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WO2002008281A2 - Proteine bstp-cad et reactifs associes et methodes d'utilisation - Google Patents

Proteine bstp-cad et reactifs associes et methodes d'utilisation Download PDF

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Publication number
WO2002008281A2
WO2002008281A2 PCT/US2001/023566 US0123566W WO0208281A2 WO 2002008281 A2 WO2002008281 A2 WO 2002008281A2 US 0123566 W US0123566 W US 0123566W WO 0208281 A2 WO0208281 A2 WO 0208281A2
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WIPO (PCT)
Prior art keywords
polypeptide
seq
sample
antibody
polynucleotide
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PCT/US2001/023566
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WO2002008281A3 (fr
Inventor
David Botstein
Patrick O. Brown
Chuck Perou
Douglas Ross
Rob Seitz
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Applied Genomics Inc
Leland Stanford Junior University
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Applied Genomics Inc
Leland Stanford Junior University
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Priority to AU2001277200A priority Critical patent/AU2001277200A1/en
Publication of WO2002008281A2 publication Critical patent/WO2002008281A2/fr
Anticipated expiration legal-status Critical
Publication of WO2002008281A3 publication Critical patent/WO2002008281A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • 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.
  • TAM tumor-node-metastasis
  • 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.
  • 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.
  • the present invention relates to the identification of genes of particular import in diagnosis, prognostication and/or therapeutic intervention in breast cancer and other tumors based on their expression profile in human breast tumor samples, their expression in other tissue and normal tissue samples, and in cell lines as assessed using cDNA microarrays.
  • the genes are identified based on their differential expression across tumor samples.
  • the invention provides a substantially purified polypeptide and fragments thereof that are encoded by an RNA molecule that is differentially expressed in human breast tumor samples and cell lines.
  • the polypeptide is referred to as BSTP-CAD.
  • the invention provides a substantially purified polypeptide whose amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO:l.
  • the invention also provides a substantially purified polypeptide whose amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the invention also provides polypeptides possessing homology to the polypeptide having the sequence of SEQ ID NO:l or to fragments of this polypeptide, wherein the polypeptides are significantly similar to the polypeptide of SEQ ID NO: 1.
  • the invention also provides polypeptides possessing homology to the polypeptide having the sequence of SEQ ID NO:2 or to fragments of this polypeptide, wherein the polypeptides are significantly similar to the polypeptide of SEQ ID NO:2.
  • the definition of "significantly similar” can vary, as described further below.
  • a significantly similar polypeptide has one or more amino acid substitutions, deletions, and/or additions with respect to the sequence of SEQ ID NO:l or SEQ ID NO:2.
  • the polypeptides are expression products of human genes.
  • the set of polypeptides or polynucleotides includes those polypeptides or polynucleotides having the particular sequence set forth in the SEQ ID NO:X in addition to other polypeptides or polynucleotides including the sequence of SEQ ID NO:X.
  • the invention provides substantially isolated and purified polynucleotides encoding the polypeptide of SEQ ID NO: 1 or SEQ ID NO:2.
  • the invention provides a substantially isolated and purified polynucleotide whose sequence comprises the sequence of SEQ ID NO:3.
  • the invention further provides substantially isolated and purified polynucleotides whose sequences comprise the sequence of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • the invention also provides polynucleotides encoding polypeptides possessing significant similarity to the polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO:2, where "significantly similar" is defined below.
  • the invention further provides a polynucleotide having a sequence that is complementary to the sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • the invention provides a polynucleotide having a sequence that is complementary to a polypeptide encoding a polypeptide possessing significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • the invention provides an isolated and purified polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising or having the amino acid sequence set forth in SEQ ID NO:l or the sequence set forth in SEQ ID NO:2.
  • the invention provides a substantially purified oligonucleotide that includes a region of nucleotide sequence that hybridizes to at least 8 consecutive nucleotides of sense or antisense sequence of a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • the invention also provides a substantially purified oligonucleotide that includes a region of nucleotide sequence that hybridizes to at least 8 consecutive nucleotides of sense or antisense sequence of a nucleotide sequence that encodes the polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO:2.
  • the oligonucleotide is attached to an oligonucleotide microarray.
  • the oligonucleotide has a sequence capable of binding specifically with an RNA molecule that encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 1 so as to prevent appropriate processing, transport, or translation of the RNA molecule.
  • the invention provides methods for detecting a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO:2, or a polypeptide having significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • One such method comprises steps of: (a) contacting the biological sample with an antibody that specifically binds to the polypeptide of SEQ ID NO: 1 or SEQ ID NO:2, or a polypeptide having significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2; and (b) determining whether the antibody specifically binds to the sample, the binding being an indication that the sample contains the polypeptide.
  • the invention also provides kits for performing these methods.
  • kits can comprise an antibody (preferably a monoclonal antibody) that binds to the polypeptide and, optionally, other materials such as suitable buffers, indicators (e.g., fluorophores, chromophores or enzymes providing same), controls (e.g., a polypeptide of this invention) and directions for using the kit.
  • a third such method comprises the step of determining whether there exists, within a cell and/or tissue of an individual, inappropriate expression of a polynucleotide encoding a polypeptide having an amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO:2, or a polypeptide having significant similarity to the polypeptide of SEQ ID NO: 1 or SEQ ID NO:2.
  • a fourth such method comprises the step of determining whether there exists, within a cell and/or tissue of an individual, inappropriate expression of a polypeptide having an amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • the method can further comprise the step of providing diagnostic, prognostic, or predictive information based on the category assigned in the assigning step.
  • the method is used to classify breast tumors.
  • the method is based on detecting expression of a single gene, e.g., a gene encoding the polypeptide of SEQ ID NO:l or SEQ ID NO:2. Detecting expression of such a gene may comprise detecting the polynucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. Detecting expression may comprise measuring either a relative or absolute level of expression.
  • the method is based on an assessment of the expression of multiple polynucleotides as described further in copending U.S. Patent Application "Reagents and Methods for Use in Managing Breast Cancer, filed July 26, 2001 and in U.S. Provisional Patent Application Ser. No. 60/220,967, filed July 26, 2000. These applications are referred to herein as "the gene subset application".
  • the multiple polynucleotides can include all of the polynucleotides disclosed therein or any subset thereof.
  • the invention provides a method of classifying a tumor by detection of the polypeptide of SEQ ID NO:l, SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO: 1 or SEQ ID NO:2 in cells and/or tissue samples obtained from the tumor or from elsewhere in the body (e.g., in blood, urine, ascites, other body fluids or secretions or excretions).
  • the method is used to classify breast tumors.
  • the invention provides a microarray for use in classifying tumors, comprising a polynucleotide whose sequence comprises or is complementary to that set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or whose sequence is of sufficient length to specifically bind to such a sequence under the microarray hybridization conditions employed.
  • a microarray for use in classifying tumors, comprising a polynucleotide whose sequence comprises or is complementary to that set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or whose sequence is of sufficient length to specifically bind to such a sequence under the microarray hybridization conditions employed.
  • Such conditions may be those described in the Examples, or any conditions appropriate for the particular microarray and detection technology employed.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • the pharmaceutical composition further preferably comprises a pharmaceutically acceptable carrier.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a substantially purified antibody that binds to a polypeptide having an amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO: 1 or SEQ ID NO:2.
  • the pharmaceutical composition further preferably comprises a pharmaceutically acceptable carrier.
  • the antibody is modified, e.g., by attaching a toxic molecule thereto.
