David L. Cooper

Impact of pharmacogenomics on the clinical laboratory

Clinical pharmacogenomics promises to increase the safety and efficacy of drug prescription, decrease the incidence of adverse drug reactions, help improve public health, and presage in an era of personalized, predictive, and prophylactic medicine. Clinical pharmacogenomics stands to be broad based and include the following laboratory components: new and expanded pharmacogenetic tests, disease profiles, chemopredictive testing, and risk profiling. There is a growing body of evidence that variable drug responsiveness is caused by polymorphisms within multiple genes, protein products of which are involved in critical metabolic and/or physiologic pathways relevant for drug action. Different pharmacogenomic approaches will be used to discover a new generation of unique and highly predictive pharmacogenetic tests that the clinical laboratory will employ to help identify patient responder populations. Disease profiling and chemopredictive testing will routinely be applied to accurately screen for disease and help guide therapeutic course of action. A growing number of risk-profiling tests will assist in predicting a patients predisposition to disease. Clinical pharmacogenomics stands to become the basis for the new millenniums practice of medicine and have a profound impact on the clinical laboratory.

Prediction of Biologic Aggressiveness in Colorectal Cancer by p53/K-ras-2 Topographic Genotyping.

Background: Topographic genotyping is a system of solid tissue molecular analysis designed to correlate microscopic alterations with specific forms of gene damage. In this system, microscopic targets are selected on the basis of cellular and immunohistochemical features. Minute tissue samples corresponding to these targets are precisely removed and analyzed for the presence and specific type of oncogene/tumor suppressor gene damage by means of polymerase chain reaction (PCR) followed by DNA sequencing. In this study, topographic genotyping was used to investigate the prognostic value of p53 and K-ras-2 mutational damage in 204 patients with colorectal cancer from two tertiary care centers. Methods and Results: The intensity and distribution of p53 immunohistochemical staining were correlated with the presence and specific type of mutational change, which resulted in a better understanding of the highly variable nature of p53 immunostaining patterns. Molecular genotype was correlated with the depth and extent of colorectal cancer spread (Dukes B and C) and with survival for follow-up periods of up to 10 years. An algorithm for p53/K-ras-2 genotyping was formulated to include p53 immunohistochemistry and DNA sequencing both of p53 exons 5-8 and of K-ras-2 exons 1 and 2. By using this algorithm, survival was shown to be significantly better in those patients whose tumors manifested normal p53 and K-ras-2 genes (P >.01). Patients with p53-mutated tumors composed a poor prognostic group, characterized by a high rate of intra-abdominal recurrence in the form of peritoneal seeding. Patients with K-ras-2-mutated tumors also composed a poor prognostic group, marked by a tendency for distant hematogenous metastasis involving lung, bone, and brain. Conclusions: Topographic genotypings molecular diagnostic approach to colorectal cancer combines immunohistochemistry, PCR, and DNA sequencing. It is informative, cost-effective, timely, and yet fully integrated with standard histopathology. The use of this approach by pathologists as a model system for molecular diagnosis of colorectal cancer and other forms of solid tumor malignancy is recommended. As new prognostic molecular lesions are documented for tumor progression and metastasis, topographic genotyping will be well suited to facilitate their clinical application.

Gene therapeutic approaches to primary and metastatic brain tumors: II. ribozyme-mediated suppression of CD44 expression.

SummaryGlioblastomas are highly invasive intracerebral tumors that are known to express the CD44 cell adhesion molecule. Human glioma cell adhesion and invasionin vitro may in part be mediated by the interaction of CD44 with extracellular matrix proteins. To suppress the growth and invasive effects of CD44 expression on primary brain tumors we have designed two hammerhead ribozymes as potential gene therapeutic agents. Both ribozymes designed to target exon 2 of CD44 exhibitedin vitro cleavage ofin vitro transcribed CD44s and CD44R1 RNAs. The anti-CD44 effect of these ribozymes results from directed RNA cleavage, requiring both a target sequence and an appropriate catalytic center. Further, following transient transfection of one of these ribozymes into the SNB-19 glioma cell line, significantin vivo cleavage activity against cellular CD44 transcripts was demonstrated by flow cytometrical analysis. These preliminary results suggest that CD44-directed hammerhead ribozymes may be useful as gene therapeutic agents.

