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  Methods of classifying, diagnosing, stratifying and treating cancer patients and their tumorsUSPTO Application #: 20060040302Title: Methods of classifying, diagnosing, stratifying and treating cancer patients and their tumors Abstract: The invention provides a variety of reagents for use in the diagnosis and management of cancer, particularly breast cancer. cDNA microarray technology was used to identify genes whose expression profile across a large group of tumor samples correlates with that of cytokeratin 5 and cytokeratin 17, markers for basal cells of the normal mammary lactation gland. The invention demonstrates that tumors that express cytokeratin 5/6 and/or 17 have a poor prognosis relative to tumors overall. The invention provides basal marker genes and their expression products and uses of these genes for diagnosis of cancer and for identification of therapies for cancer. In particular, the invention provides basal marker genes including cadherin3, matrix metalloproteinase 14, and cadherin EGF LAG seven-pass G-type receptor 2. The invention provides antibodies to the polypeptides expressed by these genes and methods of use thereof. (end of abstract) Agent: Choate, Hall & Stewart LLP - Boston, MA, US Inventors: David Botstein, Patrick O. Brown, Charles M. Perou, Brian Ring, Douglas Ross, Rob Seitz, Jan Matthijs van de Rijn USPTO Applicaton #: 20060040302 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20060040302. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a divisional application of U.S. Ser. No. 09/916,849, filed Jul. 26, 2001 which claims priority to provisional application U.S. Ser. No. 60/220,967, filed Jul. 26, 2000, each of which is incorporated herein by reference. REFERENCE TO MATERIAL PRESENTED IN APPENDICES [0003] This patent application refers to material comprising tables and data that were presented as appendices on CD-ROM in parent application U.S. Ser. No. 09/916,849, filed Jul. 26, 2001. A duplicate CD-ROM is not being presented with this divisional application; instead the entire contents of Appendices A-H are incorporated herein by reference via parent application U.S. Ser. No. 09/916,849, filed Jul. 26, 2001. A paper copy of Appendix H, Tables 3-16 are also being presented with this divisional. The 24 files on the CD-ROM are entitled Appendix A (4,651 kb), Appendix B (481 kb), Appendix C (7,810 kb), Appendix D (3,721 kb), Appendix E (1238 kb), Appendix F (540 kb), Appendix G (377 kb), AppendixH_Table1 (2,102 kb), AppendixH_Table2 (760 kb), AppendixH_Table3 (22 kb), AppendixH_Table4 (25 kb), AppendixH_Table5 (27 kb), AppendixH_Table6 (655 kb), AppendixH_Table7 (88 kb), AppendixH_Table8 (28 kb), AppendixH_Table9-1 (22 kb), AppendixH_Table9-2 (22 kb), AppendixH_Table10 (21 kb), AppendixH_Table11 (22 kb), AppendixH_Table12 (23 kb), AppendixH_Table13 (21 kb), AppendixH_Table14 (23 kb), AppendixH_Table15 (21 kb), AppendixH_Table16 (21 kb). The size of each file in kilobytes is listed following the file name. The total number of bytes is 23,164,507. All files were created on Jul. 25, 2001. The format is IBM-PC. The operating system is Windows. The 24 files on the CD-ROM are incorporated herein by reference. BACKGROUND OF THE INVENTION [0004] A major challenge of cancer treatment is to target specific therapies to distinct tumor types in order to maximize efficacy and minimize toxicity. A related challenge lies in the attempt to provide accurate diagnostic, prognostic, and predictive information. At present, tumors are described with the tumor-node-metastasis (TNM) system. This system, which uses the size of the tumor, the presence or absence of tumor in regional lymph nodes, and the presence or absence of distant metastases, to assign a stage to the tumor is described in the American Joint Committee on Cancer: AJCC Cancer Staging Manual. Philadelphia, Pa.: Lippincott-Raven Publishers, 5th ed., 1997, pp 171-180, and in Harris, J R: "Staging of breast carcinoma" in Harris, J. R., Hellman, S., Henderson, I. C., Kinne D. W. (eds.): Breast Diseases. Philadelphia, Lippincott, 1991. The assigned stage is used as a basis for selection of appropriate therapy and for prognostic purposes. In addition to the TNM parameters, morphologic appearance is used to further classify tumors and thereby aid in selection of appropriate therapy. However, this approach has serious limitations. Tumors with similar histopathologic appearance can exhibit significant variability in terms of clinical course and response to therapy. For example, some tumors are rapidly progressive while others are not. Some tumors respond readily to hormonal therapy or chemotherapy while others are resistant. [0005] Assays for cell surface markers, e.g., using immunohistochemistry, have provided means for dividing certain tumor types into subclasses. For example, one factor considered in prognosis and in treatment decisions for breast cancer is the presence or absence of the estrogen receptor (ER) in tumor samples. ER-positive breast cancers typically respond much more readily to hormonal therapies such as tamoxifen, which acts as an anti-estrogen in breast tissue, than ER-negative tumors. Though useful, these analyses only in part predict the clinical behavior of breast tumors. There is phenotypic diversity present in breast cancers that current diagnostic tools fail to detect. Therefore, there exists a need for improved methods for classifying tumors. [0006] Mutation or dysregulation of any of a large number of genes contributes to the development and progression of cancer as discussed in