  • the invention provides methods for identifying modulators of the expression or activity of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • the invention further provides agonists and antagonists capable of modulating the expression or activity of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:l or SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • the invention provides pharmaceutical compositions including such modulators and methods of use thereof for the treatment or prevention of cancer, particularly breast cancer.
  • the invention provides a method for the treatment or prevention of cancer comprising the step of administering to an individual in need thereof, a pharmaceutical composition comprising a polypeptide having an amino acid sequence comprising the sequence of SEQ ID NO: 1 or SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO: 1 or SEQ ID NO:2.
  • the invention provides a method of treating or preventing a tumor comprising steps of: (i) providing an individual in need of treatment or prevention of a tumor, (ii) administering a compound that enhances the level or activity of a polypeptide comprising the amino acid sequence of SEQ ID NO:l or SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • the compound is provided as a component of a pharmaceutical composition.
  • the invention also includes such a pharmaceutical composition.
  • the invention provides methods of inhibiting growth of a cell comprising enhancing the level or activity of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 or a polypeptide having significant similarity to the polypeptide of SEQ ID NO:l or SEQ ID NO:2 in the cell.
  • the cell can be a normal cell or a tumor cell, e.g., a breast tumor cell.
  • the level of the polypeptide is enhanced by overexpressing the polypeptide in the cell, e.g., by introducing an expression vector or other nucleotide sequence (e.g., DNA, RNA, or modified nucleotides, etc.) that encodes the polypeptide into the cell.
  • 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:l or 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.
  • the detecting step may comprise detecting the polypeptide.
  • 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.
  • the tumor is a breast tumor and the tumor subclass is a luminal 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.
  • stratification is the process or result of describing or separating a patient population into more homogeneous subpopulations according to specified criteria. Stratifying patients initially rather than after the trial is frequently preferred, e.g., by regulatory agencies such as the U.S. Food and Drag Administration that may be involved in the approval process for a medication. In some cases stratification may be required by the study design.
  • Various stratification criteria may be employed in conjunction with detection of expression of one or more basal marker genes.
  • Stratification is frequently useful in performing statistical analysis of the results of a trial.
  • 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:l or SEQ ID NO:2 in the sample, and (iii) providing diagnostic, prognostic, or predictive information based on the detecting step.
  • the detecting step may comprise detecting the polypeptide. 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.
  • 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.
  • 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.
  • the invention provides a method of testing a compound or a combination of compounds for activity against tumors comprising steps of (i) obtaining or providing tumor samples taken from subjects who have been treated with the compound or combination of compounds, wherein the tumors fall within a tumor subclass, (ii) comparing the response rate of tumors that fall within the tumor subclass and have been treated with the compound with the overall response rate of tumors that have been treated with the compound or combination of compounds or with the response rate of tumors that do not fall within the subclass and have been treated with the compound or combination of compounds and (iii) identifying the compound or combination of compounds as having selective activity against tumors in the tumor subclass if the response rate of tumors in the subclass is greater than the overall response rate or the response rate of tumors that do not fall within the subclass.
  • the tumors are breast tumors.
  • the tumor subclass is a luminal tumor subclass.
  • the tumors may be classified according to any of the inventive classification methods described above. In certain embodiments of the invention the classification is based on expression of the polypeptide of SEQ ID NO:l or SEQ ID NO:2.
  • the invention further provides a method of testing a compound or a combination of compounds for activity against tumors comprising steps of (i) treating subjects in need of treatment for tumors with the compound or combination of compounds, (ii) comparing the response rate of tumors that fall within a tumor subclass with the overall response rate of tumors or with the response rate of tumors that do not fall within the subclass, and (iii) identifying the compound or combination of compounds as having selective activity against tumors in the tumor subclass if the response rate of tumors in the subclass is greater than the overall response rate or the response rate of tumors that do not fall within the subclass.
  • the method may further comprise various additional steps.
  • the invention includes a method of testing a compound or a combination of compounds for activity against tumors comprising steps of (i) treating subjects in need of treatment for tumors with the compound or combination of compounds or with an alternate compound, wherein the tumors fall within a tumor subclass, (ii) comparing the response rate of tumors treated with the compound or combination of compounds with the response rate of tumors treated with the alternate compound; and (iii) identifying the compound or combination of compounds as having superior activity against tumors in the tumor subclass, as compared with the alternate compound, if the response rate of tumors treated with the compound or combination of compounds is greater than the response rate of tumors treated with the alternate compound.
  • the method may further comprise various additional steps.
  • the method may comprise steps of (i) providing tumor samples from subjects in need of treatment for tumors, (ii) determining whether the tumors fall within a tumor subclass, and (iii) stratifying the subjects based on the results of the determining step prior to performing the treating step.
  • the method may further comprise the steps of (i) providing tumor samples from subjects in need of treatment for tumors, (ii) detecting expression or activity of a gene encoding the polypeptide of SEQ ID NO:l or SEQ ID NO:2 in the samples, and (iii) stratifying the subjects based on the results of the detecting step prior to performing the treating step.
  • Figure IB presents the sequence of a portion of BSTP-CAD as initially determined from sequencing of I.M.A.G.E. clone 52071 (SEQ ID O:2).
  • Figure 1C presents the polynucleotide sequence of an open reading frame that encodes
  • BSTP-CAD (SEQ IDNO:3).
  • Figures ID presents a polynucleotide sequence obtained by sequencing I.M.A.G.E. clone 52071 (SEQ ID NO:4).
  • Figure 2 presents the polynucleotide sequence that encodes the polypeptide of SEQ ID NO:2.
  • Figure 3 presents a sequence map in which the four cadherin domains (indicated as CI, C2, C3, and C4) of BSTP-CAD are highlighted in gray, and the predicted transmembrane domain (indicated as TM) is also highlighted in gray. Small triangles represent exon boundaries.
  • Figure 5 A presents a plot of a TMpred prediction of the transmembrane regions and orientation for BSTP-CAD.
  • Figure 5B presents a prediction of transmembrane helices for BSTP-CAD produced using a Hidden Markov Model.
  • the tables contain the numerical data corresponding to micrarray images. Other tables provide additional information or list the individual genes in the various gene subsets.
  • each row is similarly identified by a number in the first column so that the name and Genbank accession number for the clone for which data appears in that row may be determined by consulting the reference list.
  • the column headings in the first row identify the tumor samples.
  • Each data cell in the table represents the measured Cy5/Cy3 fluorescence ratio at the corresponding target element on the appropriate array. Empty cells indicate insufficient or missing data. All ratio values are log transformed (base 2) to treat inductions or repressions of identical magnitude as numerically equal but with opposite sign.
  • Table 2 is a master data table for the 19 microarray experiments performed on cell line samples, in which rows represent I.M.A.G.E. clones that identify approximately 1753 genes whose expression varied by at least a factor of 4 and columns represent individual microarray experiments.
  • This table contains only a data portion, in which the column headings in the first row identify the cell lines.
  • Each row in the table is identified by a number which appears in the first column.
  • the same reference list that forms part of Table 1 may be consulted to determine the name and Genbank accession number for the clone for which data appears in that row.
  • Each data cell in the table represents the measured Cy5/Cy3 fluorescence ratio at the corresponding target element on the appropriate array. Empty cells indicate insufficient or missing data. All ratio values are log transformed (base 2) to treat inductions or repressions of identical magnitude as numerically equal but with opposite sign.
  • Table 3 presents a listing and description of the 11 cell lines used to create the common reference sample.
  • Table 4 presents a complete listing of the 84 experimental samples that were assayed versus the common reference sample.
