HK1015379A - Dna sequence and encoded mammary-specific breast cancer protein - Google Patents

Description

DNA sequence and breast-specific breast cancer protein encoded by same

no marking

Background of the invention (1) field of the invention

The present invention relates generally to the field of breast cancer pathology, and more particularly to a cDNA sequence and its encoded breast-specific proteins for use in the detection and treatment of breast cancer. (2) Description of the related Art

Breast cancer is one of the most common lethal cancers. Although early diagnosis and treatment can reduce the morbidity and mortality of the disease, the positive predictive value for mammography is estimated to be only 25% (Hall et al, N Engl J Med 327: 319, 1992, incorporated herein by reference). Therefore, it would be desirable to have a method for detecting cancer prior to detection using mammography, and genetic or biochemical markers would provide such a means to complement and improve the positive predictive value of mammography. (Hayes, HematolOncol Clin N Am 8: 485, 1994, referenced herein).

The development of breast cancer is accompanied by a number of genetic alterations (see Porter Jordan, Hematol Oncol Clin NAm 8: 73, 1994, incorporated herein by reference). Such alterations include gross chromosomal changes and loss of genetic markers (Devilee et al, journal of biochemistry and biophysics Acta 1198: 113, 1994; Callahan et al, J Cell Biochem Suppl 17: 167, 1993, incorporated herein by reference). The development of breast neoplasia has also been shown to result in qualitative and quantitative changes in gene expression of the identified expressing growth factors and their receptors (Zajchowski et al, Cancer research 48: 7041, 1988, referenced herein), structural proteins (Trask et al, Proc Natl Acad Sci 87: 2319, 1990, referenced herein), second messenger proteins (Ohuchi et al, Cancer research 26: 2511, 1986, referenced herein), and transcription factors (Harris, Cancer research progress (Adv Cancer Res) 59: 69: 1992, referenced herein). Changes in the expression of these genes may potentially form the basis for the development of breast cancer markers, although the precise role of changes in these genes in breast cancer pathology in patient biopsy samples is unknown.

In addition to providing genetic or biochemical markers for breast cancer for early diagnosis, it would be desirable to have a tumor marker that allows for the assessment of prognosis, i.e., a method for selecting and evaluating treatments and a method for targeting treatments. Although many tissue markers have been identified, none are sufficiently sensitive or lack tumor specificity and are therefore not suitable for diagnosis or screening of the general population (supra). Therefore, there is a need for a breast cancer marker, such as a gene and an expressed protein thereof, that can specifically and selectively detect the presence and pathological progression of breast cancer in a patient.

Differential expression sequence markers for isolation of breast cancer using modified differential display polymerase chain reaction techniques several sequence fragments were isolated that were uniquely expressed in neoplastic mammary epithelial tissues compared to normal tissue controls (Watson and Fleming, cancer research 54: 4598-4602, 1994, incorporated herein by reference). One of these sequence tags was found and identified as DEST002, from which a novel full-length cDNA and its encoded protein, now known as mammaglobin (mammaglobin), was discovered and isolated. Both the cDNA and the protein are novel. Summary of the invention

The present invention, therefore, relates generally to the identification of novel genes with increased expression in breast cancer, and the isolation of cDNA from the mRNA of these genes. Therefore, applicants have successfully discovered a new cDNA and the encoded mammary-specific secreted protein, mammaglobin. The cDNA is in purified and isolated form and is identified as SEQ ID NO: 1, the protein encoded thereby, mammaglobin, in purified and isolated form, is identified as SEQ ID NO: 2.

in stage I of primary breast cancer tumors, mammaglobin was overexpressed by 27%. This suggests that dysregulation of the mammaglobin gene is frequently associated with early stages of breast cancer. Therefore, the discovery of mammaglobin and its cDNA provides the basis for the development of novel methods and compositions for the detection and treatment of breast neoplastic disease in humans and other mammals.

The present invention therefore also relates to a novel method for detecting the presence of breast neoplasia cells in a sample. In one embodiment, a cDNA encoding mammaglobin or a derivative of said cDNA is used to detect the presence of mammaglobin mRNA in a sample. The method comprises the following steps: (a) providing a polypeptide comprising SEQ ID NO: 1 or a derivative thereof, (b) incubating the nucleotide sequence with the sample under conditions such that the sequence is capable of hybridizing to mRNA from a breast neoplasia cell, and (c) detecting the presence of a DNA-RNA hybridization complex.

Another aspect of the invention provides a kit for detecting the presence of a breast neoplastic cell in a sample. The kit comprises a section of nucleic acid containing SEQ ID NO: 1 or a derivative thereof.

In another embodiment of the invention, mammaglobin or a derivative thereof is used to detect the presence of cDNA reverse transcribed from mammaglobin mRNA in a sample. The method comprises the following steps: (a) generating cDNA from mRNA by reverse transcription in a sample taken from a patient, (b) providing two oligomers as primers for polymerase chain reaction which flank or flank the cDNA encoding mammaglobin, and (c) amplifying the cDNA encoding mammaglobin using polymerase chain reaction. The two oligomers comprise SEQ ID NO: 3 and SEQ ID NO: 4.

another embodiment of the invention provides a kit for detecting the presence of a breast neoplastic cell in a sample. The kit comprises two oligos packaged in a container as primers for polymerase chain reaction flanking or within a cDNA encoding mammaglobin. The two oligomers comprise SEQ ID NO: 3 and SEQ ID NO: 4.

in another embodiment of the invention, the presence of mammaglobin expression by tumor cells is detected in the sample using a protein, an antibody specific for mammaglobin. Such specific antibodies may be polyclonal or monoclonal antibodies.

Therefore, it is important to note in the advantages of the present invention to provide a nucleotide sequence and its encoded amino acid sequence that can be used as a marker for breast cancer cells; methods for early detection of the presence of breast neoplasia cells are provided; methods for detecting breast cancer that complement mammography and improve positive predictive value are provided; and methods capable of assessing prognosis; and providing indicia of guided therapy. Drawings

FIG. 1 shows the isolation of full-length mammaglobin cDNA using Rapid Amplification of CDNA Ends (RACE) Polymerase Chain Reaction (PCR) followed by subcloning into the vectors pGEM7Z and pCEV 27.

Figure 2 shows the human cDNA sequence SEQ ID NO: 1 (nucleotide numbering above) and the amino acid sequence of the breast-specific protein encoded thereby, mammaglobin SEQ ID NO: 2 (amino acid numbering below), the solid line represents the 403bp fragment isolated by RACE PCR (SEQ ID NO: 5), and the open line represents the 206bp DEST002 sequence (SEQ ID NO: 6).

