WO2000020044A1 - Nouveau procede pour diagnostiquer, controler, etablir le bilan d'extension, visualiser, et traiter des cancers gynecologiques - Google Patents

Nouveau procede pour diagnostiquer, controler, etablir le bilan d'extension, visualiser, et traiter des cancers gynecologiques Download PDF

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WO2000020044A1
WO2000020044A1 PCT/US1999/022753 US9922753W WO0020044A1 WO 2000020044 A1 WO2000020044 A1 WO 2000020044A1 US 9922753 W US9922753 W US 9922753W WO 0020044 A1 WO0020044 A1 WO 0020044A1
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esbpiii
cancer
levels
patient
cells
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PCT/US1999/022753
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Roberto A. Macina
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Diadexus Llc
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Priority to CA002345285A priority Critical patent/CA2345285A1/fr
Priority to US09/806,302 priority patent/US7014996B1/en
Priority to JP2000573402A priority patent/JP2002527723A/ja
Priority to EP99951679A priority patent/EP1169066A4/fr
Publication of WO2000020044A1 publication Critical patent/WO2000020044A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57411—Specifically defined cancers of cervix
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates, in part, to newly developed assays for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating cancers, particularly gynecologic cancers including endometrial, mammary, ovary and uterine cancer. BACKGROUND OF THE INVENTION In women, gynecologic cancers account for more than one- fourth of the malignancies .
  • endometrial cancer occurs at a rate of approximately 44,500 new cases per year with approximately 10,000 deaths per year. If diagnosed and treated early, when the cancer is still confined to the endometrium, cure can be achieved in approximately 95% of the cases by hysterectomy. Pap smears can show endometrial cancers but are effective in only 50% of the cases. For the remainder, abnormal vaginal bleeding is typically the first clinical sign of endometrial cancer.
  • Sarcoma of the uterus is a disease in which cancer (malignant) cells start growing in the muscles or other supporting tissues of the uterus.
  • Sarcoma of the uterus is different from cancer of the endometrium, a disease in which cancer cells start growing in the lining of the uterus .
  • Women who have received therapy with high-dose x-rays (external beam radiation therapy) to their pelvis are at a higher risk to develop sarcoma of the uterus. These x-rays are sometimes given to women to stop bleeding from the uterus.
  • sarcoma of the uterus is best treated when it is found (diagnosed) early.
  • Sarcoma of the uterus usually begins after menopause.
  • an internal pelvic examination is usually performed to detect any lumps or changes in shape of the pelvic organs.
  • a Pap test may also be performed, however because sarcoma of the uterus begins inside the organ, this cancer is not usually detected by the Pap test.
  • a dilation and curettage may also be performed and a biopsy sample taken and examined microscopically.
  • Steroid binding proteins including uteroglobin and CC10, are a class of proteins which bind steroids along with methylsulfonyl metabolites of polychlorinated biphenyls. The exact function of members of this class of protein is uncertain. However, uteroglobin has been shown to inhibit PLA 2 mediated responses.
  • hESF I, II, and III Human Endometrial Specific Steroid-Binding Factors I, II and III.
  • Methods for utilizing these genes and gene products in research and diagnostic and clinical arts are disclosed.
  • methods for detecting mutations in the hESFI, II or III gene or altered protein expression resulting from a mutant gene are indicated to be useful in diagnosing susceptibility to asthma and endometrial cancer.
  • human mammoglobin homolog A gene and gene product homologous to uteroglobin and very similar to hESF III, referred to as human mammoglobin homolog or HGH, is also described in WO 99/19487.
  • the human mammoglobin homolog is suggested to be useful for the diagnosis, prevention and treatment of neoplastic disorders and endometriosis .
  • ESBPIII hESF III
  • ESBPIII refers, among other things, to native protein expressed by the gene comprising the polynucleotide sequence of SEQ ID NO:l.
  • the amino acid sequence of a polypeptide encoded by SEQ ID NO:l is depicted herein as SEQ ID NO: 2.
  • SEQ ID NO: 2 The amino acid sequence of a polypeptide encoded by SEQ ID NO:l is depicted herein as SEQ ID NO: 2.