Differential expressions of CD44 variants in tumors affecting the central nervous system.

BACKGROUND The polymorphic cell adhesion molecule CD44 exists as a family of proteins generated by extensive alternative splicing of the CD44 pre-messenger RNA and marked posttranslational modification. The differential expression of CD44 isoforms in a variety of human cancers has been proposed to influence tumorigenesis and metastasis. In this study, CD44 gene expression was analyzed in primary and metastatic tumors and in cell lines derived from tumors that affect the central nervous system (CNS), including tumors metastatic to the spine. MATERIALS AND METHODS Fifty-four samples were subjected to semiquantitative reverse-transcriptase polymerase chain reaction with CD44-specific primers and hybridized individually with probes specific for the CD44 variant (CD44v) exons v3 to v10. RESULTS Compared with CD44v-positive breast cancer cell lines and CD44v-negative normal brain tissue, CD44v expression was weak in primary brain tumors and cell lines derived from normal brain and tumor tissue. However, high levels of isoforms encoding multiple-variant exons were shown in all metastatic brain tumors. In contrast, tumors metastatic to the spine were virtually negative for CD44v expression. Several rare CD44 isoforms composed of single-variant exons v3, v4, v6, or v9 were identified in primary brain tumors and may reflect their invasive potential or culturability in vitro. CONCLUSION These data suggest differential expression of CD44v may substantially influence the end-organ site of metastasis for tumor cells destined for the CNS.

CD44 expression in benign and neoplastic human prostates

Background: CD44, a major cell surface receptor for hyaluronic acid, is a family of ubiquitous cell surface glycoproteins. Altered levels of CD44 expression, seen in many epithelial neoplasms, have prognostic implications. Expression of standard and variant isoforms of CD44 was assessed in normal and neoplastic human prostate tissue and culture cells to evaluate as a marker for malignant transformation. Methods and Results: Expression of CD44s, CD44R, v5, v6, v7/8 and v10 was assessed in prostate tissue (benign and malignant) and cell lines (DU-145, PC-3, LNCaP, p69) and primary cultures of normal prostates and adenocarcinoma cells obtained from prostatectomies using reverse transcriptor polymerase chain reaction, Western blotting, and immunofluorescence. No CD44 expression was seen in LNCaP cells. p69, DU-145, and PC-3 cells expressed CD44s and CD44R. p69, cells demonstrated a 1000-bp-long form of CD44 mRNA, unique to this normal cell line. Both normal and neoplastic prostatic tissue demonstrated CD44s on Western blotting. Conclusions: In agreement with previous studies, prostatic adenocarcinoma cells, except LNCaP, expressed CD44s. Different patterns of CD44 expression were seen in benign and neoplastic prostate. Benign prostate exhibited higher v5 protein levels, whereas neoplastic prostates demonstrated higher CD44s expression. CD44s expression was identified in all neoplastic prostates as compared with only 50% of the benign prostates. No significant difference in expression of the other variants assessed (v6, v7, v7/8, and v10) was observed in the benign and neoplastic prostates.

Gene therapeutic approach to primary and metastatic brain tumors: I. CD44 variant pre-RNA alternative splicing as a CEPT control element

SummaryOur laboratory and others have shown alternative splicing of up to ten exons at a discrete extracellular site to be primarily responsible for the generation of CD44 variant (CD44v) isoforms. Based on clear differences in the expression of these CD44v isoforms between normal and malignant tissues, we believe that elucidation of the mechanisms underlying the regulation of CD44 alternative splicing may provide a new gene therapeutic targeting approach based on CD44 pre-mRNA processingin vivo. This strategy incorporates utilization of CD44 alternative splicing control elements into a chimeric enzyme/prodrug therapy (CEPT), a novel modification of the virus-directed enzyme/prodrug therapy (VDEPT) approach for the treatment of brain metastases from tumors of systemic origin. As initial steps towards the development of a gene therapeutic approach based on targeting tumor cell expression of specific CD44v alternatively spliced isoforms, we have: (1) developed a novelin vivo assay system that allows the rapid analyses of potentially therapeutic CD44 alternative splicing minigene constructs; and (2) cloned theE. coli cytosine deaminase (CD) gene and fused its enzymatically active domain to alternatively spliced CD44 exons (CD44/CD). Deamination of cytosine by this CD44/CD chimeric fusion protein is demonstrated inE. coli cell lysates to be equal to that of wild type cytosine deaminase.