FIG. 3 shows a comparison of the mamma-specific protein mammaglobin (hMAM) (SEQ ID NO: 2) with rat prostate steroid binding protein subunit C3(rPSE3) (SEQ ID NO: 7) and human Clara cell 10KD protein (hCC10) (SEQ ID NO: 8), identical amino acids are indicated in bold letters and double lines, and structurally similar amino acids are indicated by single lines;

FIG. 4 shows (A) a Northern blot assay of the hybridization of a human cDNA sequence encoding a breast-specific protein, mammaglobin (hMAM), to mRNA expressed in breast neoplastic, normal breast and other adult tissues; and (B) analysis of RT/PCR amplified tissue samples from breast neoplasia, normal breast tissue and other adult tissues;

FIG. 5 shows translation of breast-specific cDNA sequences in an in vitro rabbit reticulocyte lysate analysis system;

FIG. 6 shows Northern blot hybridization analysis using cDNA encoding mammaglobin in tumor 2410, in tumors from 3 out of 8 other patients (shown in bold), and in fewer two cases of normal breast tissue for comparison (shown in italics), to detect mRNA, and expression of mammaglobin in tumor tissue and normal tissue of the patients;

FIG. 7 shows a Western blot analysis of polyclonal antibodies of 16C-terminal amino acids (SEQ ID NO: 14) from conditioned medium (S) and cell lysates (C) of MDA-MB415 breast tumor cells in the absence (-) and presence (+) of peptides used to generate the polyclonal antibodies;

FIG. 8 shows Western blot analysis of cell lysates of human breast tumor cells, using a polyclonal antibody of 16C-terminal amino acids (SEQ ID NO: 14) and goat anti-rabbit antibodies to detect mammaglobin, and visualized using enzyme-linked chemiluminescence;

FIG. 9 shows in color paraffin fixed sections of breast cancer cells from patient samples, the sections were immunohistochemically stained with horseradish peroxidase-labeled polyclonal antibody of 16C-terminal amino acids (SEQ ID NO: 14) and goat anti-rabbit antibody and substrate DAB, and cells expressing mammary proteins were stained brown;

FIG. 9A shows in black and white paraffin-fixed sections of breast cancer cells from patient samples, the sections were immunohistochemically stained with horseradish peroxidase-labeled polyclonal antibody of 16C-terminal amino acids (SEQ ID NO: 14) and goat anti-rabbit antibody and substrate DAB, and cells expressing mammary proteins were stained brown. Description of the preferred embodiments

One aspect of the present invention is based on the identification of the sequence of SEQ ID NO: 1, which encodes the cDNA identified as SEQ ID NO: 2 (fig. 2) is mammaglobin. As described below, the full-length cDNA for mammaglobin was isolated by reverse transcription of mRNA from tumor cells, amplified using PCR techniques, and subcloned into an expression vector. In addition, the protein encoded by the cDNA, mammaglobin, was identified and described.

The corresponding gene product, which is not known to date but is referred to herein as mammaglobin, was demonstrated to be particularly abundant in the breast cancer tumor cell line MDA-MB-415 using an anonymous sequence tag known as DEST 002. To isolate full-length mammaglobin cDNA, mRNA from this cell line was reverse transcribed and cloned using RACE PCR technology (Edwards et al, Nucleic Acid Research 19: 5227-32, 1991, incorporated herein by reference). This technique is based on a strategy of ligating oligodeoxynucleotides to the 3' end of single stranded cDNAs. FIG. 1 illustrates this method for isolating mammaglobin cDNA. From the sequence information of the 403bp fragment isolated using this technique (SEQ ID NO: 5) (FIG. 2), in combination with the sequence information obtained from the corresponding DEST sequence in our previous studies (DEST002, SEQ ID NO: 6) (Watton and Fleming, supra), a cDNA sequence of 503bp in full length (SEQ ID NO: 1) was deduced. FIG. 2 shows a full-length mammaglobin cDNA and the polypeptide encoded thereby. Within the 503bp cDNA there is a 279bp open reading frame which encodes a 93 amino acid polypeptide with a predicted molecular weight of 10.5kD (FIG. 2). The first 19 amino acids of the open reading frame also indicate a hydrophobic peptide signal sequence. The first methionine of the open reading frame comprises a nearly identical consensus sequence of Kozak (Kozak, Cell 22: 7-8, 1980, incorporated herein by reference). The 60bp upstream of this sequence does not contain other in-frame methionine or translation endpoints. The 3' untranslated sequence of the cDNA is 163bp in total and contains a polyadenylation signal, AATAAA, which is located 12bp upstream of the initiation site of the original DEST002 sequence. The above data indicate that full-length mammaglobin cDNA was isolated.

Searching the gene bank (Genbank) for a DNA sequence similar to mammaglobin cDNA using the BLAST algorithm found no significant homologous DNA sequence. Thus, mammaglobin cDNA is believed to be a new, as yet unknown sequence.

The amino acid sequence homology between mammaglobin and other polypeptides was found by searching for sequences related to mammaglobin among other polypeptides. Mammaglobin has 42% amino acid identity (58% including conservative substitutions) to rat prostate steroid binding protein (prostasin) subunit C3(rPSC3) (fig. 3) (SEQ ID NO: 7). Rat prostate steroid binding protein is a major secreted protein in rat prostate, consisting of two distinct dimeric subunits: C3/C1 and C3/C2(Parker et al, Ann N Y Acad Sci 348: 115-124; Parker et al, J Steroid Biochem 20: 67-71, 1984, incorporated herein by reference). The C1, C2 and C3 genes all encode an approximately 6kD secreted protein and are considered to be the result of gene replication, but although C1 and C2 share very high homology, they differ greatly from C3. Therefore, mammaglobin has no sequence homology to the C1 and C2 proteins.

As described above, prostanoid binding protein (prostasin) is a major secreted protein in rat prostate and its expression is regulated by androgen steroids (Parker et al, Ann N Y Acad Sci 438: 115-. Another protein, human estramustine binding protein (hEMBP), has been reported to be expressed in human prostate, human breast Cancer and human malignant melanoma (Bjork et al, Cancer research 42: 1935-. The human estramustine binding protein is immunochemically similar to rat estramustine binding protein, which is estimated to be identical to the rat steroid binding protein, i.e. the prostate protein. As described above, mammaglobin exhibited 42% amino acid sequence identity and 58% homology, including conservative substitutions, with the C3 subunit of prostatein. Therefore, mammaglobin may be associated with hEMBP to some extent. However, although both prostasin and hEMBP are detected in the prostate, mammaglobin mRNA is completely absent from this tissue. Thus, mammaglobin is neither the same protein nor a subunit of hEMBP, and, since the sequence of hEMBP has not yet been determined, it is not even known whether mammaglobin shares any similarity with certain fragments or subunits of hEMBP.

Although recent reports have demonstrated that the rPSC3 promoter fused to the SV 40T antigen causes both prostate and breast cancer in transgenic mice (Maroulakou et al, Proc. Natl. Acad. Sci. USA 91: 11236-11240, 1994; Sandmoller et al, Oncogene (Oncogene) 9: 2805-2815, 1994, incorporated herein by reference), the exact biological function of this protein is unclear. Furthermore, although an association between rat prostate steroid binding protein and human EMBP was postulated, no human polypeptide or human gene corresponding to rPSC3 has been identified. Therefore, mammaglobin and the cDNA encoding mammaglobin represent new sequences that have not been known to date.