Hanahan, D. and Weinberg, R., The Hallmarks of Cancer, Cell, 100, 57-70, 2000. Genes that play a role in cancer can be divided into a number of broad classes including oncogenes, tumor suppressor genes, and genes that regulate apoptosis. Oncogenes such as ras typically encode proteins whose activities promote cell growth and/or division, a function that is necessary for normal physiological processes such as development, tissue regeneration, and wound healing. However, inappropriate activity or expression of oncogenes can lead to the uncontrolled cell proliferation that is a feature of cancer. Tumor suppressor genes such as Rb act as negative regulators of cell proliferation. Loss of their activity, e.g., due to mutations or decreased expression at the level of mRNA or protein, can lead to unrestrained cell division. A number of familial cancer syndromes and inherited susceptibility to cancer are believed to be caused by mutations in tumor suppressor genes. Apoptosis, or programmed cell death, plays important roles both in normal development and in surveillance to eliminate cells whose survival may be deleterious to the organism, e.g., cells that have acquired DNA damage. Many chemotherapeutic agents are believed to work by activating the endogenous apoptosis pathway in tumor cells. [0007] Although a substantial number of genes have been implicated as playing important roles in cancer, the factors responsible for the phenotypic diversity of tumors remain largely unknown. In particular, understanding of the underlying differences in gene expression that may contribute to tumor phenotype is limited. Understanding the differences in gene expression between normal and cancerous tissue and between different tumors of the same tissue type is of significant diagnostic, prognostic, and therapeutic utility. There is therefore a need for the identification of genes exhibiting differential expression between tumors. In particular, there is a need for the identification of additional genes and proteins that can be used to classify tumors, especially genes and proteins that can provide diagnostic, prognostic, and/or predictive information in cancer. There is also a need for antibodies and other reagents for the detection and measurement of such genes and proteins. [0008] Most of the commonly used chemotherapeutic agents act relatively nonselectively. Rather than specifically killing tumor cells, these agents target any dividing cell, resulting in a variety of adverse effects. In addition, current therapeutic strategies are of limited efficacy, and the mortality rate of breast cancer remains high. There is therefore a need for the identification of additional genes and proteins that can be used as targets for the treatment of cancer. There is also a need for antibodies and other reagents that can modulate, regulate, or interact with these genes and proteins to provide new method of treatment for cancer. SUMMARY OF THE INVENTION [0009] The present invention relates to the identification of markers that are useful in classifying tumors, particularly breast tumors. The markers identify a class of tumors whose cells have characteristics of basal cells of normal breast lactation ducts. The markers were identified based on their expression profiles in human breast tumor samples, normal breast tissue, and cell lines as assessed using cDNA microarrays. In particular, the basal cell markers of the present invention were identified based on the similarity of their mRNA expression patterns to the expression patterns of markers previously known to identify breast duct basal cells, e.g., cytokeratin 5 and cytokeratin 17, across a set of breast tumor samples. The basal markers include the three genes known as cadherin 3 or P-cadherin (SEQ ID NO:1; GenBank protein accession number NP.sub.--001784; GenBank cDNA accession number NM.sub.--001793), matrix metalloproteinase 14 (SEQ ID NO:2; GenBank protein accession number NP.sub.--004986; GenBank cDNA accession number NM.sub.--004995); and cadherin EGF LAG seven-pass G-type receptor 2 or EGF-Like Domain, Multiple 2 (SEQ ID NO:3; GenBank protein accession number NP.sub.--001399; GenBank cDNA accession number NM.sub.--001408). The invention further provides antibodies that specifically bind to the polypeptides encoded by the basal marker genes identified herein. The antibodies recognize basal cells of normal mammary lactation glands. [0010] The invention provides various diagnostic methods based on the reagents mentioned above. The diagnostic methods include methods for classifying a tumor. In particular, the invention allows classification of a breast tumor as belonging to a basal class of breast tumors. According to certain of the inventive methods the presence or amount of a gene product, e.g., a polypeptide or a nucleic acid, encoded by a basal marker gene is detected in a sample derived from a subject (e.g., a sample of tissue or cells obtained from a tumor or a blood sample obtained from a subject). In general the subject is a human, however the subject may also be an animal of any other kind. The subject may be an individual who has or may have a tumor. The sample may be subjected to various processing steps prior to or in the course of detection. In certain embodiments of the invention the gene product is a polypeptide that is detected using an antibody capable of binding to the polypeptide. In certain embodiments of the invention the antibody is used to perform immunohistochemical staining on a sample obtained from a subject. In certain embodiments of the invention basal marker gene mRNA expression is measured using a