WO2001088537A1 - Procede de diagnostic destine a balayer des niveaux de proteine regulatrice de complement - Google Patents

Procede de diagnostic destine a balayer des niveaux de proteine regulatrice de complement Download PDF

Info

Publication number
WO2001088537A1
WO2001088537A1 PCT/US2001/014769 US0114769W WO0188537A1 WO 2001088537 A1 WO2001088537 A1 WO 2001088537A1 US 0114769 W US0114769 W US 0114769W WO 0188537 A1 WO0188537 A1 WO 0188537A1
Authority
WO
WIPO (PCT)
Prior art keywords
crp
antibody
detectable label
patient
neoplasia
Prior art date
Application number
PCT/US2001/014769
Other languages
English (en)
Inventor
Mark G. Martens
Anil K. Kaul
Rashmi Kaul
Original Assignee
Martens Mark G
Kaul Anil K
Rashmi Kaul
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Martens Mark G, Kaul Anil K, Rashmi Kaul filed Critical Martens Mark G
Priority to AU2001261259A priority Critical patent/AU2001261259A1/en
Publication of WO2001088537A1 publication Critical patent/WO2001088537A1/fr
Priority to US10/292,130 priority patent/US20030129677A1/en

Links

Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • invasive carcinomas can only be life threatening when it become invasive, at which point it carries potential for spreading and metastasis. It is critical to distinguish invasive carcinomas from noninvasive lesions, including ductal carcinoma in situ (DCIS), which represents an early, pre-invasive stage in the development of invasive breast carcinoma. While this distinction is usually made based on histologic evaluation alone, in a small but significant number of cases, accurate diagnosis may be impossible, particularly in the context of core needle biopsies. Yaziji H, et al. Adv Anat. Pathol 2000 Mar; 7(2): 100-9. A standardized pathologic staging and grading system, however, does not exist for breast cancer.
  • DCIS ductal carcinoma in situ
  • Endomerrial cancer is the most common invasive neoplasm of the female genital tract and is ranked fourth in age-adjusted cancer incidence among women in the United States (Parker, S. L., et al, Cancer statistics, 47:5-27 (1997)). In 1997, uterine corpus cancer accounted for about 5.8% of female cancer incidence (an estimated 35,000 cases) and 2.2% of female cancer mortality (an estimated 6000 deaths) in the United States (Mass, H., Epidemiologiebgynakêtêtêtêtêtologier Tumoren, in Wulff, K. H., Schmidt-Mattiesen, H. (eds.), ceremoni der
  • estrogen and progesterone receptors The most extensively studied biologic markers in endometrial carcinoma are estrogen and progesterone receptors. It has been shown that high levels of estrogen and progesterone receptors directly correlate with better tumor differentiation, less myometrial invasion, and a lower incidence of nodal metastases and that they independently predict better survival (Zaino, R. J., et al, Gynecol. Oncol., 16, 196-208 (1983); Creasman, W. T., Cancer, 71 (Suppl.): 1467-1470 (1993)).
  • adenomatous hyperplasia As a precursor to well-differentiated endometrial cancer has increased yearly. A very careful histologic examination and regular follow- up visits are prerequisites for this method of treatment, since hyperplasia and cancer can coexist in the same patient.
  • the present invention provides a method for diagnosing a predisposition for neoplasia in a patient by contacting a biological sample potentially comprising a complement regulatory protein (CRP) from the patient with a anti- CRP antibody to form an CRP-antibody complex; and measuring the quantity of CRP-antibody complex in the biological sample as compared to a normal control level, wherein the quantity of CRP-antibody complex as compared to a normal control is indicative for a predisposition for neoplasia.
  • CRP complement regulatory protein
  • the CRP may be cell membrane-associated or secretory.
  • the neoplasia may be malignant.
  • the CRP may be CD35 (complement receptor type 1, CR1), CD46 (membrane cofactor protein, MCP), CD55 (also decay accelerating factor, DAF) or CD59 (membrane attack complex inhibitory factor, MACIF).
  • the anti-CRP antibody may be immobilized on a solid surface.
  • the anti-CRP antibody may be a detectable label or a binding site for a detectable label to form detectable complexes.
  • the detectable label may be an enzyme label, or a fluorogenic compound.
  • the binding site for the detectable label may be biotin, avidin or streptavidin.
  • the biological sample may be a physiological fluid or tissue sample, such as a tissue sample from a breast, cervical, ovarian, prostate or endometrial tumor.
  • the present invention also provides a method for diagnosing a predisposition for neoplasia in a patient by contacting a biological sample potentially comprising CRP from the patient with a solid surface having immobilized thereon anti-CRP antibodies, so that the CRP binds to the anti- CRP antibodies; contacting labeled CRP, which comprises a detectable label or a binding site for a detectable label, with the solid surface, so that the labeled CRP binds to free antibodies on the solid surface to form detectable complexes; and detecting the complexes, wherein the quantity of the complexes is inversely proportional to the amount of CRP in the biological sample, wherein the quantity of CRP-antibody complex as compared to a normal control is indicative for a predisposition for neoplasia.
  • the neoplasia may be malignant.
  • the CRP may be CD35, CD46, CD55 or CD59.
  • the detectable label may be an enzyme label or a fluorogenic compound.
  • the binding site for the detectable label may be biotin, avidin or streptavidin.