Artificially aligning other sequences with lower BLAST scores with the mammaglobin and rPSC3 sequences, we found homology to the 10kD protein of human Clara cells (hCC10) (SEQ ID NO: 8) (Peri et al, J.Clin. invest.) 92: 2099-2109, 1993, herein referenced) (FIG. 3), and to rabbit and mouse uteroglobin (Miele et al, Endocrine Rev. 8: 474-90, 1987; Cato and Beato, anticancer studies 5: 65-72, 1985; Miele et al, Endocrinol. invest. 17: 679-692, 1994, where Cys, according to species, these are 40% identical or include substitutions, especially homology is that many amino acids are fully conserved among various proteins, including those known to form disulfide bonds between the uteroglobin subunits (see also protein-69) Mammaglobin was shown to be a novel member of a small family of proteins secreted by epithelial cells (Miele et al, 1994, supra).

hCC10 is a human homolog of the rabbit and mouse uteroglobin genes (Peri et al, J. Clin. Res. 92: 2099-2109, 1993, incorporated herein by reference). Uteroglobin was first recognized as a secreted protein characteristic of that present in the uterus of rabbits, but was later found in other epithelial organs including the lungs, breast and prostate. Unlike rat prostatic protein, uteroglobin is a homodimeric protein, coupled by disulfide bonds at conserved residues Cys-2 and Cys-69 (Miele et al, 1994, supra). Although transcription of the uteroglobin gene is regulated by steroid hormones, the ability of the protein itself to bind progesterone or other steroid hormones is not known, and its exact biological function is not known (Miele et al, 1994, supra).

Expression of mammaglobin is restricted to the mammary gland. This is in contrast to the results of rPSC3 expression in rat prostate (Parker et al, Ann N Y Acad Sci 438: 115. sup. 1124, 1984), and hCC 10/uteroglobin expression in many tissues including lung, uterus, prostate and breast (Miele et al, 1987, supra; Cato and Beato, supra; Miele et al, 1994, supra). Due to sequence homology between mammaglobin and the above proteins, we determined that its expression pattern is tissue specific. The 500bp mammaglobin message was readily detectable in tumor sample 2410 (proto-sequence tag, i.e., isolated from this tissue), but much less in normal human breast tissue (FIG. 4). The mammaglobin message could not be detected in the immortalized mammary epithelial cell line B5-589. Mammaglobin expression was also undetectable in both the uterus and lung of humans with uteroglobin expression.

Mammaglobin mRNA was detected by RT/PCR amplification in both tumor 2410 and normal breast tissues, but not in the other 15 tissues tested, including tissues that normally expressed rPSC3 and uteroglobin (lung, uterus, prostate), hormone-responsive and steroid-producing tissues (ovary, testis, placenta) and other secretory epithelial organs (colon) (fig. 4B). Therefore, mammaglobin mRNA expression is relatively breast tissue specific.

According to this aspect of the study, mammaglobin is a relatively breast-specific protein. Two other genes known to be over-expressed in breast cancer are erb-B and cyclin D (Jardines et al, Pathobiology 61: 268. sup. 1116, 1994; Keyomars and Pardee, Proc. Natl. Acad. Sci. USA 90: 1112. sup. 1116, 1993, incorporated herein by reference). Unlike overexpression of erb-B and cyclin D, overexpression of mammaglobin is likely to reflect a more specific change in mammary epithelial cells rather than a general increase in growth capacity or mitotic rate. Thus, the occurrence of dysregulation of the mammaglobin gene may be more specific in suggesting therapeutic vulnerability or clinical course of the tumor.

At the sensitivity level of the one-step RT/PCR assay, mammaglobin expression could not be detected in normal lymph nodes or peripheral lymphocytes. This suggests that mammaglobin transcript analysis in peripheral lymph nodes can be used to detect underlying breast cancer metastasis as well as other epithelial-specific genes (Schoenfeld et al, cancer research 54: 2986-90, incorporated herein by reference).

To demonstrate that mammaglobin cDNA encodes a translatable protein, cDNA clones were used in an in vitro translation system. FIG. 5 shows protein products from rabbit reticulocyte lysates treated with mammaglobin cNDA. An approximately 6kD protein was produced with mammaglobin cNDA. Apparent molecular weights are lower than expected for the conceptual translational open reading frame, but this result is also frequently found in rabbit and human uteroglobin translation products.

Although we found overexpression of mammaglobin in one tumor sample (i.e., 2410), the frequency of this overexpression in other breast cancers is unclear. Therefore, we examined a panel of 15 different histological types of stage I primary breast cancer using Northern blot hybridization with mammaglobin cDNA probes. Since environmental influences may cause potential changes in expression, we have tried to directly compare tumor samples with normal breast tissue samples of the respective patients, but this is not possible in many cases. As shown in FIG. 6, 500bp of mammaglobin mRNA was again detected in normal breast tissue and tumor 2410. Mammaglobin was also detected in three other tumors, two of which were expressed little or no in normal tissues of the respective patients. In total, 4 out of 15 tumors examined (27%) overexpressed mammaglobin mRNA. The above data indicate that overexpression of mammaglobin is not limited to a single tumor sample, and in fact it is quite common in primary breast cancer. Moreover, the fact that all tumors examined were stage I indicates that this disorder occurs early in the progression of breast cancer.

Since the applicants believe mammaglobin is likely to be a secreted protein, it is expected that its presence can be detected in the serum of patients whose tumors overexpress the gene product. Therefore, mammaglobin is likely to be used clinically as a Prostate Specific Antigen (PSA) and other solid Tumor marker for the treatment of patients with breast cancer ("Tumor marker in diagnostic pathology" clinical laboratory medicine ", Clin Lab Med 10: 1-250, 1990, reference herein).

We determined the prevalence of mammaglobin as a tumor marker in the general breast cancer tumor population by detecting the expression of mammaglobin in each primary breast cancer. Although the number of samples examined in this study was small, 27% of the tumors examined overexpress mammaglobin mRNA. This percentage can be compared to other genetic alterations such as erb-B amplification and the high prevalence of the p53 mutation (Slamon et al, science 244: 707-712, 1989; Thor et al, J Nat' l Cancer Inst 84: 845-855, 1992, referenced herein). Moreover, because we have limited the analysis to stage I tumors, overexpression of mammaglobin is actually much more prevalent than other genetic alterations reported in this subset of tumors (Alllerd et al, J Nat' l Cancer Inst 85: 200-206, 1993, incorporated herein by reference).

The identification of mammaglobin as a marker for breast cancer provides the basis of a further aspect of the invention which relates to a method of detecting the presence of breast cancer in a patient. "detecting" as used herein in reference to detecting breast cancer disease is intended to include determining the presence of breast cancer in a patient, distinguishing breast cancer from other diseases, estimating prognosis, i.e., the likely outcome and prospects for recovery from disease, monitoring disease status or disease recurrence, determining a preferred treatment regimen appropriate for the patient, and directing anti-tumor therapy.

A method of detecting breast cancer includes hybridizing a polynucleotide to mRNA from a breast cancer cell. The polynucleotide comprises SEQ ID NO: 1 or a derivative thereof. Deriving a nucleotide sequence means that the derived sequence is substantially identical to the derived prosequence in that the derived sequence has sufficient sequence complementarity to the derived prosequence to be capable of hybridizing to mRNA from breast cancer cells under the same stringency conditions as the prosequence does to mRNA from breast cancer cells.

