MXPA99005962A - Immunohistochemical detection assay for carcinoma proliferative status - Google Patents

Abstract

A method of determining the proliferative status of a carcinoma is disclosed. One obtains a patient sample and then quantitatively analyzes the sample for NGAL gene expression product. The amount of NGAL expression product is compared with a standard curve to determine the S-phase value. The sample can be breast tissue or breast fluid aspirate. Alternatively, blood can be analyzed for this marker to diagnose metastasis.

Description

TEST OF IMMUNOISISCHEMICAL DETECTION OF THE PROLIFERATIVE STATE OF CARCINOMAS BACKGROUND OF THE INVENTION The present invention relates to a method for determining the proliferative status of a carcinoma. Mammary carcinoma and adjacent tissue are examined with respect to NGAL protein using immunohistochemical staining techniques. It is known that the c-erbB-2 oncogene is associated with the clinical progress of human breast cancer. In vivo models have been developed using a rodent homologue c-erbB-2, neu, to try to evaluate the function of c-erbB-2 in mammary carcinogenesis and tumor biology. In a transgenic model, mice have been generated in which the expression of activated neu is directed towards the mammary gland using specific promoters of the breast. In a second model, the activated neu oncogene has been directed and stabilized by introducing it into rat mammary epithelial cells in itself, using a defective replication retroviral vector. In both methods, neu was found to be a potent tumor inducer. Previously it has been reported "the isolation of a lipocalin only overexpressed in rat mammary carcinomas initiated by neu". S. Stoesz et al. , REF .: 30238 1994 AACR Abstract. This lipocalin was called "NRL" (for lipocalin related to neu (ne? Related lipocalin)). The description of this summary and all other publications referred to herein, are incorporated in their entirety as a reference. Since it is known that lipocalins have a wide range of functions, the specific function of the NRL is not known. A protein somewhat homologous to the rat NRL, the human NGAL, has already been isolated and sequenced. Several coding gene cDNA sequences for NGAL and the sequence of the NGAL protein have been reported in L. Kjeldsen et al. , J. Biol. Chem. 268: 10425-10432 (1993); J. Bundgaard et al. , Biochem. Biophys. Res. Comm. 202 [3]: 1468-1475 (1994); S. Bartsch et al. , FEBS Let. 37: 255-289 (1995). NGAL (also known as neutrophilic lipocalin hu ana / HNL) has been found in a variety of cell types (e.g., bone marrow, ovarian cell cancers). Once again, its specific function is unknown. Notice that Bundgaard reported that the first amino acid of the mature protein was Q of CAG, while of Kjeldsen in one place it reported an E in that position. The claims herein use the term "NGAL" to encompass both variants. The treatment and diagnosis of breast carcinoma can be improved by an accurate determination of the cancer's proliferative status. An important measure of the proliferative state is the percentage of cells in "S phase". The S phase is the phase of the cell cycle in which DNA duplication occurs. See generally F. Cross et al. , Annu. Rev. Cell Biol. 5: 341-395 (1989). The measurement of the percentage of cells in a biopsy sample, which are in S phase, is an indicator of the state of cell proliferation. It is known that a high percentage of cells in S phase is indicative of an unfavorable diagnosis for tumors, in addition to a very aggressive treatment. The percentage of carcinoma cells in S phase has been measured by staining, flow cytometry and analyzing certain markers. Known techniques have problems (e.g., high cost, time consuming) and specific type requirements, which make them unattractive for routine use in clinical laboratories. Thus, there is a need for an improved assay to determine the proliferative status of carcinomas. BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention provides a method for determining the relative degree of proliferation of a human mammary carcinoma. A sample of the carcinoma is obtained. An antibody having specificity for the NGAL protein is then used to bind to the NGAL protein in the sample. Then a marker bound to the antibody is used, to mark the extent to which the NGAL protein is present in the carcinoma. The NGAL protein, as used herein, is a protein having at least the sequence SEQ ID No: 1, sequence 21-197. In a preferred embodiment, the sample also has tissue adjacent to the carcinoma. Then, the marker also marks the extent to which the NGAL protein is present in the adjacent tissue. In one aspect, the method is an immunohistochemical staining assay wherein the marker creates a visible color to mark the presence of the NGAL protein. It will be appreciated that an objective of the present invention is to provide an assay for the proliferation of cancer in breast tissue. Another objective of the present invention is to provide a technique for minimizing false positive results, also by analyzing NGAL in the surrounding tissues. Other objects, features and advantages of the present invention will become apparent upon examination of the description and the claims that follow. DETAILED DESCRIPTION OF THE INVENTION A standard curve can be created to determine the S-phase values by flow cytometry for a panel of human breast tumor cells, then the NGAL protein levels for these same known samples are determined using, for example, the NGAL protein assay of S. Xu et al. , J. Immunol. Meth. 171: 245-252 (1994). We have found that NGAL protein levels in carcinomas are predictive of the S-phase values. Therefore, staining -punohistcchemical "stan- dards" for these known values can be developed. Obtaining Human Samples for Initial Correlation Studies. Breast tissue was obtained from women suspected of having a carcinoma, using biopsy methods known to those skilled in the art. For example, aspiration biopsy (or fine needle), which involves aspiration of cells and fragments of tissue through a needle that has been guided into the suspect tissue. Needle biopsy (or core), which involves obtaining a tissue core through a specially designed needle inserted into the suspect tissue, is another option. Incisional biopsies, where a small piece of tissue is removed from a larger tumor mass and biopsies by excision, which involve the excision of all suspect tumor tissue with a little or no normal tissue surrounding it, are other Examples of suitable tissue extraction methods to confirm the correlation with the S phase. See in general V. DeVita Jr. et al. , Cancer Principles and Practice of Oncology, Vol. 1, 4th ed., J. B. Lippincott Co., pp. 243-244 (1993). The tissue can be prepared in the following manner for protein analysis. In order to extract the proteins, the tissue can be homogenized with Polytron in PBSTDS (10 mM sodium dibasic phosphate); sodium chloride 154 mM; sodium salt of 12 mM deoxycholic acid; 1 M sodium fluoride; 3.5 mM sodium dodecyl sulfate (DSS); 31 mM sodium azide; Triton X-100 at 1%; 1 mM phenylmethylsulfonyl fluoride) at a concentration of 100 mg per ml and centrifuged at 10,000 g for 15 minutes at 4 ° C. The supernatant containing the soluble proteins is then removed. Assay The breast carcinoma tissue of the patients was examined with respect to the absolute amount of protein NGAL This test was performed using a RIA (radioimmunoassay). See, for example, S. Xu et al. , J.