  • the biological sample may be a physiological fluid or tissue sample, such as a tissue sample from a breast, cervical, ovarian, prostate or endometrial tumor.
  • the present invention also provides an article of manufacture for diagnosing a predisposition for neoplasia in a patient comprising packaging material, and a diagnostic kit and instructions within the packaging material, wherein the diagnostic kit comprises anti-CRP antibody, and a means for measuring the quantity of CRP-antibody complexes in a biological sample from a patient wherein the quantity of CRP-antibody complex as compared to a normal control is indicative for a predisposition for neoplasia, and wherein the instructions that indicate that the diagnostic kit can be used to diagnose a predisposition for neoplasia in a patient.
  • the neoplasia may be malignant.
  • the CRP may be CD35, CD46, CD55 or CD59.
  • the kit may also contain a solid substrate.
  • the anti-CRP antibody of the kit may be immobilized on a solid surface.
  • the anti-CRP antibody may be a detectable label or a binding site for a detectable label to form detectable complexes.
  • the detectable label may be an enzyme label.
  • the detectable label may be a fluorogenic compound.
  • the binding site for the detectable label may be biotin, avidin or streptavidin.
  • the biological sample may be a physiological fluid or tissue sample, such as a tissue sample from a breast, cervical, ovarian, prostate or endometrial tumor.
  • the present invention further provides a method for early diagnosis of a premalignant lesion in a patient comprising contacting a biological sample potentially comprising a cell membrane-associated complement regulatory protein (CRP) from the patient with a anti-CRP antibody to form an CRP- antibody complex; and measuring the quantity of CRP-antibody complex in the biological fluid as compared to a normal control level, wherein the quantity of CRP-antibody complex as compared to a normal control is indicative for a predisposition for developing a malignant lesion.
  • CRP complement regulatory protein
  • the present invention provides a method for determining the prognosis of a malignant lesion in a patient comprising contacting a biological sample potentially comprising a cell membrane-associated complement regulatory protein (CRP) from the patient with a anti-CRP antibody to form an CRP-antibody complex; and measuring the quantity of CRP-antibody complex in the biological fluid as compared to a normal control level, wherein the quantity of CRP-antibody complex as compared to a normal control is predictive of the likelihood of success of a particular course of treatment of the malignant lesion.
  • CRP complement regulatory protein
  • Figure 1 Graphic representation of amount of CR1 expression in benign and malignant endometrial tissue samples.
  • Figure 2 Complement MCP: Graphic representation of amount of MCP expression in benign and malignant endometrial tissues samples.
  • Figure 3 Complement DAF: Graphic representation of amount of DAF expression in benign and malignant endometrial tissue samples.
  • One of the physiologic roles of the complement system is the lysis of foreign cells, including tumor cells.
  • This cytolytic process is activated by the interaction of more than 20 plasma proteins in two pathways; the classical and alternative pathways.
  • the classical pathway is triggered by antigen-antibody complexes, while the alternative pathway is triggered by foreign surfaces including tumor cells (Kumar, S., et al, Cancer Res. 53, 348-353 (1993)).
  • Activation of either pathway leads to the formation of a biomolecular complex, designated C3 convertase (Lublin, D. M., et al. Annu. Rev. Immunol., 7, 35-58 (1989)).
  • CRPs cell membrane-associated complement regulatory proteins
  • CRPs that operate at the C3 convertase level are known as the regulators of complement activation (RCA) group (Koretz, K., et al, Br. J. Cancer. 68, 926-931 (1993)).
  • Complement receptor type 1 (CRl, CD35), membrane cofactor protein (MCP, CD46), and decay-accelerating factor (DAF, CD55) are members of the RCA group of proteins.
  • CRl is a polymorphic (190-280 kDa) receptor whose primary ligands are C3b and C4b. CRl has limited tissue distribution, and is found primarily on hematopoietic cells in peripheral blood.
  • MCP is also a widely distributed C3b/C4b-binding dimeric protein with molecular masses of 50-58 kDa (lower form) and 59-68 kDa (upper form), and serves as a cofactor for the plasma serine protease factor I, which irreversibly inactivates C3b and C4b (Bjorge, L., et al. Cancer Immunol. Tmmunother., 42, 185-192 (1996)).
  • DAF is a 70 kDa glycolipid-anchored membrane-bound CRP with a wide tissue distribution that possesses regulatory activity for the C3 convertases.
  • DAF protects the host tissue by inhibiting assembly (C2a with C4b or Bb with C3b) and/or promoting dissociation of preformed C3 convertases on the same cell (Hourcade, D., et al, Adv. Tmmunol, 45, 381-416 (1989)).
  • the CRP that operates at the MAC level is known as protectin [membrane attack complex inhibitory factor (MACIF), CD59].
  • MACIF membrane attack complex inhibitory factor
  • Protectin is an 18- to 20-kDa phosphatidylinositol-anchored glycoprotein in the cell membrane.
  • MACIF inhibits formation of terminal MAC on complement by binding to C8 and C9 molecules and disturbing the C8:C9 ratio in the MAC (Koretz, K., et /., B ⁇ JL Cancer, 68, 926-931 (1993)).
  • CRPs collectively play a leading role in the immune system both in identification of and in removal of foreign agents, including microorganisms and tumor cells (Bjorge, L., et al. , Cancer Tmmunol. Tmmunother., 42, 185-192 (1996)).