The derivative nucleotide sequence need not be physically derived from a nucleotide sequence but may be produced by any method including, for example, chemical synthesis or DNA replication or reverse transcription or transcription.

In order to detect the presence of mammaglobin-encoding mRNA in a detection system for detecting breast cancer, a sample is taken from the patient. The sample may be a tissue biopsy sample or a sample of blood, plasma, serum, or the like. The sample may be treated to extract the nucleic acids contained therein. The resulting nucleic acids are subjected to gel electrophoresis or other size separation techniques.

Detection involves contacting nucleic acids, particularly mRNA of a sample, with DNA sequences that serve as probes for hybrid double-strand formation. "Probe" refers to a structure comprising a polynucleotide sequence that forms a hybrid with a target sequence due to the complementarity of the probe sequence to a sequence in the target region.

The double strand formed is typically detected using a labeled probe. Alternatively, the probe may be unlabeled and become detectable upon specific binding, either directly or indirectly, to a labeled ligand. Suitable labels and methods for labeling probes and ligands are known in the art and include, for example, radiolabels, biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes, particularly primed dioxetanes), enzymes, antibodies, and the like, which may be added by known methods (e.g., nick translation or kinase methods).

When a cDNA encoding mammaglobin or a derivative thereof is used as a probe, high stringency conditions can be used to prevent false positives. Low stringency conditions can be used when using sequences derived from mammaglobin. The stringency of hybridization depends on many factors during hybridization and washing, including temperature, ionic strength, time, and formamide concentration. The above factors are described, for example, in Sambrook et al, "molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual, 2 nd edition, 1989).

To increase the sensitivity of detection in a sample of mammaglobin-encoding mRNA, reverse transcription/polymerase chain reaction (RT/PCR) techniques may be used to amplify cDNA transcribed from mammaglobin-encoding mRNA. RT/PCR methods are well known in the art (see, e.g., Watson and Fleming, supra).

RT/PCR can be performed as follows. Total cellular RNA is isolated and reverse transcribed using, for example, guanidinium isothiocyanate. Reverse transcription involves the synthesis of DNA using RNA as a template using reverse transcriptase and a 3' end primer. Typically, the primer comprises an oligo (dT) sequence. The cDNA thus generated is then amplified by PCR and mammaglobin-specific primers (Belyavsky et al, nucleic acids Res. 17: 2919. sup. 2932, 1989; Krug and Berger, Methods in Enzymology, Academic Press, N.Y. Vol. 152, pp.316-325, 1987, incorporated herein by reference).

Two oligonucleotide primers complementary to both sides of the DNA sequence to be amplified are used for polymerase chain reaction. The upstream and downstream primers are typically 20 to 30 base pairs in length and hybridize to the flanking regions to replicate the nucleotide sequence. The polymerization reaction is catalyzed by a DNA polymerase in the presence of deoxynucleoside triphosphates or nucleotide analogs to produce double-stranded DNA molecules. The strands are then separated by any denaturing method, such as physical, chemical or enzymatic methods. Physical methods are generally used, i.e.nucleic acids are generally heated to about 80 ℃ to 105 ℃ for about 1 to 10 minutes. The process is repeated as many cycles as desired.

Primers are selected that are substantially complementary to the amplified cDNA strands. Therefore, the primer does not necessarily completely reflect the sequence of the template, but must have sufficient complementarity to selectively hybridize to the amplified strand.

After amplification, the PCR products were detected by staining with ethidium bromide (Sambrook et al, 1989, supra).

In another embodiment of the invention, the mammaglobin cDNA sequence or derivative thereof may be used to identify any mammaglobin gene alteration (i.e., gene rearrangement, gene amplification or gene deletion) in a sample from a breast cancer patient. This provides a means by which patient samples without intact mRNA can also be examined for changes in gene structure.

In one use of the present technology, mammaglobin or a derivative thereof is hybridized to genomic DNA isolated from a tumor, normal tissue or lymphocyte of a patient and digested with one or more restriction enzymes. The assay utilizes Southern blotting, which is well known in the art, to detect whether a patient or a breast tumor in a patient has a deleted, rearranged or amplified mammaglobin gene. Detecting such changes can provide important information for predicting prognosis and for the treatment of the patient.

In a second use of the present technology, one or more pairs of oligonucleotide primers may be used in a polymerase chain reaction to amplify mammaglobin gene fragments from a patient sample. Analysis of the resulting PCR products showed whether a particular fragment of mammaglobin was deleted or rearranged. Such information is useful for prognosis and treatment of the patient.

The invention also provides methods for detecting the presence of a polypeptide, mammaglobin, in a sample taken from a patient. Any protein detection method known in the art may be used. These methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, immunochemistry, ligand binding assays, immunohistochemistry, agglutination and complementation assays (see, e.g., Basic and Clinical Immunology) Sites and Terr editions, Appleton & Lange, Norwalk, conn. pp.217-262, 1991, incorporated herein by reference). Preferred are ligand binding immunoassays which comprise reacting an antibody with one or more epitopes of mammaglobin and competitively replacing the labeled mammaglobin or derivative thereof.

Mammaglobin derivatives refer herein to polypeptides comprising amino acids or modified amino acids which cross-react with mammaglobin. Cross-reactivity refers to the reaction of an antibody with an antigen other than the antigen that induced its production.

Various competitive and non-competitive protein binding immunoassays are well known in the art. The antibodies used in such assays may be unlabeled, such as in agglutination assays, or labeled as used in a variety of assays. Labels that may be used include radionuclides, enzymes, fluorescers, chemiluminescent agents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and the like for use in Radioimmunoassays (RIA), enzymatic immunoassays such as enzyme-linked immunosorbent assays (ELISA), fluorescent immunoassays, and the like.

Polyclonal or monoclonal antibodies that produce mammaglobin or one of its epitopes can be used in immunoassays using any of a number of methods known in the art. An epitope refers to an antigenic determinant of a polypeptide. An epitope may comprise 3 amino acids in a spatial conformation unique to the epitope. An epitope typically has at least 5 such amino acids. Methods for detecting the spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

One method of preparing antibodies to a protein is to select and prepare all or part of the amino acid sequence of the protein, chemically synthesize the sequence and inject it into a suitable animal, typically a rabbit or mouse.

Methods for preparing mammaglobin or an epitope thereof include, but are not limited to, chemical synthesis, recombinant DNA techniques, or isolation from a biological sample. For example, chemical synthesis of peptides can be carried out using the classical Merrifeld method, i.e., solid phase peptide synthesis (Merrifeld, J.Am.chem.Soc., 85: 2149, 1963, referenced herein) or the FMOC method on the Rapid automated multiple peptide Synthesis System (DuPont Company, Wilmington, DE) (Caprino and Han, J.Org Chem., 37: 3404, 1972, referenced herein).

