Kristina A. Cole

Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis

Microdissection of routinely stained or unstained frozen sections has been used successfully to obtain purified cell populations for the analysis of cell-specific gene expression patterns in primary tissues with a complex mixture of cell types. However, the precision and usefulness of microdissection is frequently limited by the difficulty to identify different cell types and structures by morphology alone. We therefore developed a rapid immunostaining procedure for frozen sections followed by laser capture microdissection (LCM) and RNA extraction, which allows targeted mRNA analysis of immunophenotypically defined cell populations. After fixation, frozen sections are immunostained under RNAse-free conditions using a rapid three-step streptavidin-biotin technique, dehydrated and immediately subjected to LCM. RNA is extracted from captured tissue, DNAse I treated, and reverse transcribed. Acetone-, methanol-, or ethanol/acetone-fixed sections give excellent immunostaining after 12 to 25 minutes total processing time. Specificity, precision, and speed of microdissection is markedly increased due to improved identification of desired (or undesired) cell types. The mRNA recovered from immunostained tissue is of high quality. Single-step PCR is able to amplify fragments of more than 600 bp from both housekeeping genes such as beta-actin as well as cell-specific messages such as CD4 or CD19, using cDNA derived from less than 500 immunostained, microdissected cells. Immuno-LCM allows specific mRNA analysis of cell populations isolated according to their immunophenotype or expression of function-related antigens and significantly expands our ability to investigate gene expression in heterogeneous tissues.

Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies

Using a general strategy for evaluating clinical tissue specimens, we found that 70% ethanol fixation and paraffin embedding is a useful method for molecular profiling studies. Human prostate and kidney were used as test tissues. The protein content of the samples was analyzed by one-dimensional gel electrophoresis, immunoblot, two-dimensional gel electrophoresis, and layered expression scanning. In each case, the fixed and embedded tissues produced results similar to that obtained from snap-frozen specimens, although the protein quantity was somewhat decreased. Recovery of mRNA was reduced in both quantity and quality in the ethanol-fixed samples, but was superior to that obtained from formalin-fixed samples and sufficient to perform reverse transcription polymerase chain reactions. Recovery of DNA from ethanol-fixed specimens was superior to formalin-fixed samples as determined by one-dimensional gel electrophoresis and polymerase chain reaction. In conclusion, specimens fixed in 70% ethanol and embedded in paraffin produce good histology and permit recovery of DNA, mRNA, and proteins sufficient for several downstream molecular analyses. Complete protocols and additional discussion of relevant issues are available on an accompanying website (http://cgap-mf.nih.gov/).

Rapid protein display profiling of cancer progression directly from human tissue using a protein biochip

The complicated, changing pattern of protein expression should contain important information about the pathologic process taking place in the cells of actual tissue. Utilization of this information for the selection of druggable targets could be possible if a means existed to rapidly analyze and display changes in protein expression in defined microscopic cellular subpopulations. As a demonstration of feasibility, we show the generation of sensitive, rapid, and reproducible molecular weight protein profiles of patient‐matched normal, premalignant, malignant, and metastatic microdissected cell populations from stained human esophageal, prostate, breast, ovary, colon, and hepatic tissue sections through the application of an affinity‐based biochip. Reproducible, discriminatory protein biomarker profiles can be obtained from as few as 25 cells in less than 5 min from dissection to the generation of the protein fingerprint. Furthermore, these protein pattern profiles reveal reproducible changes in expression as cells undergo malignant transformation, and are discriminatory for different tumor types. Consistent protein changes were identified in the microdissected cells from patient‐matched tumor and normal epithelium from eight out of eight different malignant esophageal tissue sets and three out of three malignant prostate tissue sets. A means to rapidly generate a display of expressed proteins from microscopic cellular populations sampled from tissue could be an important enabling technology for pharmacoproteomics, molecular pathology, drug intervention strategies, therapeutic assessment of drug entities, disease diagnosis, toxicity, and gene therapy monitoring. Drug Dev. Res. 49:34–42, 2000. Published 2000 Wiley‐Liss, Inc.

Molecular profiling of clinical tissue specimens: feasibility and applications.

The relationship between gene expression profiles and cellular behavior in humans is largely unknown. Expression patterns of individual cell types have yet to be precisely measured, and, at present, we know or can predict the function of a relatively small percentage of genes. However, biomedical research is in the midst of an informational and technological revolution with the potential to increase dramatically our understanding of how expression modulates cellular phenotype and response to the environment. The entire sequence of the human genome will be known by the year 2003 or earlier. 1,2 In concert, the pace of efforts to complete identification and full-length cDNA sequencing of all genes has accelerated, and these goals will be attained within the next few years. 3-7 Accompanying the expanding base of genetic information are several new technologies capable of global gene expression measurements. 8-16 Taken together, the expanding genetic database and developing expression technologies are leading to an exciting new paradigm in biomedical research known as molecular profiling.

Calcium-mediated signal transduction: biology, biochemistry, and therapy.

The process of proliferation, invasion and metastasis is a complex one which involves both the autonomy of the malignant cells and their interaction with the cellular and extracellular environments. The way in which the tumor cells respond to cellular and extracellular stimuli is regulated through transduction of those signals and translation into cellular activity. Transmembrane signal transduction involves three major categories of events: ion channel activation, transmission through guanine nucleotide binding protein intermediates with production of second messengers, and phosphorylation events. A frequent common denominator of these different pathways is a cellular calcium homeostasis. Calcium may be both a result of and a regulator of many of these signal transduction pathways and has been shown to have a role in the regulation of proliferation, invasion, and metastatic potential. The understanding and application of the basic tenets of these pathways to tumor cell proliferation, invasion, and metastases opens a new target for therapeutic intervention. We have identified a novel agent, CAI, which through inhibition of stimulated calcium influx inhibits proliferation and migrationin vitro, and growth and dissemination in human cancer xenograftsin vivo. CAI offers a new approach to cancer therapy, signal transduction therapy.