  • the table includes a list of alternate names (in the column entitled Sample ID/old name) for the same tumors.
  • the alternate names are used to identify the tumor samples in certain contexts, and the table allows conversion between the two sets of names.
  • Table 5 lists the tumors used in the experiments described herein, along with clinical and pathological information about each tumor/patient.
  • each row is similarly identified by a number in the first column so that the name and Genbank accession number for the clone for which data appears in that row may be determined by consulting the reference list.
  • the column headings in the first row identify the tumor samples.
  • Each data cell in the table represents the measured Cy5/Cy3 fluorescence ratio at the corresponding target element on the appropriate array. Empty cells indicate insufficient or missing data. All ratio values are log transformed (base 2) to treat inductions or repressions of identical magnitude as numerically equal but with opposite sign.
  • Table 7 is a listing of the 374 clones that identify genes selected for the epithelial enriched gene set including Genbank accession numbers.
  • Table 8 is a listing of the clones that identify genes that comprise the luminal subset including Genbank accession numbers.
  • Tables 9-1 and 9-2 are listings of the two groups of clones that identify genes that comprise the basal subset including Genbank accession numbers.
  • Table 10 is a listing of the clones that identify genes that comprise the ErbB2 subset including Genbank accession numbers.
  • Table 13 is a listing of the clones that identify genes that comprise the B-cell gene subset including Genbank accession numbers.
  • Table 16 is a listing of the clones that identify genes that comprise the T-cell gene subset including Genbank accession numbers.
  • Genbank accession number for each clone appears in the column entitled "Name", following a brief descriptive name for the gene identified by the clone, where available. In some cases the descriptive name is a number corresponding to an I.M.A.G.E. clone ID number.
  • the Genbank accession number represents a means of definitively identifying a particular clone, since Genbank accession numbers will be maintained permanently or, if changed, the change will be accomplished in such a manner as to allow unambiguous correlation between any new numbering system and the numbering system currently in use.
  • Tables 1, 2, and 6 are provided for purposes of presenting the clone identifications and the data that was used to perform hierarchical clustering analysis, and that the format of the tables may not correspond exactly with the format required by software developed for the analysis of the data. Appropriate format will, in general, depend upon the particular computer program. See, for example, the Web site http://genome-www.stanford.edu/ ⁇ sherlock/tutorial.html for discussion of the appropriate format for one particular analysis program.
  • each entry identifies a clone.
  • the first portion of each entry is a brief descriptive name for the gene identified by the clone.
  • the Genbank accession number for the clone appears on the last line of the entry for that clone.
  • Agonist refers to a molecule that increases or prolongs the duration of the effect of a polypeptide or a nucleic acid.
  • Agonists may include proteins, nucleic acids, carbohydrates, lipids, small molecules, ions, or any other molecules that modulate the effect of the polypeptide or nucleic acid.
  • An agonist may be a direct agonist, in which case it is a molecule that exerts its effect by binding to the polypeptide or nucleic acid, or an indirect agonist, in which case it exerts its effect via a mechanism other than binding to the polypeptide or nucleic acid (e.g., by altering expression or stability of the polypeptide or nucleic acid, by altering the expression or activity of a target of the polypeptide or nucleic acid, by interacting with an intermediate in a pathway involving the polypeptide or nucleic acid, etc.)
  • Antagonist refers to a molecule that decreases or reduces the duration of the effect of a polypeptide or a nucleic acid. Antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules that modulate the effect of the polypeptide or nucleic acid.
  • an mRNA corresponds to a clone on a microarray when the mRNA (or cDNA derived therefrom) hybridizes specifically (under the experimental conditions described) to the clone or to its complement, e.g., when the sequence of the mRNA (or cDNA derived therefrom) and the sequence of the clone are sufficiently complementary to one another for specific hybridization to occur.
  • a gene corresponds to a clone on a microarray when mRNA transcribed from the gene corresponds to the clone. Note that it is not necessary that the entire mRNA, cDNA, etc. hybridize with the clone or vice versa.
  • diagnostic information is any information that is useful in determining whether a patient has a disease or condition and/or in classifying the disease or condition into a phenotypic category or any category having significance with regards to the prognosis of or likely response to treatment (either treatment in general or any particular treatment) of the disease or condition.
  • diagnosis refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have a condition (such as a tumor), information related to the nature or classification of a tumor, information related to prognosis and/or information useful in selecting an appropriate treatment.
  • the term “gene” generally refers to a portion of a nucleic acid that encodes a protein; the term may optionally encompass regulatory sequences. This definition is not intended to exclude application of the term “gene” to non-protein coding expression units but rather to clarify that, in most cases, the term as used in this document refers to a protein coding nucleic acid.
  • homologous sequences may be identified by searching databases (e.g., GenBank, EST [expressed sequence tag] databases, GST [gene sequence tag] databases, GSS [genome survey sequence] databases, organism sequencing project databases) using computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. These programs are described in Altschul, SF, et al, Basic local alignment search tool, J. Mol.
  • operably linked refers to a relationship between two nucleic acid sequences wherein the expression of one of the nucleic acid sequences is controlled by, regulated by, modulated by, etc. the other nucleic acid sequence.
  • a promoter is operably linked with a coding sequence if the promoter controls transcription of the coding sequence.
  • a nucleic acid sequence that is operably linked to a second nucleic acid sequence is covalently linked, either directly or indirectly, to such a sequence, although any effective three-dimensional association is acceptable.
  • Prognostic information and predictive information are used interchangeably and somewhat informally to refer to any information that may be used to foretell any aspect of the course of a disease or condition either in the absence or presence of treatment. Such information may include, but is not limited to, the average life expectancy of a patient, the likelihood that a patient will survive for a given amount of time (e.g., 6 months, 1 year, 5 years, etc.), the likelihood that a patient will be cured of a disease, the likelihood that a patient's disease will respond to a particular therapy (wherein response may be defined in any of a variety of ways). Prognostic and predictive information are included within the broad category of diagnostic information.
  • sample obtained from a subject may include, but is not limited to, any or all of the following: a cell or cells, a portion of tissue, blood, serum, ascites, urine, saliva, and other body fluids, secretions, or excretions.
  • sample also includes any material derived by processing such a sample.
  • Derived samples may include nucleic acids or proteins extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of mRNA, etc.
  • Specific binding refers to an interaction between a target polypeptide (or, more generally, a target molecule) and a binding molecule such as an antibody, agonist, or antagonist.
  • the interaction is typically dependent upon the presence of a particular structural feature of the target polypeptide such as an antigenic determinant or epitope recognized by the binding molecule.
  • an antibody is specific for epitope A
  • the presence of a polypeptide containing epitope A or the presence of free unlabeled A in a reaction containing both free labeled A and the antibody thereto will reduce the amount of labeled A that binds to the antibody. It is to be understood that specificity need not be absolute.
  • binding molecule may be evaluated in the context of additional factors such as the affinity of the binding molecule for the target polypeptide versus the affinity of the binding molecule for other targets, e.g., competitors. If a binding molecule exhibits a high affinity for a target molecule that it is desired to detect and low affinity for nontarget molecules, the antibody will likely be an acceptable reagent for immunodiagnostic purposes.
  • the specificity of a binding molecule is established in one or more contexts, it may be employed in other, preferably similar, contexts without necessarily re-evaluating its specificity.
  • Treating a tumor is taken to mean treating a subject who has the tumor.