microarray. In other embodiments of the invention basal marker gene mRNA expression is measured by quantitative PCR using a set of primers designed to amplify a portion of the gene. Additional detection means that may be employed in the present invention are described in U.S. Pat. No. 6,057,105. In any of the methods for tumor classification and diagnosis, it may be advantageous to detect and/or measure expression of a set of basal markers rather than expression of a single marker. [0011] By providing reagents that may reliably be used to classify tumors as belonging to a basal subclass, the invention enables a variety of methods for improving therapeutic options for patients with breast cancer. Much effort has and continues to be expended on the discovery of new chemotherapeutic agents. These agents are tested for efficacy in clinical trials. In many such trials it is noticed that a small number of patients stabilize or improve while receiving the treatment, while most patients do not appear to benefit. Most such agents are not further developed for a number of reasons. For example, the clinical trial results may not be adequate to gain approval by the Food and Drug Administration. In addition, a pharmaceutical company may determine that the potential market for the drug is too small to justify further efforts. However, if it were possible to identify those patients likely to respond to the treatment, then it would be possible to design clinical trials that would show efficacy, and it would be possible to appropriately select patients who would benefit from the treatment. In addition, the availability of markers that can be used to classify breast tumors enables the retrospective examination of the thousands of breast tumor samples archived in hospitals and pathology labs. These samples can be classified using the inventive reagents and classification scheme, and the results can be correlated with the clinical outcome, based on medical records. Thus it is possible to determine whether tumors that fall into a particular tumor class, e.g., a basal tumor class, are responsive to a particular treatment. This will enable the re-evaluation of drugs that failed in clinical trials and may identify a subset of tumors that are likely to respond to a particular drug, and thus a subset of patients that are likely to benefit from treatment with that drug. [0012] The inventors have recognized that in order to achieve these goals it is necessary to develop new and improved methods for classifying breast tumors. The inventive methods provide a molecular basis for classifying tumors, based on their underlying biology. While not wishing to be bound by any theory, the inventors postulate that tumors arising from a particular cell type within the breast are likely to display common features. Such features may include the prognosis (e.g., predicted survival time or likelihood that a patient's life expectancy exceeds a given length of time) or likelihood that a tumor will respond to a particular therapy. [0013] In particular, tumors that display characteristics of basal cells of the normal breast lactation duct (also referred to herein as breast basal cells) form a distinct subclass (referred to herein as the basal subclass). Inventors have confirmed that patients with breast tumors whose cells display characteristics of breast basal cells, e.g., expression of cytokeratin 5 and/or cytokeratin 17, have a poor clinical outcome relative to patients with breast tumors that do not express these markers. However, antibodies to these cytokeratins have been found (by the inventors and by other investigators) to give spotty, focal staining patterns when used to perform immunohistochemistry on breast tumor samples. Thus the utility of cytokeratins 5 and 17 as markers and the utility of antibodies that bind to cytokeratin 5 or 17 for determining whether a tumor is a member of the basal subclass has been limited. The inventors have therefore identified genes whose mRNA expression profiles across a large set of tumor samples correlate with, i.e. are similar to, the expression profiles of the known basal cell markers cytokeratins 5 and 17. These genes include the basal markers of the present invention mentioned above. As described in Examples 10 and 13, the inventors have generated antibodies to the proteins expressed by these genes and shown that the antibodies stain basal cells of normal mammary lactation glands. Thus detection of one or more expression products of these genes may be used to identify tumors that fall within the basal tumor subclass. [0014] The invention further provides therapeutic agents based on the identification of breast basal cell markers. The therapeutic agents include compounds that modulate these genes or that modulate polypeptides encoded by these genes. In particular, the therapeutic agents include antibodies that bind to polypeptides encoded by the basal cell marker genes. The invention further includes agonists and antagonists to the basal marker genes, to the polynucleotides transcribed from those genes, and to their encoded polypeptides. The invention also provides methods for identifying such agonists and antagonists. The invention further includes pharmaceutical compositions comprising such antibodies, agonists, and antagonists as well as methods of use of the pharmaceutical compositions in the treatment of cancer, particularly breast cancer. [0015] According to one aspect, the invention provides a method of classifying a tumor comprising the steps of (i) providing a tumor sample, (ii) detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:1 