Immunol. Meth. 171: 245-252 (1994). It can also be performed by an enzyme-linked immunosorbent assay (ELISA) In an ELISA, the sample is exposed to an antibody specific for NGAL, polyclonal antiserum to NGAL can be obtained in rabbits that were immunized with NGAL: After binding the polyclonal antibody to the NGAL protein, the mixture is exposed to a second antibody (eg, goat anti-rabbit IgG serum), which is bound to an enzyme whose substrate changes color. This enzyme label indicates the presence of the NGAL antigen More specifically, the anti-NGAL serum (monoclonal or polyclonal) captures antibodies that can be coated on microplates After the washings, patient samples containing unknown concentrations of the NGAL antigen are incubated In the plates After the unbound antigen is washed, an anti-NGAL secondary antibody is added to the wells and incubated.This antibody can be labeled with the enzyme, or is followed by the addition of a third antibody labeled with the enzyme that recognizes the secondary antibody, but does not capture it. After the unbound antibody is washed, an appropriate chromogenic substrate for the enzyme is added to the wells. The degree of color change produced by incubating the substrate is proportional to the concentration of the NGAL protein in the tumor sample and is compared with the known concentration standards of a recombinant NGAL, tested in parallel. See E. Engvall et al. , Immunochemistry 8: 871-879 (1971) for a general review of ELISA techniques. From the results of the ELISA, the level of NGAL protein present in the tissue samples of the patients can be determined and compared with a tumor panel with known histories of prognosis and S phase. Other methods can also be used, such as western blotting to analyze the amount of NGAL protein in a sample. Known Human Mammary Tumor Samples Samples of 15 randomly selected human mammary tumors were examined, with known S phase percentages (the percentage of S phase was reported by us or by other researchers). It was found that four samples indicated the presence of high levels of the NGAL expression product. The results are presented in the following Table 1.