Polyclonal antibodies were prepared by injecting antigen into the hind knee lymph node to immunize rabbits, followed by intraperitoneal injection of antigen at two week intervals to boost the immunity. The blood of the animals is discharged and serum analysis is carried out with purified mammaglobin, typically by ELISA. Monoclonal antibodies can be prepared according to the method of Milstein and Kohler, i.e., spleen cells of immunized mice fused with continuously replicating tumor cells such as myeloma cells or lymphoma cells (Milstein and Kohler, Nature 256: 495-497, 1975; Gulfre and Milstein, methods in enzymology: immunochemical techniques 73: 1-46, Langon and Banatis eds., Academic Press, 1981, incorporated herein by reference). The hybridoma cells thus formed were then cloned by limiting dilution method, and the production of antibody in the supernatant was detected by ELISA or RIA.

The unique ability of antibodies to recognize and specifically bind to target antigens expressed by tumor cells provides a method for treating cancer (see LoBuglio and Saleh, J. Med. Sci. USA 304: 214-224, 1992; Bagshawe, pharmaceutical developments 24: 99-121, 1993, incorporated herein by reference). Thus, another aspect of the present invention provides a method for the prevention and treatment of breast cancer in an animal based on the use of antibodies to mammaglobin which have been found to be overexpressed in breast cancer cells. Monoclonal or polyclonal antibodies specific for mammaglobin may be prepared by any of the methods known in the art. For example, murine or human monoclonal antibodies can be prepared using hybridoma technology. Alternatively, mammaglobin or an immunologically active fragment thereof, or an anti-idiotype antibody or fragment thereof can be administered to an animal to elicit production of antibodies capable of recognizing mammaglobin-expressing cells.

The antibodies or fragments thereof so produced are labeled with one or more tumor cell lysing substances, such as radionuclides, toxins, or cytotoxic drugs, and administered to a patient suspected of having breast cancer. Binding of the labeled antibody to mammaglobin overexpression by the breast cancer cells results in death of the cancer cells.

Any of a variety of tumor cell lysing substances known in the art can be used to produce such labeled antibodies. For example, plant and bacterial toxins can be conjugated to antibodies to produce immunotoxins. Such toxins include, for example, ricin, diphtheria toxin and pseudomonas exotoxin a. Drug-antibody conjugates of chemotherapeutic drugs linked to antibodies can also be prepared. Chemotherapeutic agents suitable for such use include, for example, tomoxifen, doxorubicin, methotrexate, chlorambucil, vinca alkaloids, and mitomycin. In addition, radioimmunoconjugates having a radionuclide stably linked to an antibody can be prepared. Radionuclides suitable for use in preparing radioimmunoconjugates include, for example, beta emitters such as¹³¹I、¹⁸⁸Re、¹⁸⁶Re、⁶⁷Cu、⁹⁰Y and⁴⁷sc; alpha emitters such as²¹¹At、²¹²Bi and²¹²pb; auger electron emitters, e.g.¹²⁵I and⁷⁷br; and fissionable nuclides such as¹⁰B。

Preferred embodiments of the present invention are described in the examples section below. Other embodiments within the scope of the following claims will be apparent to those skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

In the examples which follow, the cell lines are supplied by the American type culture Collection and cultured in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum. Tissue biopsy samples are provided by the Human Cooperative Tissue Network (LiVolsi et al, cancer 71: 1391-. Example 1

This example illustrates the isolation of mammaglobin cDNA.

Total cellular RNA from cell line MDA-MB415 was isolated using a standard guanidine isocyanate method (Belyavsky et al, supra). The RNA was used in RACEPCR procedure using Amplifinder kit (Clonetech) and following the manufacturer's protocol.

First strand cDNA was synthesized in a standard reaction format comprising 1. mu.g RNA, 10. mu.M specific mammaglobin primer D2R (5'-ATA AGA AAG AGA AGG TGT GG-3') (SEQ ID NO: 4), 4. mu.l of 5 XTT buffer (250mM TrisCl pH8.3, 375mM Kcl, 15mM MgCl) in a 20. mu.l reaction volume₂) 2. mu.l of 100mM DTT, 1. mu.l of 10mM dNTP and 200 units of Superscript^TMII reverse transcriptase (Gibco/BRL). The reaction was terminated by keeping the temperature at 45 ℃ for 1 hour and at 95 ℃ for 5 minutes. RNA was hydrolyzed with 400. mu.M NaOH at 65 ℃ for 30 minutes and then neutralized with 400. mu.M acetic acid. Then 3 volumes of 6M NaI and 10. mu.l of treated glass beads were added to the reaction.The glass beads were washed 3 times with 80% ethanol, and the nucleic acid was eluted from the glass beads into 45. mu.l of water. The nucleic acid was then pelleted and resuspended in 10. mu.l of water. The purified first strand cDNA was ligated with the manufacturer's supplied anchor oligonucleotide (SEQ ID NO: 9, 5'-CAC GAA TTC ACT ATC GAT TCT GGA ACC TTC AGA GG-3') using T4 RNA ligase at 27 ℃ for 20 hours. One tenth of the ligation reaction was taken for PCR amplification and contained 1. mu.M of the manufacturer's anchored primer (SEQ ID NO: 10, 5'-CTG GTT CGG CCC ACC TCT GAA GGT TCC AGA ATCGAT AG-3'), 1. mu.M of the mammaglobin-specific primer D2Rb (SEQ ID NO: 11, 5'-AAT CCGTAG TTG GTT TCT CAC C-3'), 200. mu.M dNTP, 5 units Vent in a 50. mu.l reaction volume^TMDNA polymerase and 1 XPase buffer (10mM KCl, 20mM TrisCl, 10mM (NH)₄)₂SO₄，2mM MgSO₄0.1% Triton X-100). The reaction was incubated at 94 ℃ for 2 minutes, then at 94 ℃ for 45 seconds, 50 ℃ for 1 minute, and 72 ℃ for 90 seconds for 40 cycles.

Two downstream mammaglobin-specific nested oligonucleotides were D2R (SEQ ID NO: 4) and D2Rb (SEQ ID NO: 11). The upstream mammaglobin-specific regulatory oligonucleotide used was also as recommended by the manufacturer, namely D2F (5'-CTT TCT GCA AGA CCT TTG GC-3') (SEQ ID NO: 12). Vent was used for all PCR amplification reactions^TMDNA polymerase (New England Biolabs). The amplified RACE product was digested with Ecori and ligated to EcoRI and SmaI sites of the plasmid vector pGEM7Z (Promega).

All sequencing was performed using the Taq DNA polymerase thermocycling sequencing kit according to the manufacturer's (Promega) protocol. The procedure used is generally as follows.

With 10pmol³²P-gamma ATP (3,000Ci/mmol and 10mCi/ml) was end-labeled with 10pmol of a sequence-specific oligonucleotide and reacted with T4 polynucleotide kinase in a volume of 10. mu.l for 30 minutes at 37 ℃. The polymerization reaction was carried out in 17. mu.l of the manufacturer's sequencing buffer containing 100ng of plasmid template, 1.5pmol of labeled sequencing primer, and 5 units of sequencing-grade Taq polymerase. Will be provided withThe reaction was divided into a set of 4 reaction tubes containing a mixture of deoxynucleotides and dideoxy-A, C, G or T as supplied by the manufacturer. This set of 4 tubes was incubated at 95 ℃ for 2 minutes, 94 ℃ for 45 seconds, 45 ℃ for 30 seconds, then at 72 ℃ for 1 minute, and so on for 30 cycles. After the reaction was complete, 3. mu.l of 80% formamide/bromophenol blue dye was added to each tube. Samples were loaded on a 6% acrylamide/7.5M urea sequencing gel after heating at 70 ℃ for 2 minutes and run at a constant pressure of 60W for 2 to 4 hours. The gel was dried and then contacted with Kodak XAR 5X-ray film for 2 to 24 hours.