  • SEQ ID NO: 2 the native mRNA encoded by the gene comprising the polynucleotide sequence of SEQ ID NO:l or levels of the gene comprising the polynucleotide sequence of SEQ ID NO:l.
  • a method of diagnosing a metastatic gynecologic cancer in a patient which is not known to have metastasized by identifying a human patient suspected of having a gynecologic cancer that has metastasized; analyzing a sample of cells, tissues, or bodily fluid from such patient for ESBPIII; and comparing the ESBPIII levels in such cells, tissues, or bodily fluid with levels of ESBPIII in preferably the same cells, tissues, or bodily fluid type of a normal human control, wherein an increase in ESBPIII levels in the patient versus the normal human control is associated with a gynecologic cancer which has metastasized.
  • Also provided by the invention is a method of staging gynecologic cancers in a human by identifying a human patient having a gynecologic cancer; analyzing a sample of cells, tissues, or bodily fluid from such patient for ESBPIII; comparing ESBPIII levels in such cells, tissues, or bodily fluid with levels of ESBPIII in preferably the same cells, tissues, or bodily fluid type of a normal human control, wherein an increase in ESBPIII levels in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of ESBPIII is associated with a cancer which is regressing or in remission.
  • the method comprises identifying a human patient having such cancer that is not known to have metastasized; periodically analyzing a sample of cells, tissues, or bodily fluid from such patient for ESBPIII; comparing the ESBPIII levels in such cells, tissue, or bodily fluid with levels of ESBPIII in preferably the same cells, tissues, or bodily fluid type of a normal human control, wherein an increase in ESBPIII levels in the patient versus the normal human control is associated with a cancer which has metastasized.
  • the method comprises identifying a human patient having a gynecologic cancer; periodically analyzing a sample of cells, tissues, or bodily fluid from such patient for ESBPIII; comparing the ESBPIII levels in such cells, tissue, or bodily fluid with levels of ESBPIII in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in ESBPIII levels in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of ESBPIII is associated with a cancer which is regressing or in remission.
  • antibodies which specifically bind ESBPIII or fragments of such antibodies which can be used to detect or image localization of ESBPIII in a patient for the purpose of detecting or diagnosing a disease or condition.
  • Such antibodies can be polyclonal, monoclonal, or omniclonal or prepared by molecular biology techniques.
  • the term "antibody”, as used herein and throughout the instant specification is also meant to include aptamers and single- stranded oligonucleotides such as those derived from an in vitro evolution protocol referred to as SELEX and well known to those skilled in the art.
  • Antibodies can be labeled with a variety of detectable labels including, but not limited to, radioisotopes and paramagnetic metals.
  • antibodies or fragments thereof can also be used as therapeutic agents in the treatment of diseases characterized by expression of a ESBPIII.
  • the antibody can be used without or with derivatization to a cytotoxic agent such as a radioisotope, enzyme, toxin, drug or a prodrug.
  • the present invention relates to diagnostic assays and methods, both quantitative and qualitative for detecting, diagnosing, monitoring, staging and prognosticating cancers by comparing levels of ESBPIII with those of ESBPIII in a normal human control.
  • levels of ESBPIII as used herein means levels of the native protein expressed by the gene comprising the polynucleotide sequence of SEQ ID NO: 1. The protein encoded by this polynucleotide is depicted in SEQ ID NO: 2.
  • levels of ESBPIII means levels of the native mRNA encoded by the gene comprising the polynucleotide sequence of SEQ ID NO: 1 or levels of the gene comprising the polynucleotide sequence of SEQ ID NO:l. Such levels are preferably measured in at least one of cells, tissues and/or bodily fluids, including determination of normal and abnormal levels.
  • a diagnostic assay in accordance with the invention for diagnosing overexpression of ESBPIII protein compared to normal control bodily fluids, cells, or tissue samples may be used to diagnose the presence of cancers, in particular gynecologic cancers including breast, uterine, ovarian and endometrial cancer.
  • All the methods of the present invention may optionally include measuring levels of other cancer markers as well as ESBPIII.
  • Other cancer markers, in addition to ESBPIII, useful in the present invention will depend on the cancer being tested and are known to those of skill in the art. Diagnostic Assays
  • the present invention provides methods for diagnosing the presence of a gynecologic cancer such as uterine, breast, endometrial or ovarian cancer by analyzing for changes in levels of ESBPIII in cells, tissues or bodily fluids compared with levels of ESBPIII in cells, tissues or bodily fluids of preferably the same type from a normal human control, wherein a change in levels of ESBPIII in the patient versus the normal human control is associated with the presence of a gynecologic cancer .