in the sample; and (iii) classifying the tumor as belonging to a tumor subclass based on the results of the detecting step. The invention also provides a method of classifying a tumor comprising the steps of (i) providing a tumor sample, (ii) detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:2 in the sample, and (iii) classifying the tumor as belonging to a tumor subclass based on the results of the detecting step. In addition, the invention provides a method of classifying a tumor comprising the steps of (i) providing a tumor sample, (ii) detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:3 in the sample, and (iii) classifying the tumor as belonging to a tumor subclass based on the results of the detecting step. The invention further includes a method of classifying a tumor comprising the steps of (i) providing a tumor sample, (ii) detecting expression or activity of at least two genes selected from the group consisting of: a gene encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the sample, and (iii) classifying the tumor as belonging to a tumor subclass based on the results of the detecting step. In any of the foregoing methods the detecting step may comprise detecting the polypeptide or polypeptides encoded by the genes. A variety of detection techniques may be employed including, but not limited to, immunohistochemical analysis, ELISA assay, antibody arrays, or detecting modification of a substrate by the polypeptide. [0016] In certain embodiments of the methods the tumor is a breast tumor and the tumor subclass is a basal tumor subclass. The methods may further comprise providing diagnostic, prognostic, or predictive information based on the classifying step. Classifying may include stratifying the tumor (and thus stratifying a subject having the tumor), e.g., for a clinical trial. The methods may further comprise selecting a treatment based on the classifying step. [0017] In another aspect, the invention provides a method of testing a subject comprising the steps of (i) providing a sample isolated from a subject, (ii) detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:1 in the sample, and (iii) providing diagnostic, prognostic, or predictive information based on the detecting step. The invention further provides a method of testing a subject comprising the steps of (i) providing a sample isolated from a subject, (ii) detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:2 in the sample (iii) and providing diagnostic, prognostic, or predictive information based on the detecting step. The invention further provides a method of testing a subject comprising the steps of (i) providing a sample isolated from a subject, (ii) detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:3 in the sample (iii) and providing diagnostic, prognostic, or predictive information based on the detecting step. The invention further includes a method of testing a subject comprising the steps of (i) providing a sample isolated from the subject, (ii) detecting expression or activity of at least two genes selected from the group consisting of: a gene encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 in the sample, and (iii) providing diagnostic, prognostic, or predictive information based on the detecting step. In any of these methods the detecting step may comprise detecting the polypeptide or polypeptides. Detection may be performed using any appropriate technique including, but not limited to, immunohistochemistry, ELISA assay, protein array, or detecting modification of a substrate by the polypeptide. [0018] The sample may comprise mRNA, in which case the detecting step may comprise hybridizing the mRNA or cDNA or RNA synthesized from the mRNA to a microarray or detecting mRNA transcribed from the gene or detecting cDNA or RNA synthesized from mRNA transcribed from the gene. In any of the above methods, the sample may be a blood sample, a urine sample, a serum sample, an ascites sample, a saliva sample, a cell, and a portion of tissue. [0019] In another aspect, the invention provides a kit for diagnosis of a tumor which may include (i) primers for amplifying an mRNA transcribed from a gene that encodes the polypeptide of any of SEQ ID NO:1, SEQ ID NO:2; and SEQ ID NO:3 (ii) instructions for use of the kit; and/or (iii) control samples for testing the primers, wherein the control samples comprise nucleic acids that hybridize to the primers. [0020] In another aspect, the invention provides an antibody that specifically binds to an epitope found in a polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:1, and wherein the antibody recognizes basal cells in normal mammary lactation glands. According to certain embodiments of the invention the antibody distinguishes basal cells from luminal cells in normal mammary lactation gland. According to certain embodiments of the invention the antibody recognizes an epitope found in a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. [0021] In another aspect, the invention provides an antibody that specifically binds to an epitope found in a polypeptide whose amino acid sequence comprises the amino acid sequence of SEQ ID NO:2, and wherein the antibody recognizes basal cells in normal mammary lactation glands. According to certain embodiments of the invention the antibody distinguishes basal cells from luminal cells in normal mammary lactation gland. According to certain embodiments of the invention the antibody recognizes an epitope found in a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. Continue reading... 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