  • Decay accelerating factor is a cell-associated complement- regulatory protein that inhibits complement activation and thus protects the autologous tissues from the cytotoxic effects of complement.
  • DAF has been previously associated with paroxysmal nocturnal hemoglobinuria (PNH), as decreased expression of DAF is correlated with presence of the disease.
  • PNH paroxysmal nocturnal hemoglobinuria
  • RBCs red blood cells
  • DAF is associated with the Cromer blood group antigens, which are located at various positions along the DAF molecule. It has been characterized as a glycosylphosphatidylinositol (GPI)-anchored membrane protein that inhibits both the classical and alternative pathways of complement activation, its chromosomal location has been identified as band q32.8,9 of human chromosome 1, and its sequence has been reported (Medof, M.E., et al. Proc. Natl. Acad. Sci. USA (1987) 84: 2007-11). In conjunction with CD59 (protectin), CD46 (membrane cofactor protein), and CD35 (complement receptor type 1 (CRl)), it participates in the regulation of complement activity in the immune response.
  • GPI glycosylphosphatidylinositol
  • CD55 CD46
  • MCP CD35
  • CD59 Membrane-associated complement regulatory proteins such as CD55 (DAF), CD46 (MCP), CD35 (CRl) and CD59, which show an important mechanism of self protection and render autologous cells insensitive to the action of complement that appears to be overexpressed on certain tumors.
  • DAF CRP-decay accelerating factor
  • CRP's like CR-1, DAF, MCP and CD59 are expressed in the human genital tract tissues including the endometrium and the cervix and these CRP's in uterine adenocarcinoma samples were found in vivo on breast tumors as well as breast cancer cell lines (Hakulinen et al, 1997, Lab, vest. : 71(6):820-827).
  • Cells expressing low levels of CRP's can be killed in the presence of complement; cells with high level of CRP's are resistant to complement-mediated killing, (Kaul et al, 1995, Tnfect. Immun., 64(2):611- 615).
  • ovariectomy and/or anti-estrogenic and antiprogestational drugs have been successfully used in the treatment of breast cancer (lino et al, 1990, Tn: Regulatory Mechanisms in Breast Cancer, Edited by Lippman ME and Dickson RB. Kluwer Acad. Pub. Norwell, MS pp 221-238).
  • the breast has a tightly regulated pattern of growth that is primarily under the control of steroid hormones.
  • High-dose progestin therapy is receiving a renewed interest for the treatment of advanced breast cancer, either as a first-line or second-line endocrine therapy. This renewed interest is due to low toxicities associated with high-dose progestins and to an efficacy similar to that of tamoxifen (Sedlacek S. Overview of megestral acetate treatment of breast cancer. Sem. Oncol., 15:3-13).
  • Progestins have been observed to be anti- proliferative (Poulin et al, 1989, Breast Cancer Res. Treatment, 13:265-276), behaving similar to antiestrogen tamoxifen by inducing cells to accumulate in the Gl of cell cycle (Sutherland et al, 1988, Cancer Res., 48:5084-5091).
  • Cytokines and Breast Cancer Multifunctional cytokines play important and only partially defined roles in mammary tumor development and progression. Over the last few years, the role of cytokines in cancer has been the subject of numerous investigations. It has been reported that, in the mammary gland, cytokines play a role in growth and differentiation, extracellular matrix production, angiogenesis and as immunomodulating factors. Recently, it has been shown that epithelial cells of the normal mammary gland produce constitutively IL-6, IL-8 and a non-secreted form of TNF.
  • TNF- ⁇ and IFN- ⁇ function as effector molecules released by activated cytotoxic CD 8+ and CD4+, lymphocytes and NK cells to reject tumor cells (Lee et al. 1996, J. Tmmunol., 157(5):1919-1925; Goedegebuure et al, 1997, Cell Tmmunol .. 175(2):150-156; Kaplan et al, 1998, Proc. Natl Acad. Sci. TTSA. 95(13):7556-7561; Seo et al, 1998, J. T munol., 161(8):4138-4145; Suzuki et al, 1 99, Cancer Lett..
  • TNF- ⁇ and IFN- ⁇ therefore might be present at various concentrations in tumor environment infiltrated by T lymphocytes and NK cells.
  • TNF- ⁇ belongs to a growing family of molecules that have fundamental roles in immune and development networks.
  • TNF- ⁇ is expressed by numerous cell types as a 26-kDa type II transmembrane cell surface proteins.
  • TNF- ⁇ is expressed by numerous cell types as a 26-kDa type II transmembrane (mTNF- ⁇ ) and as a 17-kDa soluble form (sTNF- ⁇ ) which results from the shedding of mTNF- ⁇ by metalloproteinases.
  • mTNF- ⁇ 26-kDa type II transmembrane
  • sTNF- ⁇ 17-kDa soluble form
  • sTNF- ⁇ is involved in the lysis of a wide range of normal, infected, or transformed cells.
  • mTNF- ⁇ acts in situations of juxtacrine intercellular signaling by killing tumoral and infected cells in a cell-to-cell contact-dependant manner (Caron et al, 1999, Eur. J. Tmmunol., 29:3588-3595).
  • IFN- ⁇ -producing tumor cells had reduced tumorigenicity and were rejected by syngeneic mice.
  • mice which rejected the IFN- ⁇ -producing tumor cells were challenged by mice which rejected the IFN- ⁇ -producing tumor cells.
  • Cultured lymphocytes derived from immunized mouse spleens had cytotoxic T cells activity against parental tumor cells, as well as against cells that produced IFN- ⁇ .
  • These findings indicate that the antitumor effects are mediated by cytotoxic T cells and, partly, by helper T cells, and that locally secreted IFN- ⁇ plays an important role in generating these effector cells (Teramura et al, 1993, .Tpn. J. Cancer Res., 84(6):689-696).