  • a gynecologic cancer such as uterine, breast, endometrial or ovarian cancer
  • a positive result indicating the patient being tested has cancer is one in which cells, tissues or bodily fluid levels of a cancer marker, such as ESBPIII, are at least two times higher, and most preferably are at least five times higher, than in preferably the same cells, tissues or bodily fluid of a normal human control.
  • a cancer marker such as ESBPIII
  • the present invention also provides a method of diagnosing the onset of metastasis of gynecologic cancers in a patient having a gynecologic cancer which has not yet metastasized.
  • a human cancer patient suspected of having a gynecologic cancer which may have metastasized (but which was not previously known to have metastasized) is identified. This is accomplished by a variety of means known to those of skill in the art.
  • determining the presence of ESBPIII levels in cells, tissues or bodily fluid is particularly useful for discriminating between a gynecologic cancer which has not metastasized and a gynecologic cancer which has metastasized.
  • Existing techniques have difficulty discriminating between gynecologic cancers which have metastasized and gynecologic cancers which have not metastasized.
  • proper treatment selection is often dependent upon such knowledge.
  • the cancer marker level measured in such cells, tissues or bodily fluid is ESBPIII.
  • Measure ESBPIII levels in a human patient are compared with levels of ESBPIII in preferably the same cells, tissue or bodily fluid type of a normal human control. That is, if the cancer marker being observed is ESBPIII in serum, this level is preferably compared with the level of ESBPIII in serum of a normal human control.
  • An increase in the ESBPIII in the patient versus the normal human control is associated with a gynecologic cancer which has metastasized.
  • a positive result indicating the cancer in the patient being tested or monitored has metastasized is one in which levels of a cancer marker such as ESBPIII in cells, tissues or bodily fluid from the patient are at least two times higher, and most preferably are at least five times higher, than in preferably the same cells, tissues or bodily fluid of a normal human control.
  • a cancer marker such as ESBPIII in cells, tissues or bodily fluid from the patient are at least two times higher, and most preferably are at least five times higher, than in preferably the same cells, tissues or bodily fluid of a normal human control.
  • Normal human control as used herein includes a human patient without cancer and/or non cancerous samples from the patient; in the methods for diagnosing or monitoring for metastasis, normal human control may preferably also include samples from a human patient that is determined by reliable methods to have uterine, breast, ovarian or endometrial cancer which has not metastasized. Staging
  • the invention also provides a method of staging a gynecologic cancer in a human patient.
  • the method comprises identifying a human patient having such cancer and analyzing cells, tissues or bodily fluid from the patient for ESBPIII.
  • the measured ESBPIII levels in such cells, tissues or bodily fluid from the patient are then compared with levels of ESBPIII in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in ESBPIII levels in the human patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of ESBPIII is associated with a cancer which is regressing or in remission.
  • the method comprises identifying a human patient having such cancer that is not known to have metastasized; periodically analyzing cells, tissues or bodily fluid from such human patient for ESBPIII; and comparing the ESBPIII levels in such cells, tissues or bodily fluid with levels of ESBPIII in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in ESBPIII levels in the human patient versus the normal human control is associated with a cancer which has metastasized.
  • the method comprises identifying a human patient having such cancer; periodically analyzing cells, tissues or bodily fluid from such human patient for ESBPIII; comparing the ESBPIII levels in such cells, tissues or bodily fluid with levels of ESBPIII in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in ESBPIII levels in the human patient versus the normal human control is associated with a cancer which is progressing in stage and a decrease in the levels of ESBPIII is associated with a cancer which is regressing in stage or in remission.
  • Assay Technisj ⁇ es Assay techniques that can be used to determine levels of gene expression (including protein levels), such as ESBPIII in the present invention, in a sample derived from a patient are well known to those of skill in the art.
  • Such assay methods include, without limitation, radioimmunoassays , reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in si tu hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches: two-dimensional gel electrophoresis (2D electrophoresis) and non-gel based approaches such as mass spectrometry or protein interaction profiling.