The sequence thus obtained is a 403bp fragment (SEQ ID NO: 5) represented by the solid line in FIG. 2. DEST002 marker sequences were isolated in previous work (Watson and Fleming, supra). The sequence is a206 bp fragment (SEQ ID NO: 6) as indicated by the open line in FIG. 2. The information of these two sequences was combined to derive a full-length 503bp mammaglobin cDNA (FIG. 2). Example 2

This example demonstrates that expression of mammaglobin is restricted to breast tumor cells only, and to a small extent in normal breast cells.

Total cellular RNA samples were isolated using the standard guanidine isocyanate method and treated with RNase-free DNase (Promega). For PT/PCR, oligo dT was used₂₁(SEQ ID NO: 13) and Superscript II reverse transcriptase (Gibco/BRL) 1. mu.g of the total RNA was reverse transcribed following the manufacturer's protocol.

200ng dT₂₁(SEQ ID NO: 13) and 1. mu.g of total RNA were incubated in a volume of 10. mu.l at 65 ℃ for 5 minutes. The sample was cooled in ice and 4. mu.l of 5 XTT buffer (250mM TrisCl pH8.3, 375mM KCI, 15mM MgCl) was added thereto₂) 2. mu.10 mM DTT, 1. mu.l 10mM dNTP and 200 units Superscript^TMII reverse transcriptase (Gibco/BRL). The reaction was terminated by carrying out the reaction at 45 ℃ for 1 hour and then incubating at 95 ℃ for 5 minutes.

One tenth of each RT reaction was subjected to PCR analysis using mammaglobin-specific primers D2R (5'-ATA AGA AAG AGA AGG TGT GG-3') (SEQ ID NO: 4) and D2102 (5'-CAGCGG CTT CTT TGA TCC TTG-3') (SEQ ID NO: 3) cycled 40 times under standard reaction conditions: 94 ℃ X30 seconds/55 ℃ X1 minute/72 ℃ X1 minute.

For Northern analysis, 20. mu.g of total RNA was analyzed using the full-length mammaglobin cDNA probe according to the known technique (Watson and Fleming, supra). Integrity and equal loading of each RNA sample was assessed by ethidium bromide staining.

As shown in fig. 4A, the 500bp mammaglobin message was readily detectable in tumor sample 2410 (from which the original DEST was isolated), much less in normal human breast tissue, and not in the immortalized mammary epithelial cell line B5-589 or human lung, placenta, uterus and ovary (fig. 4A). After amplification by RT/PCR analysis, mammaglobin expression was still not found in 15 tissues tested (FIG. 4B). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger (fig. 4B) and EGF receptor messenger (not shown) were detected in these reactions, indicating that no expression was not due to degradation of RNA or other minor reasons. Therefore, mammaglobin mRNA expression is relatively breast tissue specific. Example 3

This example demonstrates that mammaglobin cDNA encodes a translatable nucleotide sequence that produces a protein product having a correctly predicted molecular weight. Using TNT^TMRabbit reticulocyte translation kit using T7RNA polymerase (Promega) and³⁵s-methionine (> 1000 Ci/mmol; 10mCi/ml, Amersham) was translated in vitro according to the manufacturer' S protocol.

At 25. mu.l TNT^TMMu.l of the reaction buffer prepared by the manufacturer, T7RNA polymerase, 20. mu.M of an amino acid mixture other than methionine, 40. mu. Ci were added to the rabbit reticulocyte lysate³⁵S-methionine (1000Ci/mmol and 10mCi/ml), 40 units of ribonuclease inhibitor, 1. mu.g mammaglobin/pGEM 7 plasmid and water treated with sufficient DEPC to make the final reaction volume 50. mu.l. The reaction mixture was incubated at 30 ℃ for 60 minutes. Mu.l of the reaction mixture was added to 20. mu.l of SDS gel buffer, boiled for 2 minutes, and then loaded on 17.5% SDS polyacrylamide gel.

Rabbit reticulocytes treated with mammaglobin cDNA produced a 60kD protein, while none of the protein products produced by treatment with cDNA. Example 4

This example demonstrates the high prevalence of mammaglobin overexpression in primary breast cancer.

To determine the frequency of overexpression of mammaglobin in breast cancer, we examined a panel of 15 different histological types of stage I breast cancer using Northern blot hybridization probed with mammaglobin cDNA. Corresponding normal breast tissue samples from both patients were also compared (fig. 6). 500bp mammaglobin mRNA was found in normal breast tissue and tumors 2410 and 3 other tumors, 2 of which were expressed in patients with little or no expression in the corresponding normal breast tissue (BO15 vs. BO 16; BO22 vs. BO23) (FIG. 6). In summary, 4 (27%) of the 15 tumors examined overexpressed mammaglobin mRNA. The above data indicate that overexpression of mammaglobin is not limited to only one tumor sample, and in fact it is quite common in primary breast tumors. Moreover, the fact that all examined tumors were stage I indicates that this disorder occurs quite early in the process of breast tumorigenesis. Example 5

The following example illustrates the use of polyclonal antibodies to detect mammaglobin.

A peptide corresponding to the 16C-terminal amino acids deduced from mammaglobin cDNA (Glu-Val-Phe-Met-Gln-Leu-Ile-Tyr-Asp-Ser-Leu-Cys-Asp-Leu-Phe, SEQ ID NO: 14) was coupled with keyhole limpet hemocyanin, followed by injection into rabbits with Freund's adjuvant, thereby preparing a polyclonal antibody. Vaccinated rabbits were boosted at 3 week intervals and then bled at 12 weeks and their sera assayed for capacity to detect mammaglobin. Serum-free conditioned media (24 hour pool) were harvested from the breast cancer cell lines MDA-MB-415 and MCF-7. MDA-MB-415 has been identified as a cell line that overexpresses the mammaglobin message, while MCF-7 has been identified as a cell line that does not produce detectable mammaglobin. Conditioned media were loaded on 12% SDS polyacrylamide gels under reducing conditions and blotted onto Nytran filters, and the assay was performed according to standard Western blotting using antibodies to the C-terminal peptide as the primary antibody in the assay. After the primary antibody was bound, the blot was washed and secondary antibody (goat anti-rabbit) was added. Mammaglobin-antibody complexes were visualized using enzyme-linked chemiluminescence (ECL Western blot detection reagent, Amersham, Arlington Heights, IL). Conditioned media from the MDA-MB-415 cell line showed a mammaglobin band with an apparent molecular weight of 20kD, which was not observed in conditioned media from the MCF-7 cell line. Thus, MDA-MB-415 cells secrete mammaglobin while MCF-7 cells do not.