Expression of the cell adhesion molecule CD44 in human lung tumors and cell lines

Background: The purpose of this study was to examine the expression of the cell adhesion molecule CD44 in normal lung, primary and metastatic lung tumors, and cell lines derived from primary lung carcinomas. Methods and Results: A total of 68 lung specimens including normal tissue and primary and metastatic tumors, as well as 28 cell lines cultured from primary lung tumors with high recurrence, were examined for CD44 expression by semiquantitative reverse transcription polymerase chain reaction. Variant exon expression was confirmed by Southern blotting and hybridization of particular samples. In tumor tissues, loss of CD44 variant expression correlated with increasing tumor stage; a smaller percentage of more aggressive and poorly differentiated tumors expressed CD44v. Tumors metastatic to the lung were negative for CD44 variant expression. In primary lung cell lines, as in tumor tissue, tumors of higher histologic grade were characterized by loss of CD44 variant expression. Conclusion: CD44 isoform expression in normal lung and tumor tissues and cell lines revealed an overall decrease in CD44 alternative splicing in lung neoplasms of increased malignancy.

Molecular mechanisms regulating the hyaluronan binding activity of the adhesion protein CD44

SummaryIn the present study, we describe the isolation and characterization of a cDNA clone designated B6F1.3, that appears to ‘activate’ the hyaluronan-binding capacity of CD44 upon transfection into the murine fibroblastoid cell line MOP8. Sequence analysis indicates that the putative regulatory molecule encoded by this clone is identical to the murine interleukin-2 receptor γ chain (mIL-2Rγ), a recently described type 1 transmembrane protein that constitutes an integral component of the cell surface receptors that bind a number of cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and perhaps also IL-13. Mutations in this molecule have been shown to be responsible for X-linked severe combined immunodeficiency (XSCID) in humans. With the exception of bone marrow, the mIL-2Rγ chain was found to be expressed at high levels on all hemopoietic cell lines and tissue types examined. Non-hemopoietic tissues are generally negative. FACS analysis and Western blot analysis indicated respectively that B6F1.3 does not mediate its effects by upregulating the expression of CD44 or by altering the alternative splicing of the molecule. Removal of the cytoplasmic tail of the mIL-2Rγ chain, including a Src homology region 2 (SH2) subdomain, abolished its ability to enhance CD44-mediated binding to hyaluronan suggesting the involvement of signal transduction events triggered via the cytoplasmic domain in the ‘activation’ process. Determining whether activating molecules such as B6F1.3 are co-expressed within tumor cells may help improve the potential value of CD44 as a diagnostic marker of metastatic disease.

How do I know what I think till I see what I say

Participants will:  Be introduced to the theories of language for thinking and of language for communication  Consider the implications of talking for thinking with bilingual children and the importance of developing complex language  Explore a range of engaging strategies for developing language and thinking in early years and primary classrooms  Plan easy changes to everyday classroom life that will develop the sophistication of children’s language and the depths of their thinking

Rapid Detection of Hepatitis C Virus in Plasma and Liver Biopsies by Capillary Electrophoresis

Liver transplantation is one of the most complicated and costly medical procedures performed in hospitals and medical centers today. Candidates for liver transplantation include patients whose livers have been damaged by hepatitis C virus (HCV) infection. Since donated livers, however, sometimes carry HCV infection (Pereira et al. 1991), it would be beneficial to be able to test for HCV prior to transplantation. Because of the nature of organ transplantation, in which surgery must be performed within a few hours following organ donation, this test must be very fast but without a loss of sensitivity.