Louisville, KY 40202, USA.
The highest cancer mortality rates for women in the United States are due to lung and breast carcinomas, while the highest for both women and men is the result of lung cancer. Archival tissues (formalin-fixed and paraffin-embedded, FFPE) represent an untouched resource for studies requiring long-term clinical follow-up. Goals of this project were 1) to establish laser capture microdissection (LCM)-based procedures for isolating pure lung/breast carcinoma cells from fresh-frozen or FFPE specimens and 2) to develop protocols for extracting, isolating and processing mRNA for gene expression profiling. Formalin-fixation catalyzes cross-linking of nucleic acids while cellular heterogeneity of most tumor biopsies compounds the contamination of cancer cell RNA in the downstream genomic analyses. LCM (PixCell IIe, Arcturus Bioscience) was used to procure pure carcinoma cells for mRNA extraction, purification and amplification in preparation for microarray analyses. Total RNA was purified from each tissue specimen and quantified by RT-qPCR with a universal RNA (Stratagene) standard curve using the ß-actin house-keeping gene for relative quantification. Two rounds of amplification were used for each cDNA, with a biotin-labeling in vitro transcription step in the second round. Spectrophotometric and BioAnalyzer TM (Agilent Technologies) analyses evaluated aRNA yield, purity and transcript length for subsequent microarray analyses. RNA extraction and amplification of FFPE lung samples using the Paradise TM kit (Arcturus Bioscience) yielded 15-30 µg biotin-labeled aRNA with 260/280 ratios of 1.6-2.1 from whole tissue sections (~0.25 cm 2 ) as well as from 5,000-6,000 LCM-procured carcinoma cells. Generally, transcipt lengths of amplified mRNA were greater than 300 bases according to BioAnalyzer TM analysis. Recent advances in microarray technology have produced cDNA chips for FFPE-extracted transcripts (e.g. X3P chip, Affymetrix) that only require ~200 bases on the 3' end of the mRNA. Therefore, transcript lengths of extracted mRNA specimens were measured using multiple ß–actin primer sets (100, 200, 300 & 400 bases from the 3' end) to generate 3'/5' ratios. A representative specimen extracted by the Paradise™ kit produced values of 9.5 ± 1.4, 3.2 ± 0.3 and 1.7 ± 0.1 for amplicon ratios of 100b/400b, 100b/300b and 100b/200b from whole tissue sections compared to those from LCM-procured carcinoma cells which gave ratios of 47.3 ± 18.8, 10.4 ± 2.3 and 3.4 ± 0.5. Results indicate that although LCM-procured carcinoma cell extracts contain RNA with relatively shorter RNA transcript lengths than those of whole tissue extracts, the mRNA appears of sufficient length for competent analyses in X3P microarrays. Gene expression profiles generated are being used to identify clinically relevant molecular signatures and to discover new targets for drug design. Supported in part by grants from the Kentucky Lung Cancer Research Program and Phi Beta Psi Research Foundation, Inc. DAK & SAA are recipients of fellowships from the Integrated Programs in Biomedical Sciences, University of Louisville .
Sarah A. Andres:
Establishing Concise Molecular Signatures from Human Breast Carcinoma Specimens for Clinical Applications
Sarah A. Andres , D.Alan Kerr II, Irina A. Smolenkova & James L. Wittliff
Department of Biochemistry & Molecular Biology, James Graham Brown Cancer Center , University of Louisville Louisville , KY 40202
While certain tumor markers (e.g., estrogen & progestin receptors, HER-2/neu oncoprotein) in breast tissue biopsies have been used for evaluating a breast cancer patient's prognosis and therapy selection with limited success, the wealth of information derived from the Human Genome Project provides greater opportunities to develop more accurate tests for diagnostics, prognostics, therapy selection and monitoring. The goal of this study is t o develop a subset of genes (5-15) based on UniGene Cluster analysis and other published information using whole tissue studies as well as to exploit a larger gene set derived from breast carcinoma cells collected by laser capture microdissection (LCM), as described by Wittliff et al. (Jensen Symposium Abstracts, University of Cincinnati, 2003).
GenBank Accession numbers (NCBI) from studies of interest were entered into the UniGene database (NCBI), which separates the GenBank oligonucleotide sequences into a non-redundant set of gene-oriented clusters. There are 54,560 sequence entries from investigators worldwide for Homo sapiens . Each UniGene Cluster contains oligonucleotide sequences that represent a unique gene, which has a specific identifier. Once the appropriate UniGene identifier is known, the gene sets were sorted by UniGene identifier and analyzed. For example, epidermal growth factor receptor (EGFR), a prospective tumor marker, has a GenBank Accession Number of M34309. Entry of this Accession Number into the UniGene database identifies UniGene Cluster Hs.306251 Homo sapiens V-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) (ERBB3). Fifteen mRNA sequences have been entered including that of M34309.1. In addition, 351 expressed sequence tag (EST) sequences have been entered in the database. Using preliminary predictions of molecular signatures from breast cancer reported in numerous published studies, Unigene clusters in common are then identified by electronic sorting. Therefore, one may identify a family of sequences associated with a single gene. Using this approach and genes identified in large molecular signatures reported by other investigators and our laboratory, a subset of 25-30 candidate genes have been selected for subsequent analyses by qPCR. By correlating the expression of those candidate genes in LCM-procured carcinoma cells with clinical outcome stored in our extensive database (e.g., time to recurrence), a clinically relevant molecular signature is being derived. Supported in part by a grant from Phi Beta Psi Research Foundation. SAA & DAK II are recipients of fellowships from the Integrated Programs in Biomedical Sciences, University of Louisville .
