Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-03
2003-01-28
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S007200
Reexamination Certificate
active
06511806
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to cancer diagnosis and treatment, and specifically to the determination of a drug resistance phenotype in neoplastic cells from cancer patients. The invention specifically relates to the separation of chemotherapeutic drug resistant neoplastic cells from drug sensitive neoplastic cells and stromal cells. The invention in particular relates to the identification of genes that are differentially expressed in chemotherapeutic drug resistant neoplastic cells compared with the expression of these genes in drug sensitive neoplastic cells. As part of this identification, the invention provides a pattern of expression from a selected number of identified genes, the expression of which is increased or decreased in chemotherapeutic drug resistant neoplastic cells. The invention provides methods for identifying such genes and expression patterns of such genes and using this information to make clinical decisions on cancer treatment, especially chemotherapeutic drug treatment of cancer patients.
2. Summary of the Related Art
Cancer remains one of the leading causes of death in the United States. Clinically, a broad variety of medical approaches, including surgery, radiation therapy and chemotherapeutic drug therapy are currently being used in the treatment of human cancer (see the textbook
CANCER: Principles & Practice of Oncology
, 2d Edition, De Vita et al., eds., J. B. Lippincott Company, Philadelphia, Pa., 1985). However, it is recognized that such approaches continue to be limited by a fundamental inability to accurately predict the likelihood of clinically successful outcome, particularly with regard to the sensitivity or resistance of a particular patient's tumor to a chemotherapeutic agent or combinations of chemotherapeutic agents.
A broad variety of chemotherapeutic agents are used in the treatment of human cancer. These include the plant alkaloids vincristine, vinblastine, vindesine, and VM-26; the antibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin; the antimetabolites methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil, bis-diamminedichloroplatinum, azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSA and mitoxantrone (DeVita et al., Id.). However, some neoplastic cells become resistant to specific chemotherapeutic agents, in some instances even to multiple chemotherapeutic agents, and some tumors are intrinsically resistant to certain chemotherapeutic agents. Such drug resistance or multiple drug resistance can theoretically arise from expression of genes that confer resistance to the agent, or from lack of expression of genes that make the cells sensitive to a particular anticancer drug. One example of the former type is the multidrug resistance gene, MDR1, which encodes an integral plasma membrane protein termed P-glycoprotein that is an non-specific, energy-dependent efflux pump. (See Roninson (ed)., 1991
, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells
, Plenum Press, N.Y., 1991; Gottesman et al., 1991, in
Biochemical Bases for Multidrug Resistance in Cancer
, Academic Press, N.Y., Chapter 11 for reviews). Examples of the latter type include topoisomerase II, the expression of which makes cells sensitive to the anticancer drug etoposide. Decreased expression of this enzyme makes neoplastic cells resistant to this drug. (See Gudkov et al., 1993
, Proc. Natl. Acad. Sci. USA
90:3231-3235). Although these are just single examples of the way that modulation of gene expression can influence chemotherapeutic drug sensitivity or resistance in neoplastic cells, these examples demonstrate the diagnostic and prognostic potential for identifying genes the expression of which (or the pattern of gene expression modulation thereof) are involved in mediating the clinical effectiveness of anticancer drug treatment.
Thus, there is a need in this art for developing methods for identifying genes and gene expression patterns that are predictive of the clinical effectiveness of anticancer drug treatment therapies, in order to make more informed decisions for treating individual cancer patients with anticancer drugs having greatest likelihood of producing a positive outcome.
SUMMARY OF THE INVENTION
The present invention provides methods identifying genes and gene expression patterns that are predictive of the clinical effectiveness of anticancer drug treatment therapies.
In a first aspect the invention provides a method for separating living neoplastic cells from dead cells and living stromal cells in a mixed population of cells from a tumor sample, the method comprising the steps of:
a) contacting the mixed population of cells with a vital stain or fluorescent dye;
b) contacting the mixed population of cells with a detectably-labeled immunological reagent that specifically binds to neoplastic cells; and
c) selecting the cells in the mixed population of step (b) that are not stained with the vital stain and that bind the immunological reagent.
In a preferred embodiment, the vital stain is propidium iodide. Most preferably, the immunological reagent is a tumor-specific antibody that is detectably labeled with a fluorescent label and the cells are separated by fluorescence activated cell sorting. In certain embodiments, the tumor sample is a solid tumor sample and the mixed cell population is a disaggregated tumor sample. In other embodiments, the tumor sample is a hematopoietic tumor sample and the mixed cell population is a nucleated hematopoietic cell sample.
In a second aspect, the invention provides a method for separating living neoplastic cells that are resistant to a cytotoxic compound from dead cells, living stromal cells and living neoplastic cells that are sensitive to the cytotoxic compound in a mixed population of cells from a tumor sample, the method comprising the steps of:
a) contacting the mixed population of cells with the cytotoxic compound for a time and at a concentration wherein the stromal cells and neoplastic cells that are sensitive to the cytotoxic compound undergo apoptosis;
b) contacting the mixed population of step (a) with a vital stain or fluorescent dye;
c) contacting the mixed population of cells of step (b) with a discrimination compound that specifically binds to apoptotic cells;
d) contacting the mixed cell population of step (c) with a detectably-labeled immunological reagent that specifically binds to the apoptotic cell discrimination compound; and
e) selecting the cells in the mixed population of step (c) that are not stained with the vital stain and that do not bind the immunological reagent.
In a preferred embodiment, the vital stain is propidium iodide. Most preferably, the apoptosis discrimination reagent is Annexin V and the immunological reagent is an Annexin V-specific antibody that is detectably labeled with a fluorescent label , wherein the cells are separated by fluorescence activated cell sorting. In preferred embodiments, the mixed population is contacted with the cytotoxic compound under in vitro cell culture conditions whereby the cells cannot attach to a solid substrate. In certain embodiments, the tumor sample is a solid tumor sample and the mixed cell population is a disaggregated tumor sample. In other embodiments, the tumor sample is a hematopoietic tumor sample and the mixed cell population is a nucleated hematopoietic cell sample.
In yet a third aspect, the invention provides a method for detecting a gene expression profile of living neoplastic cells that are resistant to a cytotoxic compound and distinguishing such a profile from the gene expression profile of living neoplastic cells that are sensitive to the cytotoxic compound in a mixed population of cells from a tumor sample, the method comprising the steps of:
a) contacting the mixed population of cells with the cytotoxic
Fruehauf John
Mechetner Eugene
Chunduru Suryaprabha
Fredman Jeffrey
McDonnell & Boehnen Hulbert & Berghoff
Oncotech, Inc.
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