  • RT-PCR reverse transcriptase PCR
  • immunohistochemistry assays in si tu hybridization assays
  • competitive-binding assays Western Blot analyses
  • ELISA assays and proteomic approaches two-dimensional gel electrophoresis (2D electrophoresis) and non-gel based approaches such as mass spectrometry or protein interaction profiling.
  • ELISAs are frequently preferred to diagnose a gene's expressed protein in biological fluids.
  • An ELISA assay initially comprises preparing an antibody, if not readily available from a commercial source, specific to ESBPIII, preferably a monoclonal antibody.
  • a reporter antibody generally is prepared which also binds specifically to ESBPIII.
  • the reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent.
  • a detectable reagent such as radioactive, fluorescent or enzymatic reagent.
  • horseradish peroxidase enzyme or alkaline phosphatase are routinely used as detectable reagents .
  • antibody specific to ESBPIII is incubated on a solid support, e.g. a polystyrene dish, that binds the antibody. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin.
  • a non-specific protein such as bovine serum albumin.
  • the sample to be analyzed is incubated in the dish, during which time ESBPIII binds to the specific antibody attached to the polystyrene dish. Unbound sample is washed out with buffer.
  • a reporter antibody specifically directed to ESBPIII and linked to a detectable reagent such as horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to ESBPIII.
  • Unattached reporter antibody is then washed out.
  • Reagents for peroxidase activity including a colorimetric substrate are then added to the dish.
  • Immobilized peroxidase, linked to ESBPIII antibodies, produces a colored reaction product.
  • the amount of color developed in a given time period is proportional to the amount of ESBPIII protein present in the sample. Quantitative results typically are obtained by reference to a standard curve.
  • a competition assay can also be employed wherein antibodies specific to ESBPIII are attached to a solid support and labeled ESBPIII and a sample derived from the host are passed over the solid support. The amount of label detected which is attached to the solid support can be correlated to a quantity of ESBPIII in the sample.
  • Nucleic acid methods can also be used to detect ESBPIII mRNA as a marker for gynecologic cancers such as uterine, breast, endometrial and ovarian cancer.
  • Polymerase chain reaction (PCR) and other nucleic acid methods such as ligase chain reaction (LCR) and nucleic acid sequence based amplification (NASABA) , can be used to detect malignant cells for diagnosis and monitoring of various malignancies.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASABA nucleic acid sequence based amplification
  • RT-PCR reverse-transcriptase PCR
  • RT-PCR is a powerful technique which can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species.
  • RT-PCR an mRNA species is first reverse transcribed to complementary DNA (cDNA) with use of the enzyme reverse transcriptase; the cDNA is then amplified as in a standard PCR reaction.
  • cDNA complementary DNA
  • RT-PCR can thus reveal by amplification the presence of a single species of mRNA. Accordingly, if the mRNA is highly specific for the cell that produces it, RT-PCR can be used to identify the presence of a specific type of cell.
  • Hybridization to clones or oligonucleotides arrayed on a solid support can be used to detect the expression of and quantitate the level of expression of that gene.
  • a cDNA encoding the ESBPIII gene is fixed to a substrate.
  • the substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon or plastic.
  • At least a portion of the DNA encoding the ESBPIII gene is attached to the substrate and then incubated with the analyte, which may be RNA or a complementary DNA (cDNA) copy of the RNA, isolated from the tissue of interest.
  • Hybridization between the substrate bound DNA and the analyte can be detected and quantitated by several means including, but not limited to, radioactive labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid. Quantitation of the level of gene expression can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards. The standards can be obtained by in vi tro transcription of the target gene, quantitating the yield, and then using that material to generate a standard curve.
  • 2D electrophoresis is a technique well known to those in the art. Isolation of individual proteins from a sample such as serum is accomplished using sequential separation of proteins by different characteristics usually on polyacrylamide gels. First, proteins are separated by size using an electric current. The current acts uniformly on all proteins, so smaller proteins move farther on the gel than larger proteins . The second dimension applies a current perpendicular to the first and separates proteins not on the basis of size but on the specific electric charge carried by each protein. Since no two proteins with different sequences are identical on the basis of both size and charge, the result of a 2D separation is a square gel in which each protein occupies a unique spot. Analysis of the spots with chemical or antibody probes, or subsequent protein microsequencing can reveal the relative abundance of a given protein and the identity of the proteins in the sample.
  • Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof.
  • Blood can include whole blood, plasma, serum or any derivative of blood.
  • Antibodies against ESBPIII can also be used in vivo in patients suspected of suffering from gynecologic cancers such as ovarian, breast, endometrial and uterine cancer. Specifically, antibodies against a ESBPIII can be injected into a patient suspected of having a gynecologic cancer for diagnostic and/or therapeutic purposes .
  • the use of antibodies for in vivo diagnosis is well known in the art.
  • antibody-chelators labeled with Indium-Ill have been described for use in the radioimmunoscintographic imaging of carcinoembryonic antigen expressing tumors (Sumerdon et al . Nucl. Med. Biol . 1990 17:247-254).
  • these antibody-chelators have been used in detecting tumors in patients suspected of having recurrent colorectal cancer
  • Antibodies with paramagnetic ions as labels for use in magnetic resonance imaging have also been described (Lauffer, R.B. Magnetic Resonance in Medicine 1991 22:339-342).
  • Antibodies directed against ESBPIII can be used in a similar manner. Labeled antibodies against ESBPIII can be injected into patients suspected of having a gynecologic cancer for the purpose of diagnosing or staging of the disease status of the patient. The label used will be selected m accordance with the imaging modality to be used.
  • radioactive labels such as Indium-Ill, Technet ⁇ um-99m or Iod ⁇ ne-131 can be used for planar scans or single photon emission computed tomography (SPECT) .
  • Positron emitting labels such as Fluor ⁇ ne-19 can be used m positron emission tomography.
  • Paramagnetic ions such as Gadlinium (III) or Manganese (II) can be used in magnetic resonance imaging (MRI) . Localization of the label permits determination of the spread of the cancer. The amount of label within an organ or tissue also allows determination of the presence or absence of cancer m that organ or tissue.
  • an antibody against ESBPIII can also have a therapeutic benefit.
  • the antibody may exert its therapeutic effect alone.
  • the antibody is conjugated to a cytotoxic agent such as a drug, toxin or radionuclide to enhance its therapeutic effect.
  • Drug monoclonal antibodies have been described m the art for example by Garnett and Baldwin, Cancer Research 1986 46:2407-2412. The use of toxins conjugated to monoclonal antibodies for the therapy of various cancers has also been described by Pastan et al . Cell 1986 47:641-648.
  • Yttr ⁇ um-90 labeled monoclonal antibodies have been described for maximization of dose delivered to the tumor while limiting toxicity to normal tissues (Goodwin and Meares Cancer Supplement 1997 80:2675-2680) .
  • Other cytotoxic radionuclides including, but not limited to Copper-67, Iodine- 131 and Rhen ⁇ um-186 can also be used for labeling of antibodies against ESBPIII.
  • Antibodies which can be used in these in vivo methods include both polyclonal, monoclonal or omniclonal antibodies and antibodies prepared via molecular biology techniques. Antibody fragments and aptamers and single-stranded oligonucleotides such as those derived from an in vi tro evolution protocol referred to as SELEX and well known to those skilled in the art can also be used.
  • Real-Time quantitative PCR with fluorescent Taqman probes is a quantitation detection system utilizing the 5'- 3' nuclease activity of Taq DNA polymerase.
  • the method uses an internal fluorescent oligonucleotide probe (Taqman) labeled with a 5' reporter dye and a downstream, 3' quencher dye.
  • Taqman internal fluorescent oligonucleotide probe
  • the 5' -3' nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) .
  • Amplification of an endogenous control is used to standardize the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency.
  • Either cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S ribosomal RNA (rRNA) is used as this endogenous control.
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • rRNA 18S ribosomal RNA
  • Table 1 The absolute numbers depicted in Table 1 are relative levels of expression of ESBPIII in 12 normal different tissues. All the values are compared to normal mammary gland (calibrator) . These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.
  • Table 1 The absolute numbers in Table 1 were obtained analyzing pools of samples of a particular tissue from different individuals. They can not be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 2.