To further demonstrate the specificity of this protein, conditioned media and cell lysates of the MDA-MB-415 cell line were analyzed by Western blotting using antibodies to the C-terminal peptide, in the presence and absence of competing peptides for antibody production. Mammaglobin-antibody complexes were visualized as described previously. As shown in FIG. 7, in the absence of competing peptides (-), conditioned media had a20 kD band representing mammaglobin. Cell lysate (C) has multiple bands at molecular weights of 14kD, 20kD and higher. The 14kD band may represent mammaglobin in its raw form. The cDNA of mammaglobin has a consistent N-glycosylation site, so the observed 20kD secreted protein represents a partially processed form of the protein. When Western blots were performed in the presence of competing peptides (+), no secreted and intracellular forms of mammaglobin were visualized, indicating that these proteins comprise the peptide to which the synthetic antibody was directed.

Antibodies to this C-terminal peptide also detected a similar band in cell lysates of primary breast tumor samples (fig. 8). In addition, the antibody also showed reactivity with breast tumor cells by immunohistochemical staining of paraffin-fixed patient breast cancer sections (fig. 9). Immunohistochemical staining was performed with horseradish peroxidase-labeled antibody and goat anti-rabbit antibody, using tetrahydrochysene 3, 3' -Diaminobenzene (DAB) as substrate. Cells expressing mammaglobin were stained brown.

Based on the above results, we considered mammaglobin as a secreted protein, and the mammaglobin as synthesized was a precursor protein, and before secretion, post-translational modification increased its necessary apparent molecular weight; mammaglobin can be detected in a human breast tumor sample. In cancer diagnosis using mammaglobin as a breast cancer marker, in evaluating the deterioration of breast tumors, in detecting whether autologous bone marrow/stem cells are contaminated with tumor cells, in breast cancer vaccines, in directionally therapeutically interfering breast cancer tumor cells using antibody-mediated complexes, the detection of mammaglobin is feasible.

From the foregoing, it can be seen that the present invention has many advantages, and other advantageous effects are obtained.

While the above methods and compositions are susceptible to various modifications within the scope of the invention, the specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Sequence listing (1) general information: (i) the applicant: WASHINGTON UNIVERSITY (ii) invention name: DNA sequence and number of breast-specific breast cancer protein (iii) sequences encoded thereby: 14(iv) communication address:

(A) the addressee: HOWELL & HAFERKAMP, L.C.

(B) Street: 7733 Forsyth BOULEVARD, SUITE 1400

(C) City: louis, st

(D) State: MISSOURI

(E) The state is as follows: USA

(F) And E, postcode: 63105-1817(v) computer readable form:

(A) type of recording medium: flexible disk

(B) A computer: IBM PC compatible

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patentln Release #1.0, Version #1.25(vi) material of the present application:

(A) application No.:

(B) application date:

(C) and (4) classification: (viii) lawyer/attorney information:

(A) name: HOLLOD, DONALD R.

(B) Registration number: 35, 197

(C) Reference/case No.: 964796(ix) communication information:

(A) telephone: (314)727-5188

(B) Faxing: (314)727-6092(2) SEQ ID NO: 1, information: (i) sequence characteristics:

(A) length: 503 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cdna for mRNA (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 1: GACAGCGGCT TCCTTGATCC TTGCCACCCG CGACTGAACA CCGACAGCAG CAGCCTCACC 60ATGAAGTTGC TGATGGTCCT CATGCTGGCG GCCCTCTCCC AGCACTGCTA CGCAGGCTCT 120GGCTGCCCCT TATTGGAGAA TGTGATTTCC AAGACAATCA ATCCACAAGT GTCTAAGACT 180GAATACAAAG AACTTCTTCA AGAGTTCATA GACGACAATG CCACTACAAA TGCCATAGAT 240GAATTGAAGG AATGTTTTCT TAACCAAACG GATGAAACTC TGAGCAATGT TGAGGTGTTT 300ATGCAATTAA TATATGACAG CAGTCTTTGT GATTTATTTT AACTTTCTGC AAGACCTTTG 360GCTCACAGAA CTGCAGGGTA TGGTGAGAAA CCAACTACGG ATTGCTGCAA ACCACACCTT 420CTCTTTCTTA TGTCTTTTTA CTACAAACTA CAAGACAATT GTTGAAACCT GCTATACATG 480TTTATTTTAA TAAATTGATG GCA 503

(2) SEQ ID NO: 2, information: (i) sequence characteristics:

(A) length: 93 amino acid

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: protein (iii) hypothesis: no (xi) sequence description: SEQ ID NO: 2:

Met Lys Leu Leu Met Val Leu Met Leu Ala Ala Leu Ser Gln His Cys

1 5 10 15

Tyr Ala Gly Ser Gly Cys Pro Leu Leu Glu Asn Val Ile Ser Lys Thr

20 25 30

Ile Asn Pro Gln Val Ser Lys Thr Glu Tyr Lys Glu Leu Leu Gln Glu

35 40 45Phe Ile Asp Asp Asn Ala Thr Thr Asn Ala Ile Asp Glu Leu Lys Glu

50 55 60

Cys Phe Leu Asn Gln Thr Asp Glu Thr Leu Ser Asn Val Glu Val Phe

65 70 75 80

Met Gln Leu Ile Tyr Asp Ser Ser Leu Cys Asp Leu Phe

8590 (2) SEQ ID NO: 3, information: (i) sequence characteristics:

(A) length: 21 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cdna (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 3: CAGCGGCTTC CTTGATCCTT G21 (2) SEQ ID NO: 4: (i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cdna (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 4: ATAAGAAAGA GAAGGTGTGG 20(2) SEQ ID NO: 5, information: (i) sequence characteristics:

(A) length: 403 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cDNA to mRNA (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 5: GACAGCGGCT TCCTTGATCC TTGCCACCCG CGACTGAACA CCGACAGCAG CAGCCTCACC 60ATGAAGTTGC TGATGGTCCT CATGCTGGCG GCCCTCTCCC AGCACTGCTA CGCAGGCTCT 120GGCTGCCCCT TATTGGAGAA TGTGATTTCC AAGACAATCA ATCCACAAGT GTCTAAGACT 180GAATACAAAG AACTTCTTCA AGAGTTCATA GACGACAATG CCACTACAAA TGCCATAGAT 240GAATTGAAGG AATGTTTTCT TAACCAAACG GATGAAACTC TGAGCAATGT TGAGGTGTTT 300ATGCAATTAA TATATGACAG CAGTCTTTGT GATTTATTTT AACTTTCTGC AAGACCTTTG 360GCTCACAGAA CTGCAGGGTA TGGTGAGAAA CCAACTACGG ATT 403(2) SEQ ID NO: 6: (i) sequence characteristics:

(A) length: 206 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cdna for mRNA (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 6: TTTATGCAAT TAATATATGA CAGCAGTCTT TGTGATTTAT TTTAACTTTC TGCAAGACCT 60TTGGCTCACA GAACTGCAGG GTATGGTGAG AAACCAACTA CGGATTGCTG CAAACCACAC 120CTTCTCTTTC TTATGTCTTT TTACTACAAA CTACAAGACA ATTGTTGAAA CCTGCTATAC 180ATGTTTATTT TAATAAATTG ATGGCA206(2) SEQ ID NO: 7, information: (i) sequence characteristics:

