Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-09-02
2001-03-20
Smith, Lynette R. F. (Department: 1641)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C422S068100, C435S004000
Reexamination Certificate
active
06204030
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods and devices for the analysis of cellular samples. More particularly, the present invention relates to methods and devices for the microdissection and analysis of cellular samples which may be used in combination with a number of different technologies that allow for analysis of enzymes, MRNA and DNA from pure populations or subpopulations of particular cell types.
BACKGROUND ART
Many diseases are now understood at the molecular and genetic level. Analysis of such molecules is important for disease diagnosis and prognosis. Previous methods for direct extraction of cellular tissue material from a tissue sample are limited because the extraction reflects only the average content of disease associated markers. In reality, tissues are very heterogeneous, and the most diagnostic portions of the tissue may be confined to a few hundred cells or less in a lesion.
Normal tissue samples contain a variety of cell types surrounding and adjacent to the pre-invasive and invasive tumor cells. A region of the tumor tissue subject to biopsy and diagnosis as small as 1.0 mm can contain normal epithelium, pre-invasive stages of carcinoma, in-situ carcinoma, invasive carcinoma, and inflammatory areas. Consequently, routine scraping and cutting methods will gather all of these types of cells, and hence, loss of an allele will be masked by presence of a normal copy of the allele in the contaminating non-malignant cells. Existing methods for cutting away or masking a portion of tissue do not have the needed resolution. Hence the analysis of genetic results by those previous are always plagued by contaminating alleles from normal cells, undesired cells or vascular cells.
The molecular study of human tumors is currently limited by the techniques and model systems available for their characterization. Studies to quantitatively or qualitatively asses proteins or nucleic acid expression in human tumor cells are compromised by the diverse cell populations present in bulk tumor specimens. Histologic fields of invasive tumor typically show a number of cell types including tumor cells, stromal cells, endothelial cells, normal epithelial cells and inflammatory cells. Since the tumor cells are often a relatively small percentage of the total cell population it is difficult to interpret the significance of net protein or nucleic acid alterations in these specimens.
The processes of tumor invasion and metastasis depend upon increased proteolytic activity of invading tumor cells. Matrix metalloproteinases, cathepsins B, D, and L, and plasminogen activator have been implicated in the metastatic cascade. Cathepsin D has been suggested to be an independent marker of prognosis in breast cancer. Several lines of correlation evidence support the concept that proteases are important in tumor invasion including: increased protease activity and/or altered subcellular distribution of proteases in highly metastatic tumor cell lines, increased protease expression in invasive human tumors as determined by both immunohistochemistry and assays of tumor tissue homogenates, and increased MRNA levels in human tumors. All of these techniques have generated important information regarding protease expression in human tumors, however, they have not provided definitive evidence that proteases are up-regulated in specific Legions where tumor invasion is occurring.
Studies of human tumor cells in culture do not account for the complex interactions of the tumor cells with host cells and extracellular matrix, and how they may regulate tumor cell protease productivity or is activation. Immunohistochemical staining allows one to examine enzyme distribution in regions of tumor invasion, however, results vary with tissue fixation and antibody-antigen affinity, and provide only a semi-quantitative assessment of protein levels. Furthermore, quantitative interpretation of staining results is complicated by the variability of staining patterns within tissue sections, subjective evaluation of staining intensity, and the difficulty in interpreting the significance of stromal staining. In addition, many antibodies utilized in the study of proteases do not differentiate pro-enzyme from active enzyme species. Assays of enzyme or MRNA levels from homogenates of human tumors does not account for either the mixed population of cells within the specimens, or the concomitant pathophysiologic processes which may be occur in the tissue.
Human tumors accumulate genetic abnormalities as they develop from a single transformed cell to invasive and metastatic carcinoma. Identification and characterization of the genes which are mutated, lost or abnormally regulated can provide important insights for cancer diagnosis, prognosis, and therapy. Furthermore, identification of such genetic lesions may facilitate early diagnosis by definitive identification of premalignant lesions so they can be treated before they progress to invasive cancer.
A general dictum of cancer progression states that cells can be transformed after acquiring two separate alterations in the tumor suppressor gene. Subsequent tumors progress stepwise from dysplastic lesions to in-situ, to invasive and metastatic neoplasms. In-situ carcinomas are frequently observed arising in association with a spectrum of epithelial hyperplasias and larger invasive tumors are often associated with regions of carcinoma in-situ at the tumor periphery.
Pathologists have historically interpreted a side-by-side association of atypical hyperplasia, in-situ carcinoma, and invasive tumors as evidence of a cause and effect relationship among the entities. However, little indirect evidence existed previously which supports this model.
Prior methods of study have not allowed investigators to specifically examine genetic alterations in pre-invasive lesions. The present invention provides a novel improved means to specifically examine genetic alterations in pre-invasive lesions of common epithelial tumors such as breast and prostate carcinoma. In particular the present invention permits the microsampling of five or less cells with RNA and DNA extraction of the sampled cells. This method has been demonstrated to be extremely sensitive and to surpass previous and current technologies by more than two orders of magnitude. It has allowed the sensitive detection of loss of heterozygosity in early pre-invasive lesions being a gateway to the discovery of a new genetic loci on chromosome 11 for breast cancer and a new genetic loci on chromosome 8 for prostate carcinoma.
DISCLOSURE OF THE INVENTION
It is according one object of the present invention to provide a method of identifying specific cells in cellular tissue sample.
Another object of the present invention is to provide a method of direct extraction of specific cells from a cellular tissue sample.
It is a further object of the present invention to provide an automated method of identifying specific cells in cellular tissue sample.
A further object of the present invention is to provide an automated method of direct extraction of specific cells from a cellular tissue sample.
A still further object of the present invention is to provide a method of obtaining pure cell populations from a cellular tissue samples.
According to these and further objects of the present invention which will become apparent as the drescription thereof proceeds, the present invention provides for a method of direct extraction of cellular material from a tissue sample which involves:
providing a tissue sample;
contacting the tissue sample with a transfer surface which can be activated to provide selective regions thereof with adhesive characteristics;
identifying at least one portion of the tissue sample which is to be extracted;
activating a region of the transfer surface which is in contact with the at least one portion of the tissue sample so that the activated region of the transfer surface adheres to the at least one portion of the tissue sample; and
separating the transfer surface from the tissue sample while maintaining adhesion between the
Bonner Robert F.
Buck Michael E.
Liotta Lance A.
Weiss Rhonda Ann
Zhuang Zhengping
Portner Ginny Allen
Smith Lynette R. F.
The United States of America as represented by the Secretary of
Townsend and Townsend / and Crew LLP
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