  • Vascular endothelium plays an important role in the pathophysiology of tumor metastasis.
  • ER-positive breast cancer cells lines such as MCF-7, produce growth factors that may act in an autocrine and/or paracrine fashion to influence the proliferation and responsiveness of breast cancer may actually be mediated by hormone-induced growth factors.
  • Progestins in the cytoplasm have been found to regular several intracellular effectors by increasing the levels of Stat5 and control the activity of key genes involved in breast cell fate (Jurianz et al, Mol. Tmmunol., Sept-Oct; 36(13-14):929-939).
  • cytokine regulation also needs to be studied during cytokine response to tumor cells.
  • TNF- ⁇ is a macrophage-derived cytokine that causes necrosis of tumors in experimental models. Although, TNF- ⁇ is well known for its antiproliferative action, the mechanisms explaining these phenotropic effects have not been well characterized. Important regulatory proteins such as CRP's and cytokines, whose expression may vary in tissue-specific ways, seem to work in concert with hormones to decide cell fate. It is becoming critically important that the subtleties of the mechanisms of action of hormones be clearly understood in breast tissues.
  • the present invention provides a method for assaying the presence or the level of CRP in a biological sample containing CRF to diagnose tumors that will likely progress to malignancy, as certain levels of CRF are predictive of tumor transformation into a malignant tumor.
  • the present invention further provides a method for determimng the prognosis of a malignant lesion in a patient, as tumors with certain CRF levels are more responsive to certain types of treatment.
  • Examples of immunoassays that can be employed to determine the relative or absolute amount of CRF in a biological sample include those assay methods, formats and kits disclosed in U.S. Pat. No. 5,516,639.
  • Examples of immunoassays that can be employed to determine the relative or absolute amount of CRF in a biological sample include those assay methods, formats and kits disclosed in U.S. Pat. No. 5,516,639.
  • CRF analytes may be distinguished from other sample components by reacting the analyte with a specific receptor for that analyte. Assays that utilize specific receptors to distinguish and quantify analytes are often called specific binding assays.
  • the analyte of the present invention may be detected using a variety of specific binding assay formats.
  • various direct-binding assays may be employed.
  • receptors such as antibodies or other binding proteins
  • the immobilized chemically cross- linked protein complexes are contacted with a sample containing the analyte of interest, which may be distinguished from other components found in the sample.
  • an antibody specific for a CRF can be immobilized on the surface of a solid substrate and used as a capture antibody to specifically bind to CRF in a biological fluid.
  • Suitable substrates include particulate substrates such as polystyrene beads, the wells of plastic microtiter plates, paper or synthetic fiber test strips and the like.
  • the immobilized antibody can then be contacted with the test sample to be assayed, e.g., with a biological fluid such as plasma, serum, tears, urine or the like.
  • a biological fluid such as plasma, serum, tears, urine or the like.
  • the resulting antibody-CRF binary complex can then be contacted with an anti-CRF antibody, such as rabbit anti-CRF serum.
  • the solid phase may be washed and then contacted with an indicator, such as a labeled conjugate.
  • the conjugate comprises an antibody, antibody fragment, binding protein or analyte depending on assay format, and the label is a florescent, enzymated, colorimetric, radiometric or other labeling molecule that is associated either directly or indirectly with the conjugate.
  • the label may be comprised of an enzymatic compound that produces florescence upon contact with a substrate.
  • an anti-CRF monoclonal antibody can be itself coupled to a detectable label of a binding site for a detectable label.
  • the antibodies can be labeled radioisotopically, e.g., by 125 I, or conjugated directly to a detector enzyme, e.g., alkaline phosphatase or horse radish peroxidase, or can be labeled indirectly with a binding site for a detectable label, e.g. , via biotinylation.
  • a detector enzyme e.g., alkaline phosphatase or horse radish peroxidase
  • the biotinylated antibody can then be detected by its ability to bind to a an avidin- linked enzyme. If the second antibody is biotinylated, a detector enzyme conjugated to avidin will be subsequently added.
  • the final step for detecting enzymes conjugated to monoclonal antibody or to avidin its the addition of a substrate appropriate for the enzyme to allow quantitative colorimetric detection of reaction product.
  • the value can be converted to frnol of CRF by reference to a standard curve generated in a control assay in which a standard extract of detergent-solubilized CRF is added in graded concentrations to the immobilized anti-CRF monoclonal antibody.
  • the present invention may use many other assay formats, such as competitive immunoassays, bead agglomeration assays and sandwich-type immunoassays, such as ELISA, as would be recognized by the art.
  • the solid phase containing immobilized chemically cross-linked protein complexes with specificity for a selected analyte is contacted with a sample presumably containing such analyte and with a specific competitive reagent.