TABLE 1 A + or ++ symbol indicates that the NGAL expression product was detected at an expressed or strongly expressed level, respectively. The statistical analysis shows that the association of the S phase with the NGAL is p = 0.0051. Generation and Purification of Polyclonal Antibodies Three pathogen-free New Zealand albino rabbits (Hazelton, Kalamazoo, MI) were initially immunized with 400 μg of purified recombinant NGAL protein. The protein was emulsified in an equal volume of Freund's complete adjuvant (Sigma, St. Louis, MO), using 2 syringes connected by a Luer fitting. The mixture was administered intradermally in 10-20 sites. At 4-week intervals, booster injections were administered subcutaneously, using 100-300 μg of diluted protein in Dulbecco's phosphate buffer (D-PBS) (Life Technologies, Gaithersburg, MD) and emulsified in incomplete adjuvant. of Freund (Sigma). 30 ml of whole blood was collected 12 days after the reinforcement, allowed to clot and centrifuged at 3000 g for 15 minutes at 4 ° C and the antiserum was frozen at -80 ° C. The relative antibody production, specificity and background reactions were determined by an enzyme-linked immunosorbent assay (ELISA) indirect (Pierce), using bovine serum albumin and pre-immune rabbit serum as negative controls. An affinity column was constructed using an AminoLink immobilization package (Pierce) and 3 mg of purified recombinant NGAL that had previously been concentrated in a volume of 0.5 ml with a Centriplus concentrator, with a cut-off value of 10 kD (Amicon, Beverly, MA). 8 mg of IgG, purified from an antisewas loaded using a HiTrap column with 1 ml protein A (Pharmacia Biotech) and washed on the affinity column with 50 mM Tris, pH 7.5 and eluted with 0.1 M glycine-HCl. , pH 2.9. Fractions of the column were monitored with respect to the IgG peak by reading absorbance at 280 nm. Go ----? Ur? 3hist-quip ca Fresh tissues were obtained for iniramc-h-i-stokinin d-e breast biopsy samples; and a small sample of tumor tissue and adjacent normal breast tissue was soaked with OCT compound (Miles, Elkhart, IN), frozen in an airtight container and stored at -70 ° C. Frozen sections of 5 μm were mounted on slides coated with poly-L-lysine and fixed in 70% ethanol for 5 minutes. The activity of the endogenous peroxidase was mitigated with 3% H2O2 in methanol for 10 minutes. After blocking with the Powerblack reagent (DAKO, Carpintería, CA), for 5 minutes, the slides were incubated with the anti-NGAL rabbit polyclonal antibody, purified by affinity chromatography, at a concentration of 18 μg / ml. Preimmune IgG was used at the same protein concentration as a negative control. The binding was visualized using the detection system LSAB + (DAKO) and the substrate of the peroxidase was 3,3-diaminobenzidine (DAB). The intensity of brown coloration was proportional to the concentration of NGAL protein. Alternatively, sections soaked in paraffin can be prepared. In some of the experiments, paraffin blocks of mammary carcinomas obtained from the pathology archives of the University of Wisconsin were routinely processed. Such preparations were dehydrated in an oven (55-60 ° C) before being dewaxed in various xylene changes and hydrated by a series of graded alcohols to water. A known positive control was treated identically to the cases that were being studied. For the recovery of the antigen, the preparations were subjected to a microwave treatment in 1 mM EDTA (pH 8.0) for 20 minutes, followed by a treatment with running water for 20 minutes. Then, the preparations were assigned a bar code and placed in APK lx wash solution (all equipment and reagents from Ventana Biotek Systems, Tucson, AZ, unless otherwise specified). The preparations were loaded in an automatic immunostaining apparatus (Gen Window) for standardized incubation times and temperatures. Anti-NGAL rabbit antiserum, purified by affinity chromatography, or pre-immune rabbit IgG as a negative control, was used at a concentration of 1.75 μg / ml for 30 minutes at 37 ° C. The biotin-avidin-horseradish peroxidase method with thiaminobenzidine substrate was used for the detection of the antigen. The preparations were counterstained with hematoxylin (Sigma, St. Louis, MO). All preparations were removed from the staining apparatus and rinsed in water with detergent to remove the oil coating. The preparations were then dehydrated by a series of graded alcohols and purified with various xylene changes before being coated with a synthetic mounting medium. Immunostaining of NGAL