  • Table 2 The absolute numbers depicted in Table 2 are relative levels of expression of ESBPIII in 55 pairs of matching samples, ovarian cancer samples from 5 different individuals, and normal ovarian samples from 5 different individuals. All the values are compared to normal mammary gland (calibrator) . A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. Table 2: Relative Levels of ESBPIII Expression in Pooled Samples
  • the levels of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for the cancer stage (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent) .
  • Table 2 shows overexpression of ESBPIII in 6 primary endometrial cancer tissues compared with their respective normal adjacent (endometrium samples #2, 3, 4, 7, 9 and 10) . There was overexpression in the cancer tissue for 54.54% of the endometrial matching samples tested (total of 11 endometrium matching samples) .
  • ESBPIII is differentially expressed in the four matching samples for uterine cancer. All four samples analyzed showed overexpression in cancer. Of twelve breast cancer matching samples analyzed, five showed underexpression of ESBPIII (#2, 6, 9, 10 and 12) in cancer, whereas five had higher levels of ESBPIII in cancer compared to the normal adjacent tissue (#1, 3, 7, 8, and 11) . Two of the breast matching samples do not show expression of ESBPIII mRNA.
  • ESBPIII is differentially expressed in the two matching samples for ovarian cancer.
  • Sample #1 shows upregulation for the mRNA of ESBPIII in cancer, whereas sample #2 shows overexpression in the normal adjacent tissue.
  • ten additional samples for ovary were analyzed including five cancer samples and five normal ovary tissue samples from different individuals. Expression of ESBPIII mRNA was observed in three of the cancer samples (#1, 2, and 3) . The median expression in the ovary cancer samples was 28.1, whereas expression in the normal ovary samples was 0.
  • ESBPIII being a diagnostic marker for gynecologic cancers including uterine, endometrial, breast, and ovarian cancer.

Abstract

La présente invention concerne un nouveau procédé permettant de détecter, de diagnostiquer, de contrôler, d'établir le bilan d'extension, de pronostiquer, de visualiser, et de traiter des cancers gynécologiques, et notamment les cancers de l'utérus, du sein, de l'endomètre, et des ovaires.
PCT/US1999/022753 1998-10-02 1999-09-30 Nouveau procede pour diagnostiquer, controler, etablir le bilan d'extension, visualiser, et traiter des cancers gynecologiques WO2000020044A1 (fr)

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CA002345285A CA2345285A1 (fr) 1998-10-02 1999-09-30 Nouveau procede pour diagnostiquer, controler, etablir le bilan d'extension, visualiser, et traiter des cancers gynecologiques
US09/806,302 US7014996B1 (en) 1998-10-02 1999-09-30 Method of diagnosing, monitoring, staging, imaging and treating gynecologic cancers
JP2000573402A JP2002527723A (ja) 1998-10-02 1999-09-30 婦人科癌を診断、監視、病期分類、イメージング及び治療する新規な方法
EP99951679A EP1169066A4 (fr) 1998-10-02 1999-09-30 Nouveau procede pour diagnostiquer, controler, etablir le bilan d'extension, visualiser, et traiter des cancers gynecologiques

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US7014996B1 (en) 1998-10-02 2006-03-21 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating gynecologic cancers
EP1700867A2 (fr) * 2000-06-02 2006-09-13 Genentech, Inc. Polypeptide secrétée et transmembranaire, ainsi que les acides nucléiques qui les codent

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US7014996B1 (en) 1998-10-02 2006-03-21 Diadexus, Inc. Method of diagnosing, monitoring, staging, imaging and treating gynecologic cancers
EP1700867A2 (fr) * 2000-06-02 2006-09-13 Genentech, Inc. Polypeptide secrétée et transmembranaire, ainsi que les acides nucléiques qui les codent
EP1700867A3 (fr) * 2000-06-02 2006-09-20 Genentech, Inc. Polypeptide secrétée et transmembranaire, ainsi que les acides nucléiques qui les codent
WO2002068645A2 (fr) * 2000-11-20 2002-09-06 Diadexus, Inc. Compositions et procedes concernant des genes et des proteines specifiques pour le sein
WO2002068645A3 (fr) * 2000-11-20 2003-10-30 Diadexus Inc Compositions et procedes concernant des genes et des proteines specifiques pour le sein

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JP2002527723A (ja) 2002-08-27
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