  • the specific competitive reagent may be a labeled analog of the analyte.
  • the labeled analog competes with the sample analyte for binding to a receptor immobilized on the solid phase.
  • an analyte may be coupled to a solid phase and contacted with a sample and with a specific competitive cross-linked protein reagent, for example, a labeled receptor for the analyte.
  • sample analyte competes with solid phase analyte for binding with soluble labeled cross- linked receptor.
  • the amount of label bound to the solid phase after washing provides an indication of the levels of analyte in the sample. That is, the amount of analyte in a sample is inversely proportional to the amount of analyte in the sample.
  • kits for detecting or determining the presence of CRF in a biological sample.
  • Immobilized antibodies and labeled antibodies are conveniently packaged in kit form, wherein two or more of the various immunoreagents will be separately packaged in preselected amounts, within the outer packaging of the kit, which may be a box, envelope, or the like.
  • the packaging also preferably comprises instruction means, such as a printed insert, a label, a tag, a cassette tape and the like, instructing the user in the practice of the assay format.
  • one such diagnostic kit for detecting or determining the presence of CRF comprises packaging containing, separately packaged: (a) a solid surface, such as a fibrous test strip, a multi-well microliter plate, a test tube, or beads, having bound thereto antibodies to CRF; and (b) a known amount of antibodies specific to CRF, wherein said antibodies comprise a detectable label, or a binding site for a detectable label.
  • a solid surface such as a fibrous test strip, a multi-well microliter plate, a test tube, or beads, having bound thereto antibodies to CRF
  • a known amount of antibodies specific to CRF wherein said antibodies comprise a detectable label, or a binding site for a detectable label.
  • a solid support useful in the present invention is a matrix of material in a substantially fixed arrangement.
  • Exemplary solid supports include glasses, plastics, polymers, metals, metalloids, ceramics, organics, etc.
  • Solid supports can be flat or planar, or can have substantially different conformations.
  • the substrate can exist as particles, beads, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc.
  • Magnetic beads or particles such as magnetic latex beads and iron oxide particles, are examples of solid substrates that can be used in the methods of the invention. Magnetic particles are described in, for example, U.S. Pat. No. 4,672,040, and are commercially available from, for example, PerSeptive Biosystems, Inc. (Framingham Mass.), Ciba Corning (Medfield Mass.), Bangs Laboratories (Carmel Ind.), and BioQuest, Inc. (Atkinson N.H.).
  • the labels used in the assays of invention can be primary labels (where the label comprises an element which is detected directly) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling).
  • Primary and secondary labels can include undetected elements as well as detected elements.
  • Useful primary and secondary labels in the present invention can include spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red, tetramethylrhodamine isothiocyanate (TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA, CyDyesTM, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, 32 P, 33 P), enzymes (e.g., horse-radish peroxidase, alkaline phosphatase) spectral colorimetric labels such as colloidal gold or colored glass or plastic (e.g.
  • fluorescent dyes e.g., fluorescein and derivatives such as fluorescein isothiocyanate (FITC) and Oregon GreenTM
  • rhodamine and derivatives e.
  • the label may be coupled directly or indirectly to a component of the detection assay (e.g., the labeling nucleic acid) according to methods well known in the art.
  • a component of the detection assay e.g., the labeling nucleic acid
  • a detector which monitors an analyte-receptor complex is adapted to the particular label which is used.
  • Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill.
  • an optical image of a substrate comprising bound analyte is digitized for subsequent computer analysis.
  • Preferred labels include those which utilize 1) chemiluminescence (using Horseradish Peroxidase and/or Alkaline Phosphatase with substrates that produce photons as breakdown products) with kits being available, e.g., from Molecular Probes, Amersham, Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2) color production (using both Horseradish Peroxidase and/or Alkaline Phosphatase with substrates that produce a colored precipitate) (kits available from Life Technologies/Gibco BRL, and Boehringer- Mannheim); 3) hemifluorescence using, e.g., Alkaline Phosphatase and the substrate AttoPhos (Amersham) or other substrates that produce fluorescent products, 4) Fluorescence (e.g., using Cy-5 (Amersham), fluorescein, and other fluorescent tags); 5) radioactivity using kinase enzymes or other approaches. Other methods for labeling and detection will be readily apparent to
  • Fluorescent labels are highly preferred labels, having the advantage of requiring fewer precautions in handling, and being amendable to high- throughput visualization techniques (optical analysis including digitization of the image for analysis in an integrated system comprising a computer).
  • Preferred labels are typically characterized by one or more of the following: high sensitivity, high stability, low background, low environmental sensitivity and high specificity in labeling.