  • Tumor sample is taken broadly to include cell or tissue samples removed from a tumor, cells (or their progeny) derived from a tumor that may be located elsewhere in the body (e.g., cells in the bloodstream or at a site of metastasis), or any material derived by processing such a sample. Derived tumor samples may include nucleic acids or proteins extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of mRNA, etc.
  • Tumor subclass A tumor subclass, also referred to herein as a tumor subset or tumor class, is the group of tumors that display one or more phenotypic or genotypic characteristics that distinguish members of the group from other tumors.
  • a vector as used herein, is a nucleic acid molecule that includes sequences sufficient to direct in vivo or in vitro replication of the molecule. These may either be self-replication sequences or sequences sufficient to direct integration of the vector into another nucleic acid present in a cell (either an endogenous nucleic acid or one introduced into the cell by experimental manipulation), so that the vector sequences are replicated during replication of this nucleic acid.
  • Preferred vectors include a cloning site, at which foreign nucleic acid molecules may be introduced.
  • Vectors may include control sequences that have the ability to direct in vivo or in vitro expression of nucleic acid sequences introduced into the vector.
  • control sequences may include, for example, transcriptional control sequences (e.g., promoters, enhancers, terminators, etc.), splicing control sequences, translational control sequences, etc.
  • Vectors may also include a coding sequence, so that transcription and translation of sequences introduced into the vector results in production of a fusion protein.
  • Nucleic acids encoding a portion of BSTP-CAD were identified based on expression profiles gathered in a series of cDNA microarray experiments. As described in more detail in Examples 1, 2, and 4, cDNA microarrays each representing the same set of approximately 8100 different human genes were produced.
  • the human cDNA clones used to produce the microarrays contained approximately 4000 named genes, 2000 genes with homology to named genes in other species, and approximately 2000 ESTs of unknown function.
  • An mRNA sample was obtained from each of a set of 84 tissue samples or cell lines. The expression levels of the approximately 8100 genes were measured in each mRNA sample by hybridization to an individual microarray, yielding an expression profile for each gene across the experimental samples.
  • cDNA Microarray Technology cDNA microarrays consist of multiple (usually thousands) of different cDNAs spotted (usually using a robotic spotting device) onto known locations on a solid support, such as a glass microscope slide. After spotting, the cDNAs are usually cross-linked to the support, e.g., by UV irradiation.
  • the cDNAs are typically obtained by PCR amplification of plasmid library inserts using primers complementary to the vector backbone portion of the plasmid or to the gene itself for genes where sequence is known.
  • PCR products suitable for production of microarrays are typically between 0.5 and 2.5 kB in length.
  • Full length cDNAs, expressed sequence tags (ESTs), or randomly chosen cDNAs from any library of interest can be chosen. ESTs are partially sequenced cDNAs as described, for example, in L. Hillier, et al. Generation and analysis of
  • ESTs correspond to known genes, frequently very little or no information regarding any particular EST is available except for a small amount of 3' and/or 5' sequence and, possibly, the tissue of origin of the mRNA from which the EST was derived.
  • the cDNAs contain sufficient sequence information to uniquely identify a gene within the human genome.
  • the cDNAs are of sufficient length to hybridize specifically to cDNA obtained from mRNA derived from a single gene under the hybridization conditions of the experiment. In a typical microarray experiment, a microarray is hybridized with differentially labeled RNA or DNA populations derived from two different samples.
  • RNA either total RNA or poly A + RNA is isolated from cells or tissues of interest and is reverse transcribed to yield cDNA. Labeling is usually performed during reverse transcription by incorporating a labeled nucleotide in the reaction mixture. Although various labels can be used, most commonly the nucleotide is conjugated with the fluorescent dyes Cy3 or Cy5. For example, Cy5- dUTP and Cy3-dUTP can be used.
  • cDNA derived from one sample (representing, for example, a particular cell type, tissue type or growth condition) is labeled with one fluor while cDNA derived from a second sample (representing, for example, a different cell type, tissue type, or growth condition) is labeled with the second fluor.
  • Each microarray experiment can provide tens of thousands of data points, each representing the relative expression of a particular gene in the two samples. Appropriate organization and analysis of the data is of key importance.
  • Various computer programs have been developed to facilitate data analysis.
  • One basis for organizing gene expression data is to group genes with similar expression patterns together into clusters.
  • a method for performing hierarchical cluster analysis and display of data derived from microarray experiments is described in Eisen, M, Spellman, P, Brown, P, and Botstein, D, Cluster analysis and display of genome- wide expression patterns, Proc. Natl. Acad. Sci. USA, 95: 14863-14868, 1998.
  • clustering can be combined with a graphical representation of the primary data in which each data point is represented with a color that quantitatively and qualitatively represents that data point.
  • this process facilitates an intuitive analysis of the data. Additional information and details regarding the mathematical tools and/or the clustering approach itself may be found, for example, in Sokal, R.R. & Sneath, P.H.A. Principles of numerical taxonomy, xvi, 359, W. H. Freeman, San Francisco, 1963; Hartigan, J.A. Clustering algorithms, xiii, 351, Wiley, New York, 1975; Paull, K.D. et al.
  • microarray hardware e.g., arrayers and scanners
  • microarray hardware e.g., arrayers and scanners
  • the present invention encompasses the realization that genes that are differentially expressed in tumors are of use in tumor classification and are targets for the development of diagnostic and therapeutic agents. Differentially expressed genes are likely to be responsible for the different phenotypic characteristics of tumors.
  • the present invention identifies one such gene.
  • a differentially expressed gene is a gene whose transcript abundance varies between different tumor samples.
  • the transcript level of a differentially expressed gene may vary by at least fourfold from its average abundance in a given sample set in at least 1 sample, at least two samples, at least three samples, etc. Of course other criteria for differential expression may be employed.
  • cDNA microarrays each representing the same set of approximately 8100 different human genes were produced.
  • the human cDNA clones used to produce the microarrays contained approximately 4000 named genes, 2000 genes with homology to named genes in other species, and approximately 2000 ESTs of unknown function.
  • An mRNA sample was obtained from each of a set of 84 tissue samples or cell lines. The expression levels of the approximately 8100 genes were measured in each mRNA sample by hybridization to an individual microarray, yielding an expression profile for each gene across the experimental samples.
  • Variation in patterns of gene expression were characterized in 62 breast tumor samples from 40 different patients, 3 normal breast tissue samples, and 19 samples from 17 cultured human cell lines (one of which was sampled 3 times under different conditions). Twenty of the tumors had been sampled twice, before and after a 16 week course of doxorubicin chemotherapy, and two tumors were paired with a lymph node metastasis from the same patient. The other 18 tumor samples were single samples from individual tumors.
  • a detailed listing of the tumor samples and various characteristics including clinical estrogen receptor and Erb-B2 status as assessed using antibody staining, estrogen receptor and Erb-B2 status as assessed by microarray result, tumor grade, differentiation, survival status and time, age at diagnosis, doxorubicin response, and p53 status is presented in Table 5 of the gene subset application.
  • a listing of the cell lines including description and ATCC (American Tissue Culture Collection) number or reference is presented in Table 3 of the gene subset application. The cell lines provided a framework for interpreting the variation in gene expression patterns seen in the tumor samples and included gene expression models for many of the cell types encountered in tumors.
  • Comparative expression measurements were made by separately mixing Cy5- labeled experimental cDNA derived from each of the 84 samples with a portion of the Cy3 -labeled reference cDNA, and hybridizing each mixture to an individual cDNA microarray. The ratio of Cy5 fluorescence to Cy3 fluorescence measured at each cDNA element on the microarray was then quantitatively measured. The use of a common reference standard in each hybridization allowed the fluorescence ratios to be treated as comparative measurements of the expression level of each gene across all the experimental samples.
