Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-06-24
2002-01-22
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06340563
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the detection of diseases, including cancers or heredity based defects in patients. More specifically, the present invention relates to detecting diseases in patients from a biological specimen based on histopathologic characteristics of the specimens as observed on the microscopic level.
BACKGROUND OF THE INVENTION
Recent advances in genetic research, especially those focused upon cancer or inherited disorders, has led to the identification of new genes having specific patterns of DNA sequence alterations directly related to pathologic disease states (Weinberg R A, Oncogenes, Antioncogenes, and the Molecular Basis of Multistep Carcinogenesis. Cancer Res., 49:3713-3721, 1989). Central to this research is the Human Genome Project, a monumental world-wide scientific effort to fully map and sequence the human genome (Watson J D, The Human Genome Project: Past, Present, and Future. Science, 248:44-49, 1990). Together the results will provide a detailed blueprint of the normal Human genome together with a understanding of DNA damage upon which the diagnosis and treatment of many conditions may be formulated. Using this information, genetic based therapies have already been instituted, consisting of the introduction into selected cells of normal or modified human genes designed to integrate and function as part of the host genome (Anderson W F, Human Gene Therapy. Science, 256:808-813, 1992). These initiatives provide a strong stimulus for tissue based methods which can characterize in detail DNA sequence alterations in selected cellular components of normal and disease affected human tissues.
To realize the potential of the expanding database of DNA sequence information, it has become necessary to have available methods which can detect and characterize DNA sequence alterations in tissue specimens such as those routinely obtained during the medical management of patients. Presently, in clinical practice, genetic analysis usually requires a fresh and relatively large tissue sample secured independently of other specimens for diagnostic purposes. Realistically, many clinical specimens, for which genetic sequence information would be vitally needed, are of small size obtained through biopsy procedures. Moreover there exists a priority of tissue management in that proper histopathologic diagnosis is paramount demanding that adequate tissue first be secured and placed into appropriate fixative solutions to preserve morphologic integrity for accurate histopathologic evaluation. Standard practices of genetic analysis are generally ineffective on specimens exposed to fixative agents (Ben Ezra J, Johnson D A, Rossi J, Cook N, Wu A, Effect of Fixation on the Amplification of Nucleic acids from Paraffin-Embedded Material by the Polymerase Chain Reaction. J. Histochem. Cytochem., 39:351-354, 1991). This has led some to the collection of fresh tissue in Freezer Banks, a process that is inconvenient, costly and very often not carried out in practice. In contrast stands the vast bulk of archival tissue specimens in the form of fixative treated, paraffin embedded tissue blocks. These tissue archives are present in all medical centers and contain valuable specimens from patients whose disease has been followed for many years as part of the normal clinical management. These informative specimens await a simple, effective means for their genetic analysis. Clinical practice is very much in need of easily applicable techniques for DNA sequence analysis from routinely prepared tissue blocks (Antonarakis S E, Diagnosis of Genetic Disorders at the DNA Level. N. Engl. J. Med., 320:153-163, 1989). The methods to be used should not be destructive to the blocks and must take advantage of the important insights gained through detailed histopathologic analysis. The techniques should be independent of archival storage time permitting the opportunity for long-term retrospective study. Finally the approach should be cost effective and timely to participate in everyday clinical decision making.
Topographic Genotyping (TG) is a novel system of tissue management comprehensive in scope specifically developed to address these specific issues enabling full DNA analysis within the context of traditional pathology. TG permits tissue specimens, routinely fixed in standard fixative chemical agents, of any size including minute needle biopsy specimens and cell blocks of cytology material, and of any age including those stored in paraffin for over thirty years, to be both fully available for standard histopathology examination as well as DNA sequence analysis. Furthermore, TG has been specifically designed to incorporate procedures for tissue and information handling allowing quick and easy clinical as well as research application. In essence TG is designed to allow the user to simply and effectively sample minute morphologic targets within fixative treated tissue specimens based on histopathologic and topographic considerations, which in turn may serve as the basis for detailed DNA sequence analysis. The results of TG is an integration of genetic and histopathologic features in a simple, reliable and cost effective manner for clinical application. Solid tissue specimens, removed at surgery or through biopsy procedures, are exposed to fixative agents designed to prevent tissue breakdown and preserve morphologic integrity for microscopic analysis and archival storage. Fixatives, the most common being a 4% buffered solution of formaldehyde, cause their tissue preserving effect by a process of chemical crosslinking of cellular constituents including proteins, sugars and nucleic acids. Much of the tissue stabilizing effect of tissue fixatives is chemically irreversible (Greer C E, Oeterson S L, Kiviat N B, Manos M M, PCR Amplification from Paraffin-Embedded Tissues. Effect of Fixative and Fixation Time. Am. J. Clin. Path., 95:117-124, 1991). This tissue stabilizing and preserving chemical interaction, essential for microscopic analysis, greatly interferes with the manipulation of DNA for genetic investigation representing a major deterrent for general application of molecular analysis on fixed tissue specimens.
In order to meet the need for up to date genetic analysis, current medical practice recommends obtaining separate tissue specimens, not subject to chemical fixation, exclusively for the purpose of genetic analysis. When this involves a fluid specimen of homogeneous character such as a blood sample or bone marrow aspirate, division of the specimen for separate microscopic and molecular biologic analysis is usually accomplished fairly easily without involving interfering with traditional pathologic diagnosis. For many needle biopsy procedures, however, and in a variety of other circumstances of limiting tissue availability, a solid tissue sample will not be able to be appropriately divided and thus molecular examination would not be performed. Even in the case of large specimens, which might appear at first to provide generous amounts of tissue sample for genetic study, appropriate subdivision may not be feasible in as much as cellular heterogeneity cannot be fully appreciated until full tissue fixation and histopathologic examination is first performed. To derive the greatest benefit from genetic analysis, it is highly desirable to focus molecular analysis on selected tissue targets reflecting the cellular basis of disease processes. This in turn can only be achieved following thorough histopathologic examination. This is the essential condition that must be met if true and effective integration of pathology and molecular biology is to be achieved.
These realities provide a strong impetus to define new ways in which fixative treated tissue specimens should be handled to allow DNA structure analysis. Current protocols in this regard, while they may be available, are, in general, highly inefficient, difficult to apply widely in clinical practice and do not take histological considerations fully into account (Shibata D K, Arnheim N, Martin W J, Detection of Human Papillo
Finkelstein Patricia Anne
Finkelstein Sydney David
Arthur Lisa B.
Schwartz Ansel M.
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