Mammary tumors showed a high positive reactivity (brown staining) in the carcinomas, with little color outside them except in the adjacent normal canals. This confirmed that the common inflammation not associated with the S phase, was not the cause of the NGAL. UTILITY It is thought that the present invention is useful for medical analysis and as a diagnostic tool. Although some existing biopsy procedures already involve the creation of frozen or paraffin-coated sections for analysis, the present invention can be easily implemented as an adjunct diagnostic tool. Note that the previously preferred embodiments are only examples of the present invention. Many other variations will fall within the scope of the claims. For example, it has been determined that low levels of NGAL also correlate with low levels of the estrogen receptor protein and low levels of progesterone receptor protein. By using samples with known levels of estrogen / progesterone (for example determined by known methods), a standard curve can easily be created for the immunohistochemical assay of NGAL vs. the levels of estrogen / progesterone. The present invention also provides a technique for verifying false positive results in other NGAL assays. In our tests, up to 4% of the samples positive for NGAL showed a significant amount of NGAL in tissue not associated with the carcinoma.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) Applicant: Wisconsin Alumni Research Foundation (ii) Title of the invention: IMMUNEHISTOCHEMICAL DETECTION TEST FOR THE PROLIFERATIVE STATE OF CARCINOMA (iii) SEQUENCE NUMBER: 1 (iv) POSTAL ADDRESS: (A) RECIPIENT: Quarles & Brady (B) STREET: 411 East Wisconsin Avenue (C) CITY: Milwakee (D) STATE: Wisconsin (E) COUNTRY: E.U.A. (F) POSTAL CODE: 53202-4497 (v) LEGIBLE FORM IN COMPUTER (A) TYPE OF MEDIUM: Diskette (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Schwartz, Cari R. (B) REGISTRATION NUMBER: 29,437 (C) REFERENCE NUMBER / CEDULA: 960296.93618 (ix) INFORMATION BY TELECOMMUNICATIONS: (A) PHONE: (414) 277-5715 (B) TELEFAX: (414) 271 -3552 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 197 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Met Pro Leu Gly Leu Leu Trp Leu Pro Ser Leu Leu Gly Ala Leu His 1 5 10 15 Wing Giii Wing Gln Asp Ser Thr Ser Asp Leu Lie Pro Wing Pro Pro Leu 20 25 30 Ser Lys Vai Pro Leu Gln Glr. Asn Phe Gln Asp Aan Glrt Phe Gln Gly 35 40 45 Lys Trp Tyr Val Val Gly Lau Ala Gly Asn Ala lie Leu Arg Glμ Asp 50 55 60 Lys Asp Pro Gln Lya Met Tyr Ala Thr He Tyr Glu Leu Lys Glu Asp 65 70 75 SO i-ys Ser Tyr Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp fiS 90 55 Tyr Trp lie Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr 100 '-.05 110 Leu Gly Aan lie Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg 115 120 125 Val Val Ser Thr Asii Tyr Asn Gln His Wing Met Val Phe Phe Lys Lys 130 135 140 Val Ser Gln Asn Arg Glu Tyr Phe Lya lie Thr Leu Tyr Gly Arg Thr 145 150 155 160 Lys Glu Leu Thr Ser Glu Leu Lyß Glu Asn Phe He Arg Phe Ser Lys 1 (J5 170 175 Ser Leu Gly Leu Pro Glu Asn His He Val Val Phe Pro Pro Asp 1B0 165 190 Gln Cys He Asp Gly 195 It is noted that in relation to this date, the best method known by the applicant to bring the said invention into practice is that which is clear from the present description of the invention.

Claims (3)

1. CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. A method for determining the relative degree of proliferation of a human mammary carcinoma, characterized in that it comprises: (a) obtaining a sample of the carcinoma with tissue that surrounds it; "(b) use an antibody that has specificity for the NGAL protein to bind to the NGAL protein in the sample, and (c) use a label attached to the antibody to indicate the relative extent in which the NGAL protein is present in the the carcinoma, in comparison with the surrounding tissue
2. 2. The method according to claim 1, characterized in that the method is a -U-Mnunohistoq --- i-p-ica staining test in which the sample is a section, the marker creates a visible color indicating the presence of the NGAL protein and the intensity of color associated with the carcinoma is compared to the intensity of color associated with the surrounding tissue
3. 3. The method in accordance with the claim 1, characterized in that the marker creates a visible color and the intensity of the color is compared to a known standard, in order to determine the extent to which the estrogen receptor protein and the progesterone receptor protein are present in the carcinoma.