  • a hierarchical clustering method (Eisen, et al, 1998) was used to group genes based on similarity in the pattern with which their expression varied over all experimental samples.
  • the same clustering method was used to group the experimental samples (tissue and cell lines separately) based on the similarity in their patterns of expression.
  • Interpretation of the data obtained from the clustering algorithm was facilitated by displaying the data in the form of tumor and gene dendrograms.
  • the pattern and length of the branches reflects the relatedness of the tumor samples with respect to their expression of genes represented on the microarray.
  • Microarray images, tumor and gene dendrograms are available in Perou, et al, Nature, 2000, and at inventors' Web site (http://genome- www.stanford.edu/molecularportraits/).
  • the similarity of the gene expression profiles of individual tumor samples or groups of tumor samples to one another is inversely related to the length of the branches that connect them.
  • adjacent tumor samples connected to one another by short vertical branches descending from a common horizontal branch are more closely related to one another in terms of their gene expression profiles than adjacent tumor samples connected to one another by longer vertical branches descending from a common horizontal branch (e.g., tumor samples Norway 100-BE and Norway 100-AF at the left side of the tumor dendrogram).
  • the expression patterns of the genes were also displayed using a matrix format, with each row representing all of the hybridization results for a single cDNA element on the array and each column representing the measured expression levels for all genes in a single sample.
  • this format tumor samples with similar patterns of expression across the gene set are close to each other along the horizontal dimension.
  • genes with similar expression patterns across the set of samples are close to each other along the vertical dimension.
  • the normalized expression value of each gene was represented by a colored box, using red to represent expression levels greater than the median and green to represent expression levels less than the median.
  • the brightest red color represents transcript levels at least 16-fold greater than the median
  • the brightest green color represents transcript levels at least 16-fold below the median.
  • clone 52071 was identified based on the expression pattern of its corresponding mRNA among the 84 samples analyzed by microarray hybridization. The polynucleotide corresponding to clone 52071 was differentially expressed among the tumor samples, indicating its potential utility for classifying tumors. The expression level of mRNA corresponding to clone 52071 varied significantly among tumor samples and was particularly high in certain tumors.
  • mRNA corresponding to clone 52071 was particularly highly expressed in tumor samples Stanford 24 (BC24), Norwayl8-BE (BC118B), and Norway 18-AF (BC118A) (In the foregoing, names of tumors in parentheses represent an alternate nomenclature used in some contexts.)
  • the presence of mRNA corresponding to clone 52071 reflects the expression of the gene from which the mRNA is transcribed, and measurement of the expression level differentiates between different breast tumors.
  • the expression pattern of clone 52071 was noteworthy in that it is expressed in a manner very similar to a few other genes, some of which have features suggesting that they are appropriate targets for drug discovery.
  • clone 52071 may identify a tumor subclass that can be targeted for particular therapies.
  • GenBank A search of GenBank revealed that only a small portion at the 3' end and a small portion at the 5' end of clone 52071 had been sequenced. Therefore, clone 52071 was sequenced and a full length sequence (SEQ ID NO:4) was obtained.
  • a search of GenBank revealed the presence of a sequence homologous to a portion of clone 52071 in a human BAC clone (GenBank accession number AC004836), a portion of which was identified as a likely gene using the program Genefinder. The 3' portion of this predicted gene overlaps with the 5' portion of SEQ ID NO:4.
  • the consensus sequence (SEQ ID NO:5) was used as input for a search of GenBank with the BLASTX program (which translates a nucleotide sequence in each of the possible six reading frames and then searches for homologous amino acid sequences). The search indicated that the translated amino acid sequence in one reading frame was homologous to members of the cadherin superfamily of proteins.
  • SEQ ID NO:6 includes both a start and a stop codon and is believed, based on results obtained using RT-PCR, to be transcribed and include a full length coding sequence.
  • the amino acid sequence of the predicted protein (BSTP-CAD) encoded by this nucleotide sequence is presented in SEQ ID NO:l.
  • the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide is 747 amino acids in length.
  • a search of the GenBank database using the BLASTP computer program (Altschul, Stephen F, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI- BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) performed with this sequence confirmed the homology to members of the cadherin superfamily, consistent with the information obtained from the BLASTX search described above.
  • the cadherins are a large family of proteins with critical roles in the regulation of cell-cell adhesion. Generally expressed in development- or tissue-specific manners, these factors have been shown to have important roles in development, cellular proliferation, and differentiation.
  • the cadherin superfamily include classic cadherins, desmogleins, desmocolhns, protocadherins, CNRs, Fats, and seven-pass transmembrane cadherins (for review see Nollet et al. 2000).
  • transmembrane proteins the cadherins are characterized by the unique cadherin, or EC, domain.
  • cadherin domains which are involved in Ca ++ binding (Takeichi 1990), are repeated in the extracellular region of all of the family members.
  • the amino acid sequences of other regions shows significant divergence among members, suggesting functional diversity amongst the various cadherin proteins.
  • the cytoplasmic domains are conserved.
  • this region interacts with catenin pl20 ctn , and plakoglobin or ⁇ -catenin. The latter binds to ⁇ -catenin, and this molecular complex further associates with -actinin, F-actin and other cytoskeletal proteins.
  • cadherin genes Consistent with their roles in regulating cell-cell adhesion events, altered expression of cadherin genes has been associated with human cancer. Alteration of cadherin function likely leads to subsequent metastasis by disaggregation of tumor cells, and one proposed role of many cadherins studied to date is as tumor- and invasion-suppressors. Mutations inactivating the E-cadherin/catenin protein complex were found in sporadic lobular breast carcinoma and in both familial and sporadic diffuse gastric carcinoma (for reviews see Nollet el al, 1999 and Berx et al, 1998).
  • Figure 3 presents a sequence map in which the four cadherin domains (indicated as CI, C2, C3, and C4) of BSTP-CAD are highlighted in gray, and the predicted transmembrane domain (indicated as TM) is also highlighted in gray.
  • Figure 4 presents an alignment of the four cadherin domains of BSTP-CAD (CDla, CD2a, CD3a, and CD4a) with a cadherin domain consensus sequence. Identical amino acids are shaded dark gray; similar amino acids are shaded light gray. Analysis of the BSTP-CAD coding sequence using several different techniques indicated the presence of a putative transmembrane domain between amino acids 574 and 604. In the sequence map presented in Figure 4 the predicted transmembrane domain is highlighted in gray.
  • Figure 5A presents a prediction of transmembrane regions and orientation for BSTP-CAD obtained using the program TMpred (K. Hofmann & W. Stoffel (1993) TMbase - A database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler 347,166).
  • Figure 5B presents a prediction of transmembrane helices for BSTP-CAD produced using a hidden Markov model (Erik L.L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences.
  • TMpred K. Hofmann & W. Stoffel (1993)
  • Figure 5B presents a prediction of transmembrane helices for BSTP-CAD produced using a hidden Markov model (Erik L.L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein
  • Determining the expression of BST-CAD may include detecting and/or measuring the amount of RNA transcribed from BST-CAD, e.g., mRNA comprising a nucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or a variant of any of these seuqences.
  • Determining the expression of BST-CAD may include detecting (qualitatively or quantitatively) a translation product of BST- CAD, e.g., a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:l, SEQ ID NO:2, or a fragment or variant of either of these polypeptides.
  • Determining the expression of BST-CAD may include detecting mRNA encoding a polypeptide selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, or a fragment or variant of either of these polypeptides.