Molecular Profiling of Clinical Tissue Specimens : Feasibility and Applications

The relationship between gene expression profiles and cellular behavior in humans is largely unknown. Expression patterns of individual cell types have yet to be precisely measured, and, at present, we know or can predict the function of a relatively small percentage of genes. However, biomedical research is in the midst of an informational and technological revolution with the potential to increase dramatically our understanding of how expression modulates cellular phenotype and response to the environment. The entire sequence of the human genome will be known by the year 2003 or earlier. 1, 2 In concert, the pace of efforts to complete identification and full-length cDNA sequencing of all genes has accelerated, and these goals will be attained within the next few years. 3, 4, 5, 6, 7 Accompanying the expanding base of genetic information are several new technologies capable of global gene expression measurements. 8, 9, 10, 11, 12, 13, 14, 15, 16 Taken together, the expanding genetic database and developing expression technologies are leading to an exciting new paradigm in biomedical research known as molecular profiling.

Histopathology and molecular biology of ovarian epithelial tumors

Carcinogenesis in the ovary presents special features related to that organ. First, the preinvasive or even invasive lesions are difficult to detect, which explains why most cases are diagnosed at an advanced stage. Second, the group of tumors of low malignant potential (borderline tumors) are still a controversial category of ovarian lesions. Finally, familial ovarian tumors represent an interesting hereditary model of carcinogenesis at the molecular level. Flow cytometry and immunohistochemistry for proliferative markers or oncogenes provide important prognostic information in patients with ovarian tumors. Molecular data, such as loss of heterozygosity at specific genetic loci, also have been correlated with prognosis. Clonality studies in patients with multiple ovarian/pelvian lesions analyzing chromosome X inactivation patterns and genetic deletions or mutations have contributed to the understanding of the origin of these lesions. New technologies to study gene expression patterns, such as cDNA library construction and DNA microarray technologies, are being applied to study histologic phases of tumor progression, such as normal, preinvasive, and tumor tissues. It is hoped that these studies will contribute important information not only for a better understanding of the process of carcinogenesis, but also for assessing the biology and behavior of individual tumors, determining patient prognosis, and eventually influencing therapy.

Analysis of mRNA Quality in Freshly Prepared and Archival Papanicolaou Samples

OBJECTIVE To study the feasibility of utilizing mRNA recovered from cytologic Papanicolaou (Pap) specimens as a resource for gene expression studies of normal and diseased cells. STUDY DESIGN To assess the effects of fixation on mRNA recovery and analysis, fresh Pap samples were processed by three separate methods: (1) routine cytologic fixation (2) 70% ethanol fixation, and (3) air drying without fixation. One-week-old, 1-month-old, 1-year-old and 10-year-old samples were studied to determine the quality of mRNA in archival samples. mRNA quality was analyzed by RT-PCR for the HPRT gene, and by complete transcript amplification. Both heterogeneous (whole slide scrapes) and microdissected cell populations were studied. RESULTS Reverse transcriptase-polymerase chain reaction (RT-PCR) for the hypoxanthine guanine phosphoribosil transferase gene was positive in all fresh and archival samples and was not affected by fixative, processing methodology or microdissection. Complete transcript amplification followed by gel electrophoresis showed cDNA smears in all fresh samples with a maximum intensity between 1 and 2 kilobases (kb). Amplification of mRNA was not affected by fixation. Smaller cDNA smears were seen in archival specimens with a maximum intensity between 0.5 and 1.5 kb in both one-week-old and one-month-old samples. Smears of approximately 500 base pairs were observed in the 1-year-old and 10-year-old samples. Successful mRNA amplification was possible from microdissected cell populations. CONCLUSION Messenger RNA recovery and analysis is possible from archival cytologic specimens, suggesting that they can serve as a useful template for RT-PCR analysis of individual genes as well as newly developing high-throughput gene expression methodologies, such as microarrays. Cytologic samples may be particularly useful for study of archival samples as well as diseases from which tissue samples amenable to mRNA-based studies are not available.

Molecular analysis of microdissected tissue: Laser capture microdissection

Publisher Summary At the microscopic level, tissues are composed of complicated interacting and interdependent cell populations, which are regulated by the local extracellular matrix. Thus, analyzing critical gene expression patterns in development, normal function, and disease progression depends on the extraction of specific cells from their complex tissue milieu. Laser capture microdissection (LCM) is developed to provide a rapid, reliable method to procure pure populations of selected cells from specific microscopic regions of tissue sections for molecular analysis. This chapter discusses the molecular analysis of microdissected tissue. Further, it describes protocols for preparing the tissue before microdissection and conducting the PCR amplification after microdissection. Finally, it outlines the applications. DNA obtained from cells procured by LCM from clinical specimens is analyzed by a variety of methods, including loss of heterozygosity, clonal analysis, and direct sequencing. Similarly, LCM is used to study expression differences in various tissues using RNA-based techniques, such as cDNA library construction, microarray hybridization, and differential gene expression.

Genetic Alterations in Prostatic Intraepithelial Neoplasia (PIN)

Prostatic intraepithelial neoplasia (PIN) is the histologic lesion most strongly associated with prostate cancer, and has been postulated to be a premalignant lesion. However, much of the natural history of PIN remains unknown. A more fundamental understanding of the relationship between PIN and invasive tumors at the molecular level is critically needed, and represents an important future challenge for investigators. This chapter reviews the clinical, pathologic, and genetic studies addressing the relationship between PIN and cancer. The final section presents newly developing techniques and research approaches in molecular pathology and describes how these methods can be used to study PIN.