  • Fluorescent moieties which are incorporated into the labels of the invention, are generally are known, including Texas red, dixogenin, biotin, 1- and 2-aminonaphthalene, p,p'- diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p,p'-diaminobenzophenone i ines, anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene, bis- benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol, bis-3- aminopyridinium salts, hellebrigenm, tetracycline, sterophenol, benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthen, 7- hydroxycoumarin, phenoxazine, cal
  • fluorescent tags are commercially available from the SIGMA Chemical Company (Saint Louis, Mo.), Molecular Probes, R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka ChemicaBiochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.
  • the analyte is measured by quantifying the amount of label fixed to the solid support by the capture of the linked complex between analyte and receptor.
  • the presence in the reaction mixture of an analyte-receptor complex will increase or decrease the amount of label fixed to the solid support relative to a control reaction which does not comprise the analyte.
  • Means of detecting and quantifying labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • typical detectors include microscopes, cameras, phototubes and photodiodes and many other detection systems which are widely available. Biological Samples
  • Biological samples that can be used in the present invention include physiological fluids or tissue samples.
  • Physiological fluids from patients include plasma, serum, tears, urine, and the like.
  • a tissue sample may be obtained such as by biopsy. The following examples are intended to illustrate but not limit the invention.
  • Endometrial tissue samples were collected from 54 patients between October 1994 and January 1997 after obtaining proper consent. Thirty-one of the fifty-four patients had final diagnosis of benign endometrium, and provided the basis for the control group. Twenty-three of the fifty-four patients had biopsy-proven diagnosis of adenocarcmoma of the endometrium, and underwent complete surgical staging. Surgical staging included pelvic washings, exploration of the upper abdomen, total abdominal hysterectomy, bilateral salpingo-oopherectomy, and sampling of pelvic and paraaortic lymph nodes. This group of patients constituted our experimental group. The collected endometrial tissue was frozen at -70°C.
  • Mouse monoclonal antibodies for human CRl (CD 35), MACIF (CD 59), and DAF (CD 55) were purchased commercially.
  • Anti- DAF (clone 1C6) was purchased from WAKO BioProducts (Richmond, VA) and diluted to 1:1000, for a final concentration of 1 ⁇ g/ml.
  • MCP (CD 46) mouse monoclonal antibody was generously provided by Dr. John P. Atkinson at Washington University School of Medicine (St. Louis, MO). The final dilution used in IHC staining of anti-MCP was 1:1000, for a concentration of 1 ⁇ g/ml. Immunohistochemical Staining
  • Frozen sections of endometrial tissues were stained with antibodies to CRl, MCP, DAF, and MACIF using the avidin-biotin-peroxidase complex (ABC) as described before (Kaul, A. et al, Am. J. Reprod. Tmmunol., 34, 236- 240 (1995)).
  • the slides were removed from the -20°C freezer and brought to room temperature over 5 min.
  • the tissue sections were fixed in ice-cold acetone for 10 min. After a rinse in PBS, the tissue samples were blocked at room temperature with normal goat serum (50 ⁇ l/slide) for 20-25 min.
  • DAB diaminobenzidine
  • Stained tissue sections were quantitated for CRP content using a microcomputer-based image analysis system as described earlier (Kaul, A. et al, Am T Reprnrl. Tmmunol.. 34, 236-240 (1995)).
  • the Samba 4000 system (Imaging Products International, San Francisco, CA) was used to measure the stained endometrial tissue sections for integrated optical density (OD).
  • OD optical density
  • a total of five fields at 20x magnification were measured in stained and unstained serial sections. The average OD per each stained and unstained section was computed. The difference in OD between antibody-stained and unstained parallel sections was used as a measure of the protein content in the benign and malignant tissue samples.
  • the 27 benign endometrial tissue samples provided our control group for the study.
  • the mean age was 51 years (range 34-80).
  • the distribution of histologic diagnoses was 14 proliferative, 4 secretory, and 8 atrophic endometrial samples.
  • Five of twenty-seven patients were on hormone replacement therapy. The following were the clinicopathologic characteristics of the 48 evaluable patients in this study.
  • the benign endometrial specimens had a mean OD of 12.76 ⁇ 6.93, and the mean OD for the carcinoma specimens was 27.96 ⁇ 7.98, reaching statistical significance (P ⁇ 0.0001).
  • a graphic representation of the amount of protein expression observed in the tissue is shown in Figure 3.
  • the benign endometrial specimens had a mean
  • Each of the CRPs inhibits the complement activation cascade at a different point in the scheme of the complement cascade.
  • CRl, DAF, and MCP inhibit the complement system at the C3 convertase level, while protectin inhibits the formation of MAC, thereby inhibiting cell lysis.
  • the proposed mechanism of the CRP is as follows: high levels of protein expression on tumor cells protect these cells from complement-mediated cytotoxicity.
  • complement activation can impact indirectly on tumor growth through effects on vessel permeability, cell trafficking, and possibly sensitization of tumors to cellular effects.