  • BSTP-CAD The discovery of BSTP-CAD and the discovery of the differential expression of the gene encoding BSTP-CAD satisfy a need in the art by providing compositions useful in the diagnosis, treatment, and prevention of cancer, particularly breast cancer, and by providing methods useful in the classification of cancer and the provision of prognostic information to patients with cancer. Furthermore, BSTP-CAD is likely to be a transmembrane protein, indicating that it will likely be accessible to therapeutic agents such as antibodies and/or small molecules. These results suggest that BST- CAD and BSTP-CAD may be useful targets for therapeutic intervention in subsets of breast cancer and useful to distinguish between different subsets of breast cancer.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO: 1 OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 60 or a % positive greater than 70 encompassing at least 25% of the length of SEQ ID NO:l OR SEQ ID NO:2, or both (where "both” refers to a situation in which both the % positive and % identity criteria are met).
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO: 1 OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 60 or a % positive greater than 70 encompassing at least 75% of the length of SEQ ID NO:l OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO: 1 OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 60 or a % positive greater than 70 encompassing at least 95% of the length of SEQ ID NO: 1 OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO:l OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 70 or a % positive greater than 80 encompassing at least 75% of the length of SEQ ID NO:l OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO:l OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 70 or a % positive greater than 80 encompassing at least 95% of the length of SEQ ID NO.T OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO:l OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 80 or a % positive greater than 90 encompassing at least 25% of the length of SEQ ID NO:l OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO:l OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 80 or a % positive greater than 90 encompassing at least 75% of the length of SEQ ID NO:l OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO: 1 OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 80 or a % positive greater than 90 encompassing at least 90% of the length of SEQ ID NO:l OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO: 1 OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 80 or a % positive greater than 90 encompassing at least 95% of the length of SEQ ID NO: 1 OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO:l OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 90 or a % positive greater than 95 encompassing at least 25% of the length of SEQ ID NO:l OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO:l OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 90 or a % positive greater than 95 encompassing at least 75% of the length of SEQ ID NO: 1 OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO.T OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 90 or a % positive greater than 95 encompassing at least 90% of the length of SEQ ID NO:l OR SEQ ID NO:2, or both.
  • a polypeptide is considered significantly similar if, when the amino acid sequence of the polypeptide is compared with the amino acid sequence of the polypeptide of SEQ ID NO:l OR SEQ ID NO:2 using the BLAST algorithm and the BLOSUM substitution matrix with default parameters, the result is a % identity greater than 90 or a % positive greater than 95 encompassing at least 95% of the length of SEQ ID NO: 1 OR SEQ ID NO:2, or both.
  • the invention also encompasses variants of the polypeptide of SEQ ID NO: 1 OR SEQ ID NO:2 and variants of significantly similar polypeptides, wherein the variants have one or more altered or modified amino acids. Alterations and modifications may include the replacement of an L- amino acid with a D-amino acid, or various modifications including, but not limited to, phosphorylation, carboxylation, alkylation, etc. Certain polypeptides having significant similarity to the polypeptide of SEQ ID NO: 1 OR SEQ ID NO:2 and variants of significantly similar polypeptides, wherein the variants have one or more altered or modified amino acids. Alterations and modifications may include the replacement of an L- amino acid with a D-amino acid, or various modifications including, but not limited to, phosphorylation, carboxylation, alkylation, etc. Certain polypeptides having significant similarity to the polypeptide of SEQ ID NO: 1 OR SEQ ID NO:2 and variants of significantly similar polypeptides, wherein the variants have one or more altered
  • the invention also includes polynucleotides having a complementary nucleotide sequence to any of the inventive polynucleotides described above.
  • Such complementary polynucleotides are useful as probes, e.g, to detect expression of the inventive polynucleotides at the RNA level.
  • Such complementary polynucleotides are also useful as antisense reagents, to inhibit the expression of the corresponding genes at the protein level, e.g, by interfering with mRNA translation. Inhibiting gene expression has a variety of applications, e.g, it may be used to gain information about the function of the encoded protein. In addition, antisense inhibition of gene expression may be used therapeutically.
  • In vitro transcription systems are well known in the art as are vectors containing appropriate genetic control elements for directing transcription of an inserted polynucleotide sequence and host cells in which such vectors are maintained.
  • the invention encompasses the production of either DNA or RNA having the sequence of an inventive polynucleotide.
  • inventive polynucleotides and fragments thereof can also be synthesized entirely through chemical means. Techniques and machines for chemical synthesis of polynucleotides are well known in the art.
  • the polynucleotides can be labeled or conjugated with detectable moieties including radionuclides, enzymes, chromogenic substrates, fluorescent substances, etc, using any of a variety of techniques.
  • a labeled secondary antibody that recognizes the primary antibody (i.e., the antibody that binds to the polypeptide being detected).
  • appropriate methods include, but are not limited to, immunohistochemistry, radioimmunoassay, ELISA, immunoblotting, and FACS analysis.
  • immunohistochemistry is a particularly appropriate detection method. Techniques for obtaining tissue and cell samples and performing immunohistochemistry and FACS are well known in the art. Such techniques are routinely used, for example, to detect the ER in breast tumor tissue or cell samples.
  • One such method comprises steps of: (a) hybridizing a nucleic acid complementary to the polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:l, or a fragment thereof, to at least one nucleic acid in the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex indicates the presence of a polynucleotide encoding the polypeptide in the biological sample.
  • a second such method comprises steps of:
  • Expression of BST-CAD can also be measured by a variety of other techniques that make use of a polynucleotide corresponding to part or all of the BST- CAD gene rather than an antibody that binds to a polypeptide encoded by the gene.
  • Appropriate techniques include, but are not limited to, in situ hybridization, Northern blot, and various nucleic acid amplification techniques such as PCR, quantitative PCR, and the ligase chain reaction.
  • the detection of mutations and allelic variants can be performed using any of a variety of methods well known in the art ranging from use of microarrays (e.g, oligonucleotide arrays) to detect single nucleotide polymorphisms (SNPs) associated with a particular allele, use of microarrays (e.g,oligonucleotide arrays) to detect substitutions, deletions, etc, detection of restriction fragment length polymorphisms (RFLPs), direct sequencing of DNA isolated from an individual, etc.
  • microarrays e.g, oligonucleotide arrays
  • SNPs single nucleotide polymorphisms
  • RFLPs restriction fragment length polymorphisms
  • antibodies suitable for use as therapeutics exhibit high specificity for the target polypeptide and low background binding to other polypeptides.
  • monoclonal antibodies are employed for therapeutic purposes.
  • antibodies against the HER2/neu/ErbB2 polypeptide represent a paradigm in terms of the development of therapeutic antibodies.
  • Ribozymes catalytic RNA molecules that are capable of cleaving other RNA molecules
  • Such ribozymes can be designed to cleave specific mRNAs corresponding to a gene of interest. Their use is described in U.S. Patent No. 5,972,621, and references therein.
  • the invention encompasses the delivery of antisense and/or ribozyme molecules via a gene therapy approach in which vectors or cells expressing the antisense molecules are administered to an individual.
  • Small molecule modulators e.g, inhibitors or activators
  • BST-CAD gene expression are also within the scope of the invention and may be detected by screening libraries of compounds using, for example, cell lines that express the polypeptide or a version of the polypeptide that has been modified to include a readily detectable moiety.
  • Methods for identifying compounds capable of modulating gene expression are described, for example, in U.S. Patent No. 5,976,793.