(A) length: 95 amino acid

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: protein (iii) hypothesis: no (xi) sequence description: SEQ ID NO: 7:

Met Lvs Leu Val Phe Leu Phe Leu Leu Val Thr Ile Pro Ile Cys Cys

1 5 10 15

Tyr Ala Ser Gly Ser Gly Cys Ser Ile Leu Asp Glu Val Ile Arg Gly

20 25 30

Thr Ile Asn Ser Thr Val Thr Leu His Asp Tyr Met Lys Leu Val Lys

35 40 45

Pro Tyr Val Gln Asp His Phe Thr Glu Lys Ala Val Lys Gln Phe Lys

50 55 60

Gln Cys Phe Leu Asp Gln Thr Asp Lys Thr Leu GIu Asn Val Gly Val

65 70 75 80

Met Met Glu Ala Ile Phe Asn Ser Glu Ser Cys Gln Gln Pro Ser

859095 (2) SEQ ID NO: information of 8: (i) sequence characteristics:

(A) length: 91 amino acid

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: protein (iii) hypothesis: no (xi) sequence description: SEQ ID NO: 8:

Met Lys Leu Ala Val Thr Leu Thr Leu Val Thr Leu Ala Leu Cys Cys

1 5 10 15

Ser Ser Ala Ser Ala Glu Ile Cys Pro Ser Phe Gln Arg Val Ile Glu

20 25 30

Thr Leu Leu Met Asp Thr Pro Ser Ser Tyr Glu Ala Ala Met Glu Leu

35 40 45

Phe Ser Pro Asp Gln Asp Met Arg Glu Ala Gly Ala Gln Leu Lys Lys

50 55 60

Leu Val Asp Thr Leu Pro Gln Lys Pro Arg Glu Ser Ile Ile Lys Leu

65 70 75 80

Met Glu Lys Ile Ala Gln Ser Ser Leu Cys Asn

8590 (2) SEQ ID NO: 9, information: (i) sequence characteristics:

(A) length: 35 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cdna (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 9: CACGAATTCA CTATCGATTC TGGAACCTTC AGAGG 35(2) SEQ ID NO: 10, information: (i) sequence characteristics:

(A) length: 38 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cdna (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 10: CTGGTTCGGC CCACCTCTGA AGGTTCCAGA ATCGATAG 38(2) SEQ ID NO: 11, information: (i) sequence characteristics:

(A) length: 22 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cdna (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 11: AATCCGTAGT TGGTTTCTCA CC 22(2) SEQ ID NO: 12, information: (i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cdna (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 12: CTTTCTGCAA GACCTTTGGC 20(2) SEQ ID NO: 13, information: (i) sequence characteristics:

(A) length: 21 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: cDNA to mRNA (iii) hypothesis: (iii) no (iv) antisense: no (xi) sequence description: SEQ ID NO: 13: TTTTTTTTTT TTTTTTTTTT T21 (2) SEQ ID NO: 14, information: (i) sequence characteristics:

(A) length: 16 amino acid

(B) Type (2): amino acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: peptide (iii) hypothesis: no (xi) sequence description: SEQ ID NO: 14: glu Val Phe Met Gln Leu Ile Tyr Asp Ser Ser Leu Cys Asp Leu Phe 151015

Claims (23)

1. A purified and isolated polynucleotide comprising the sequence of SEQ ID NO: 1 or a derivative thereof.

2. The purified and isolated polynucleotide of claim 1, wherein the polynucleotide is SEQ id no: 1 or a derivative thereof.

3. The purified and isolated polynucleotide of claim 1, wherein the polynucleotide comprises a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 sequence.

4. A purified and isolated peptide comprising SEQ ID NO: 2 or a derivative thereof.

5. The purified and isolated peptide of claim 4, wherein the peptide is SEQ ID NO: 2 or a derivative thereof.

6. A method for detecting whether a patient has breast cancer, the method comprising detecting and/or quantifying in a sample from the patient a nucleic acid encoding SEQ ID NO: 2 or a derivative thereof, the level of which in excess of that in a normal individual is indicative of the presence of breast cancer cells.

7. The method of claim 6, wherein the detecting and/or quantifying step further comprises:

(a) providing a polypeptide comprising SEQ ID NO: 1 or a derivative thereof, or a pharmaceutically acceptable salt thereof,

(b) incubating the polynucleotide with the sample under conditions such that the polynucleotide hybridizes to mRNA of the breast neoplasia cells, and

(c) detecting the presence of the NDA-RNA hybridization complex.

8. The method of claim 7, wherein the polynucleotide is SEQ ID NO: 1.

9. the method of claim 7, wherein the polynucleotide comprises a sequence encoding a polypeptide comprising the amino acid sequence of SEQ id no: 2 sequence.

10. A kit for detecting the presence of a breast neoplasia cell in a sample comprising a nucleic acid sequence having SEQ ID NO: 1 or a derivative thereof.

11. The kit of claim 10, wherein the polynucleotide sequence is SEQ ID NO: 1.

12. the kit of claim 10, wherein the polynucleotide comprises a sequence encoding a polypeptide having the sequence set forth in SEQ id no: 2 sequence.

13. The method of claim 6, wherein the detecting and/or quantifying step further comprises:

(a) producing cDNA from mRNA in a sample taken from a patient by reverse transcription,

(b) two oligonucleotides are provided as primers for polymerase chain reaction, which are located in a sequence encoding SEQ ID NO: 2 or wherein either side of the cDNA of (2),

(c) amplification of cDNA encoding mammaglobin by polymerase chain reaction, and

(d) detecting the presence or absence of a nucleic acid encoding SEQ ID NO: 2.

14. The method of claim 13, wherein one of the two oligonucleotides comprises SEQ id no: 3, and the other segment comprises SEQ ID NO: 4.

15. a kit for detecting the presence of a mammary neoplasia cell in a sample comprising two oligonucleotide segments packaged in a container, which are polymerase chain reaction primers and are located within a nucleic acid sequence encoding SEQ ID NO: 2 or flanking it.

16. The kit of claim 15, wherein one of said two oligonucleotides comprises SEQ id no: 3 and the other stretch comprises SEQ ID NO: 4.

17. a method of detecting whether a patient has breast cancer, the method comprising detecting and/or quantifying in a sample from the patient a nucleic acid comprising SEQ ID NO: 2, the concentration of said protein exceeding that of a normal individual is indicative of the presence of breast cancer.

18. The method of claim 17, wherein the detecting and/or quantifying step further comprises contacting the purified antibody with a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or an epitope thereof, and detecting a protein comprising SEQ ID NO: 2 or an epitope thereof with an antibody.

19. The method of claim 18, wherein the purified antibody is a polyclonal antibody.

20. The method of claim 19, wherein the purified antibody is a monoclonal antibody.

21. A kit for detecting the presence of a breast neoplasia cell in a sample comprising a purified antibody packaged in a container, the antibody capable of detectably hybridizing to a polypeptide comprising SEQ ID NO: 2 or an epitope thereof.

22. The kit of claim 21, wherein the purified antibody is a polyclonal antibody.

23. The method of claim 22, wherein the purified antibody is a monoclonal antibody.