  • Local formation of C3a can increase both blood flow and diffusion of proteins into tumor-containing tissues.
  • the generation of C5a may also increase the influx of phagocytes to tumor sites.
  • iC3b or C3b
  • the deposition of iC3b (or C3b) on target cell surfaces has been demonstrated to promote cytotoxic activity of lymphocytes (Perlmann, H. et al, J. Exp. Med., 153, 1592-1603 (1981)).
  • the increase in recruitment of effector cells to tumor sites and the enhancement in the efficiency of cell-dependent modalities of tumor killing should contribute to cytotoxicity.
  • TNF- ⁇ modulation of DAF on MCF-7 Breast Cancer Cells Estrogen plays an integral role in the growth of most estrogen receptor positive mammary carcinomas.
  • the proliferative effects of steroids in normal and neoplastic tissues seen in breast cancer may actually be mediated by hormone-induced growth factors.
  • Aberration in growth factors signaling pathways are a common element in the endocrine resistant phenotype and thus affect therapeutic response rates.
  • TNF can alter estrogen-regulated metabolic processes in breast cancer cells leading to growth inhibition.
  • Estrogen primed and unprimed MCF-7 cells were treated with different concentrations of recombinant TNF- ⁇ for 18 hours. Estrogen primed cells were treated overnight with 20 ng/ml ⁇ estradiol. DAF protein was extracted by lysis treatment of cells and quantitated, run on SDS-PAGE followed by western blot analysis using anti DAF antibody. Bands were visualized by ECL method (Amersham). DAF levels were quantitated densitometrically and expressed as densitometric units. In MCF-7 cells growth under nonestrogenized conditions, TNF- ⁇ downregulated expression of CD55 in a dose dependent manner.
  • TNF- ⁇ upregulated CD55 in a dose dependent manner (Figure 5).
  • DAF CD55
  • the downregulation under nonestrogenized conditions by TNF- ⁇ indicates important interaction between immune and endocrine system ⁇ perhaps modulated by estrogen and progesterone receptor expression (Jurianz et al, Mol. Immunol., Sept-Oct; 36(13-14):929-939).
  • EXAMPLE 3 Analysis and Quantification of the Levels of DAF in Normal and Neoplastic Human Breast Tissue The regulation of normal breast development, breast carcinogenesis and growth progression of breast cancer seem to depend upon response to hormonal factors. Elevated levels of DAF are seem in endometrial cancers and is under hormonal control in the human endometrium (see example 1).
  • tissue samples About 60 normal and 60 neoplastic breast tissue samples are collected. Complete patient history is evaluated including the hormonal status, diet, menopause status, and if menstruating, the timing of the menstrual cycle for an correlation with the laboratory findings.
  • Breast tissues are collected after each prospective patient is approached by an attending physician. The procedures, risks, and long-range benefits of the tissue donation and the project are described to the patient. If for any reason a patient is hesitant to participate, she is excluded. Collected tissue is immediately placed into a plastic container on wet crushed ice and transported at +4°C to surgical histopathology laboratory. Upon arrival (no longer than 20 minutes) tissue samples are frozen in liquid nitrogen and placed in a -70 °C freezer. Tissue sections (5 ⁇ m) are prepared and examined histopathologically. The tissues are fixed in acetone, and used for immunohistochemical staining.
  • CRP's such as DAF are under the control of hormonal influence in the human endometrium (Kaul et al, 1995, Tnfect Immun., 64(2):611-615). Malignant and benign breast tissues are also studied to determine the role of CRP's such as DAF.
  • the expression of DAF is correlated with that of estrogen and progesterone receptors in the collected tissue samples.
  • the estrogen and progesterone receptors are currently used as an important diagnostic tool in the prognosis of breast cancers.
  • the ER/PR in the collected breast tissue samples are quantitated using immunohistochemical staining procedures.
  • ER and PR The antibodies for ER and PR are obtained from Dako Corp., Carpentaria, CA.
  • the value of ER PR is then correlated with the values obtained by the immunohistochemical staining of parallel tissue section using specific antibodies to identify DAF in these tissues. This correlation is an important factor in comparing DAF levels with either ER or PR or both.
  • DAF as a Prognostic Marker in Ductal Carcinoma in situ rPCIS
  • DAF can be used as an important immunohistochemical marker to help predict the potential stromal invasion.
  • Over-expression of DAF by breast cancer cell can be used as an early prognostic tool in patients diagnosed with DCIS of the breast.
  • DAF levels on breast tissue samples form patients who have been diagnosed with DCIS had have had multiple breast tissue samples (at least 3) taken over a period of time are analyzed and quatitated. Tissue sections from at least 25 patients previously diagnosed with DCIS are obtained on glass slides from paraffin embedded breast tissue blocks.
  • Immunohistochemical assay for DAF protein in human breast carcinoma along with tissue from normal breast and benign diseases are performed.
  • Breast tissues are stained with specific antibody (positive control) and isotype control of the same antibody (as the negative control), by the avidin-biotin peroxidase complex techniques. Stained section are measured for integrated optical density (OD).
  • OD integrated optical density