  • the screening methods described therein are particularly appropriate for identifying compounds that do not naturally occur within cells and that modulate the expression of genes of interest whose expression is associated with a defined physiological or pathological effect within a multicellular organism.
  • the invention encompasses compounds that modulate the activity of a polypeptide encoding BSTP-CAD.
  • Methods of screening for such interacting compounds are well known in the art and depend, to a certain degree, on the particular properties and activities of the polypeptide encoded by the gene. Representative examples of such screening methods may be found, for example, in U.S. Patent No. 5,985,829, U.S. Patent No. 5,726,025, U.S. Patent No. 5,972,621, and U.S. Patent No. 6,015,692.
  • the skilled practitioner will readily be able to modify and adapt these methods as appropriate for BSTP-CAD.
  • the mechanism of modulation need not be direct.
  • the modulator may act on an enzyme that may modify BSTP-CAD.
  • each agent will be administered at a dose and on a time schedule determined for that agent.
  • the invention encompasses the delivery of the inventive pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce or modify their metabolism, inhibit their excretion, or modify their distribution within the body.
  • the invention encompasses treating cancer, particularly breast cancer, by administering the pharmaceutical compositions of the invention.
  • the pharmaceutical compositions of the present invention can be used for treatment of any subject (e.g, any animal) in need thereof, they are most preferably used in the treatment of humans.
  • compositions of this invention can be administered to humans and other animals by a variety of routes including oral, intravenous, intramuscular, intraarterial, subcutaneous, intraventricular, transdermal, rectal intravaginal, intraperitoneal, topical (as by powders, "ointments, or drops), bucal, or as an oral or nasal spray or aerosol.
  • routes including oral, intravenous, intramuscular, intraarterial, subcutaneous, intraventricular, transdermal, rectal intravaginal, intraperitoneal, topical (as by powders, "ointments, or drops), bucal, or as an oral or nasal spray or aerosol.
  • routes of administration will depend upon a variety of factors including the nature of the compound (e.g, its stability in the environment of the gastrointestinal tract), the condition of the patient (e.g, whether the patient is able to tolerate oral administration), etc.
  • the intravenous route is most commonly used to deliver therapeutic antibodies and nucleic acids.
  • the invention encompasses the delivery of the inventive pharmaceutical
  • the term "pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference.
  • the salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy- ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
  • pharmaceutically acceptable ester refers to esters that hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
  • Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.
  • suitable esters includes formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
  • prodrugs refers to those prodrugs of the compounds of the present invention that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
  • prodrug refers to compounds that are rapidly transformed in vivo to yield a particular active compound, for example by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems", Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed, Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.
  • compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.
  • sterile injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non- irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.
  • the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
  • Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • the present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, and in certain embodiments, includes an additional approved therapeutic agent for use as a combination therapy.
  • an additional approved therapeutic agent for use as a combination therapy can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Instructions for use of the compound(s) may also be included.
  • the total daily dose of the compounds of this invention administered to a human or other mammal in single or in divided doses can be in amounts, for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1 to 25 mg/kg body weight.
  • Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
  • treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 0.1 ⁇ g to about 2000 mg of the compound(s) of the invention per day in single or multiple doses.
  • the human cDNA clones used in this study were obtained from Research Genetics (Huntsville AB, USA) as bacterial colonies in 96-well microtiter plates. The clones were chosen from a set of 15,000 cDNA clones that corresponded to the Research Genetics Human Gene Filters sets GF200-202 (http://www.resgen.com/). These clones form part of a set of clones assembled by the I.M.A.G.E. consortium (Lennon, G.G, Auffray, C, Polymeropoulos, M, Soares, M.B. The I.M.A.G.E. Consortium: An Integrated Molecular Analysis of Genomes and their Expression.
  • the protocol includes steps of (1) cleaning the glass slides onto which the DNAs (e.g, products of PCR reactions) are to be spotted; (2) spotting the DNAs onto the glass slides with an arrayer; (3) Post processing to prepare arrays containing spotted DNAs for hybridization. All procedures are done at room temperature and with double distilled water unless otherwise stated. Unless otherwise stated, in this Example and the following Examples, reagents are prepared according to protocols available in Maniatis, T, Sambrook, J. and Fritsch, E, Molecular Cloning: A Laboratory Manual (3 Volume Set), Cold Spring Harbor Laboratory Press, Cold -Spring Harbor, 1989, the contents of which are herein incorporated by reference.
  • Microarrays were prepared according to the above protocol using the 8498 cDNA clones described above. All microarrays used in the experiments described herein were from a single print run batch of microarrays.
  • the 11 mRNA samples were then mixed together in equal amounts, aliquoted in lOmM Tris (7.4), and stored at -80 C until use (2 micrograms of common reference sample was used per microarray hybridization and was always labeled using Cy3).
  • H&E hematoxylin and eosin
  • mRNA was isolated from breast tissue, breast tumor samples, and cell lines as described in Example 2. Fluorescently labeled cDNA was synthesized from the mRNA using a reverse transcriptase reaction that included dUTP labeled with either Cy3 or Cy5. For each hybridization experiment differentially labeled cDNA samples (an experimental sample and a reference sample) were pooled and hybridized to a cDNA microarray, which was then scanned as described in Example 4. The protocol below provides details of the steps performed for cDNA synthesis and labeling and for microarray hybridization.
  • RT reverse transcriptase
  • Vacuum dryer Box is from Labconco, Kansas City, MO; Pump is from Alcatel, Laurel MD).
  • the sequence of the desired peptide was provided to the peptide synthesizer.
  • the C- terminal residue was determined and the appropriate Wang Resin was attached to the reaction vessel.
  • the peptides were synthesized C-terminus to N-terminus by adding one amino acid at a time using a synthesis cycle. Which amino acid is added was controlled by the peptide synthesizer, which looks to the sequence of the peptide entered into its database.
  • Step 1 Resin Swelling: Added 2 mL DMF, incubated 30 minutes, drained DMF. Step 2 - Synthesis cycle
  • the Collection of Rabbit Serum The rabbits were bled (30 to 50 mL) from the auricular artery. The blood was allowed to clot at room temperature for 15 minutes and the serum was separated from the clot using an IEC DPR-6000 centrifuge at 5000 x g. Cell-free serum was decanted gently into a clean test tube and stored at -20°C for affinity purification.
  • the plates were blocked by completely filling each well with BBS-TW containing 1 % BSA and 0.1 % gelatin (BBS-TW-BG) and incubating for 2 hours at room temperature.
  • BBS-TW-BG BBS-TW containing 1 % BSA and 0.1 % gelatin
  • the plates were emptied and sera of both pre- and post- immune serum were added to wells.
  • the first well contained sera at 1 :50 in BBS.
  • the sera were then serially titrated eleven more times across the plate at a ratio of 1 : 1 for a final (twelfth) dilution of 1:204,800.
  • the plates were incubated overnight at 4°C.
  • the plates were emptied and washed three times as described.
  • Biotinylated goat anti-rabbit IgG (100 ⁇ L) was added to each microtiter plate test well and incubated for four hours at room temperature. The plates were emptied and washed three times. Horseradish peroxidase-conjugated Streptavidin (100 ⁇ L diluted 1:10,000 in BBS-TW-BG) was added to each well and incubated for two hours at room temperature. The plates were emptied and washed three times.
  • the ABTS was prepared fresh from stock by combining 10 mL of citrate buffer (0.1 M at pH 4.0), 0.2 mL of the stock solution (15 mg/mL in water) and 10 ⁇ L of 30% H 2 0 2 . The ABTS solution (lOO ⁇ L) was added to each well and incubated at room temperature. The plates were read at 414 ⁇ , 20 minutes following the addition of substrate.
  • the affinity column was prepared by conjugating 5 mg of peptide to 10 mL of cyanogen bromide-activated Sepharose 4B, and 5 mg of peptide to hydrazine- Sepharose 4B. Briefly, 100 uL of DMF was added to peptide (5 mg) and the mixture was vortexed until the contents were completely wetted. Water was then added (900 ⁇ L) and the contents were vortexed until the peptide dissolved.
  • the conjugated sepharose was pooled and loaded onto fritted columns, washed with 10 mL of BBS, blocked with 10 mL of 1 M glycine, and washed with 10 mL 0.1 M glycine adjusted to pH 2.5 with HC1 and re- neutralized in BBS. The column was washed with enough volume for the optical density at 280 ⁇ to reach baseline.
  • the peptide affinity column was attached to a UV monitor and chart recorder.
  • the titered rabbit antiserum was thawed and pooled.
  • the serum was diluted with one volume of BBS and allowed to flow through the columns at 10 mL per minute.
  • the non-peptide immunoglobulins and other proteins were washed from the column with excess BBS until the optical density at 280 ⁇ reached baseline.
  • the columns were disconnected and the affinity purified column was eluted using a stepwise pH gradient from pH 7.0 to pH 1.0. The elution was monitored at 280 nM, and fractions containing antibody (pH 3.0 to pH 1.0) were collected directly into excess 0.5 M BBS. Excess buffer (0.5 M BBS) in the collection tubes served to neutralize the antibodies collected in the acidic fractions of the pH gradient.
  • the entire procedure was repeated with "depleted" serum to ensure maximal recovery of antibodies.
  • the eluted material was concentrated using a stirred cell apparatus and a membrane with a molecular weight cutoff of 30 kD.
  • the concentration of the final preparation was determined using an optical density reading at 280 nM.
  • extracts are made from a variety of different cell lines and subjected to SDS-PAGE followed by immunoblotting according to the protocol below, using an affinity purified polyclonal antibody to BSTP-CAD prepared as described in Example 6.
  • Bovine Serum Albumin (LP) (Cat. No. 100-350, Boehringer Mannheim,
  • Hybond ECL (Cat. No. RPN303D, Amersham Pharmacia Biotech)
  • Nonfat dry milk (Kroger Co, Cincinnati, OH) Ponceau-S (Cat. No. P-07170, Sigma)
  • Trizma® Base (Cat. No. T-6066, Sigma)
  • Tissue Tearor tissue homogenizer (Cat. No. 985370-07, BioSpec Products Inc, Bartletsville, OK)
  • Sample Preparation Preferably a variety of cell lines known in the art are used for the experiment, including cell lines derived from breast tumors, cell lines derived from normal breast tissue, cell lines derived from other cancer types, etc.
  • a selection of appropriate cancer cell lines for investigation of the expression of BSTP-CAD is found in reference 21.
  • a selection of noncancer cell lines appropriate for investigation of the expression of BSTP-CAD is found in Perou, et al. Molecular portraits of human breast tumours, Nature, 406(6797):747-52, 2000.
  • Appropriate cell lines include MCF7, Hs578T, OVCAR3, HepG2, NTERA2, MOLT4, RPMI-8226, NB4+ATRA, UACC-62, SW872, and Colo205: also see Table 2 for more details).
  • Cell lines are maintained under standard growth conditions and in standard tissue culture media as appropriate for the particular cell line. Cells are collected according to standard techniques (e.g, trypsinization in the case of adherent cells), and the resulting cell suspension is prepared as follows:
  • the cell suspension is pelleted by centrifugation at 3000 RPM for 10 minutes, and the supernatant was discarded.
  • the pellet is washed with 1ml PBS, centrifuged at 10000 RPM for 10 minutes, and the supernatant was discarded.
  • M-PerTM Reagent a volume of M-PerTM Reagent is added to the cell pellet and mixed gently for 10 minutes in an ice bath. The mixture is centrifuged at 13200 RPM for 15 minutes, and the supernatant is saved. The protein concentration in the supernatant is measured according to standard techniques.
  • Standard SDS-PAGE stacking and running gels are prepared and placed in an electrophoresis apparatus. After filling the upper and lower chambers with running buffers the samples (60 Dg/lane) are loaded. The inner core is placed in the lower chamber and the lid placed on top. The apparatus is connected to the power supply and recirculating system. The temperature setting is 10°C The stacking gel is run at 14mA per gel for 1 hour. The separating gel is run at 0.58mA per gel per hour for 16 hours.
  • the assembly for transfer is as follows: cathode pre-soaked blotting paper gel pre-wetted nitrocellulose pre-soaked blotting paper anode
  • the transfer is performed at 20V for 25 minutes, then 25V for 20 minutes. After the transfer is complete, the gel is stained with Coomassie and the blot is stained with Ponceau-S.
  • One wash cycle is performed.
  • One wash cycle consists of: Wash 5 min, rinse Wash 5 min, rinse Wash 10 min, rinse
  • the blots are placed in a Ziploc® bag. Equal volumes of ECL western blotting detection reagents are mixed and distributed evenly over the blots. The blots are placed in an autoradiography cassette, covered with a piece of film, and exposed.
  • a breast cancer tissue microarray consisting of tissue samples from a large number of breast cancer biopsies is prepared essentially as described in Kononen, J, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens, Nature Medicine, 4(7), 844-847, 1998. Briefly, several hundred archival paraffin- embedded breast tumor samples were obtained from the Pathology Department at Stanford University Medical Center. The samples were reviewed by a pathologist (applicant MVR) to ensure that they met pathological criteria for breast cancer. Small tissue cores were removed from the samples and embedded in a single paraffin block to produce a tissue array. Immunohistochemistry is performed as described previously (Perou, C, et al, 1999; Bindl, J.
  • H-cadherin expression inhibits in vitro invasiveness and tumor formation in vivo. Carcinogenesis, 19, 1157-1159.
  • Cadherin-6 a cell adhesion molecule specifically expressed in the proximal renal tubule and renal cell carcinoma. Cancer Res. 57, 2741-2748. 60. Sato M, Mori Y, Sakurada A, Fujimura S, Horii A (1998) The H-cadherin (CDH13) gene is inactivated in human lung cancer. Hum Genet 103:1 96-101

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Abstract

L'invention concerne un polypeptide (BSTP-CAD), des polypeptides associés, des fragments et des variantes de ceux-ci, et des polynucléotides identifiant et codant pour BSTP-CAD et les autres polypeptides. L'invention concerne également des vecteurs d'expression, des cellules hôte, des anticorps, des agonistes et des antagonistes. De plus, l'invention concerne des méthodes de traitement ou de prévention des troubles associés à la prolifération cellulaire, en particulier le cancer du sein, consistant à administrer une composition pharmaceutique comprenant un polypeptide, un polynucléotide, ou un anticorps de l'invention. L'invention concerne également des méthodes de classification des maladies, en particulier du cancer du sein, par détection de l'expression de BSTP-CAD ou d'un polynucléotide codant pour BSTP-CAD, et de génération d'informations diagnostiques, pronostiques, et/ou prédictives sur un patient, basées sur la détection et/ou la mesure de BSTP-CAD ou d'un polynucléotide codant pour BSTP-CAD.
PCT/US2001/023566 2000-07-26 2001-07-26 Proteine bstp-cad et reactifs associes et methodes d'utilisation Ceased WO2002008281A2 (fr)

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