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé destiné au diagnostic précoce d'une lésion prémaligne, au pronostic d'une lésion maligne, ainsi qu'un kit destiné à être utilisé pour l'identification plus rapide d'une prédisposition à une malignité.
PCT/US2001/014769 2000-05-12 2001-05-09 Procede de diagnostic destine a balayer des niveaux de proteine regulatrice de complement WO2001088537A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2001261259A AU2001261259A1 (en) 2000-05-12 2001-05-09 Diagnostic method for screening complement regulatory protein levels
US10/292,130 US20030129677A1 (en) 2000-05-12 2002-11-12 Diagnostic method for screening complement regulatory protein levels

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US20396700P 2000-05-12 2000-05-12
US60/203,967 2000-05-12

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/292,130 Continuation US20030129677A1 (en) 2000-05-12 2002-11-12 Diagnostic method for screening complement regulatory protein levels

Publications (1)

Publication Number Publication Date
WO2001088537A1 true WO2001088537A1 (fr) 2001-11-22

Family

ID=22756024

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/014769 WO2001088537A1 (fr) 2000-05-12 2001-05-09 Procede de diagnostic destine a balayer des niveaux de proteine regulatrice de complement

Country Status (2)

Country Link
AU (1) AU2001261259A1 (fr)
WO (1) WO2001088537A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7148038B2 (en) 2001-10-16 2006-12-12 Raven Biotechnologies, Inc. Antibodies that bind to cancer-associated antigen CD46 and methods of use thereof
WO2012031273A2 (fr) 2010-09-03 2012-03-08 Stem Centrx, Inc. Nouveaux modulateurs et leurs procédés d'utilisation
US9778264B2 (en) 2010-09-03 2017-10-03 Abbvie Stemcentrx Llc Identification and enrichment of cell subpopulations
US9945842B2 (en) 2010-09-03 2018-04-17 Abbvie Stemcentrx Llc Identification and enrichment of cell subpopulations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAKULINEN J.: "Expression and function of the complement membrane attack complex inhibitor protectin (CD59) on human breast cancer cells", LABORATORY INVESTIGATION, vol. 71, no. 6, 1994, pages 820 - 827, XP002943681 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7148038B2 (en) 2001-10-16 2006-12-12 Raven Biotechnologies, Inc. Antibodies that bind to cancer-associated antigen CD46 and methods of use thereof
US7744878B2 (en) 2001-10-16 2010-06-29 Raven Biotechnologies, Inc. Antibodies that bind to cancer-associated antigen CD46 and methods of use thereof
WO2012031273A2 (fr) 2010-09-03 2012-03-08 Stem Centrx, Inc. Nouveaux modulateurs et leurs procédés d'utilisation
US9458231B2 (en) 2010-09-03 2016-10-04 Stemcentrx, Inc. Modulators and methods of use
US9778264B2 (en) 2010-09-03 2017-10-03 Abbvie Stemcentrx Llc Identification and enrichment of cell subpopulations
US9945842B2 (en) 2010-09-03 2018-04-17 Abbvie Stemcentrx Llc Identification and enrichment of cell subpopulations
US10017565B2 (en) 2010-09-03 2018-07-10 Abbvie Stemcentrx Llc Modulators and methods of use

Also Published As

Publication number Publication date
AU2001261259A1 (en) 2001-11-26

Similar Documents

Publication Publication Date Title
Al-Haddad et al. Psoriasin (S100A7) expression and invasive breast cancer
Miyake et al. Motility-related protein-1 (MRP-1/CD9) reduction as a factor of poor prognosis in breast cancer
EP1859273B1 (fr) Méthodes de détection du cancer de l'ovaire
Murray et al. Expression of complement regulatory proteins—CD 35, CD 46, CD 55, and CD 59—in benign and malignant endometrial tissue
Morsi et al. The patterns of expression of an apoptosis-related CK18 neoepitope, the bcl-2 proto-oncogene, and the Ki67 proliferation marker in normal, hyperplastic, and malignant endometrium
US9110064B2 (en) Methods for diagnosis and treatment of endometrial cancer
JP2005508144A (ja) 卵巣癌の診断方法、卵巣癌のモジュレーターをスクリーニングする組成物及び方法
JP6216732B2 (ja) 癌の診断方法ならびに癌患者の全生存期間および無病生存期間の決定方法
WO2013104104A1 (fr) Marqueur de diagnostic et d'indication pour le cancer du sein
EP2739290A2 (fr) Procédé de sélection d'agents chimiothérapeutiques pour un adénocarcinome
Said et al. Carboxypeptidase A1 and regenerating islet-derived 1α as new markers for pancreatic acinar cell carcinoma
US7560230B2 (en) Method for determining susceptibility of tumor to treatment with anti-neoplastic agent
US20030129677A1 (en) Diagnostic method for screening complement regulatory protein levels
US6656684B1 (en) Method for predicting tumor recurrence
WO2004109286A2 (fr) Procedes de detection et de traitement du cancer
WO2001088537A1 (fr) Procede de diagnostic destine a balayer des niveaux de proteine regulatrice de complement
Hemstreet III et al. Intermediate endpoint biomarkers for chemoprevention
US7566541B2 (en) Diagnostic method for screening complement regulatory protein levels to predict spontaneous abortion
EP3652541B1 (fr) Combinaison de marqueurs pour diagnostiquer un cancer
AU2006315651A1 (en) P66-Shc as predictive marker in cancer treatment
Baildam et al. The expression of milk fat globule antigens within human mammary tumours: relationship to steroid hormone receptors and response to endocrine treatment
Stanley et al. Peanut lectin binding in breast carcinoma: Lack of correlation with estrogen receptor content
KR102401005B1 (ko) 고위험군 자궁내막증의 예방 또는 치료용 조성물
El-Sarha et al. Serum sFas and tumor tissue FasL negatively correlated with survival in Egyptian patients suffering from breast ductal carcinoma
US7238492B2 (en) Method for predicting tumor recurrence

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWE Wipo information: entry into national phase

Ref document number: 10292130

Country of ref document: US

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP