CA2347906A1 - A novel method of diagnosing, monitoring, staging, imaging and treating breast cancer - Google Patents

Abstract

The present invention provides a new method for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating breast cancer.

Description

A NOVEL METHOD OF DIAGNOSTNG, MONITORJ:NG, STAGING, IMAGING AND TREATING BREAST CANCER
FIELD OF T:HE INVENTION
This invention relates, in part, to newly developed assays for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating cancers, particularly breast cancer.
BACKGROUND OF THE INVENTION
One of every nine American women will develop breast cancer sometime during her life based on a lifespan of 85 years. Annually, over 180,000 women in the United States will be diagnosed with bz'east cancer and approximately 46,000 will die of the disease.
Every woman i.s at risk for breast cancer. A woman's chances of developing breast cancer increase as she grows older; 80 percent of: all cancers are found in women over the age of 50. There are also several risk factors that can increase a woman's chances of developing cancer. A woman may be at increased risk: if she has a family history of the disease, if she had her first child after the age of 30 or has no children, or if :she began menstruating early.
However, more than 70 percent of women who develop breast cancer have n« known risk factors. Less than 10 percent of breast cancer case; are thought to be related to the BRCA1 gene disco~rered in LS~94. Researchers are now investigating the role other factor's such as nutrition, alcohol, exercise, smoking, and oral contraceptives may play in cancer prevention.
As with many other cancers, the best chance for successful treatment occurs when breast cancer is found early.

WO 00/08210 PCT/I:fS99/16811 Mammograms, special x:-rays of the breast, can detect more than 90 percent: of all lareast cancer's. If breast cancer is found early, tl-..e chance of cure i:> greater than 90 percent.
Treatment options include surgery, chemotherapy, and radiation therapy depending on tree stage of the cancer.
Procedures used for detect:ing, diagnosing, monitoring, staging, :~rognostica.t:ing and imaging breast cancer are of critical importance r_o the outcome of the patient. Patients diagnosed with early breast cancer generally have a much greater five-year survival rate as compared to the survival rate for ~~atients diagnosed with distant metastasized breast cancer. New di_agno:~t.ic methods which are more sensit=ive and specific for detect:i.ng early breast cancer are clearly needed.
Bre~~st cancer ~at.ients az~e closely monitored following initial therapy arid during ac~juvant therapy to determine response too therapy and to detect persistent or recurrent disease of metastasis. There is clearly a need for a breast cancer marker whir.~h is more sensitive and specific in detecting breast cancer and its recurrence and progression.
Another impoi:t<~nt step in managing breast cancer is to determine the stage of the patient's disease. Stage determination has potential prognostic value and provides criteria for designing optimal therapy. Generally, pathological staging of breast cancer is preferable over clinical staging because the former gives a more accurate prognosis. However, clinical staging would be preferred were it at lea:~t as accurate as pathological staging because it does not depend on an invasive procedure to obtain tissue for pathological evaluation. Staging of breast cancer would be improved b~~ detecting new markers in cells, tissues, or bodily fluids which could c:ii.f_ferentiate between different stages of invasion.
In the present invention methods are provided for detecting, diagnosing, moni~oring, staging, prognosticating, imaging anc3 treating breast cancer via 9 Breast Specific Genes (BSGs). The 9 BSGs refer, among other things, to native proteins expressed by the genes comprising the polynucleotide sequences of any of SEQ ID NO: 1-9. In the alternative, what is meant by the 9 BSCxs as used herein, means the native mRNAs encoded b:~ the genes comprising any of the polynucleotide sequences of SEQ ID NO: 1--9 or it can refer to the actual genes comprising arty of the polynucleotide sequences of SEQ
ID NO: 1-9.
Other object~~, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the following description. It should be understood., however, that t:he Following description and the specific examples, while indicating preferred embodiments of the invention, are rliven by way cf illustration only. Various changes and modificat:.ions within the spirit and scope of the disclosed inventiorv will. become readily apparent to those skilled in the art t:rom reading the following description and from reading the other parts of the present disclosure.
SUMMARY OF THE INVENTION
Toward these ends, and others, it is an object of the present invention to provide a method for diagnosing the presence of breast cancer by analyzing for changes in levels of BSG in cells, tissues or bodily fluids compared with levels of BSG in ~~referably the same cells, tissues, or bodily fluid type of a normal human control, wherein a change in levels of BSG in tree patient versus the normal human control is associated with brea~ct: cancer.
Further provided is a method of diagnosing metastatic breast cancer in a patient having such cancer which is not known to Have metastasized by identifying a human patient suspected of havin_~ breast. cancer that has metastasized;
analyzing a sample of cells, tissues, or. bodily fluid from such patient for BSC~; comparing the BSG levels in such cells, tissues, or bodily fluid with levels of BSG in preferably the same cell:, tissue;, or bodily fauid type of a normal human control, wherein a ch<~nge i:n BSG levels in the patient versus the normal human control is associated with a cancer which has metastasized.
Also provided by the invention is a method of staging breast cancer in a human which has such cancer by identifying a human patient having such cancer; analyzing a sample of cells, ti:~sues, or bodily fluid from such patient zor BSG;
comparing BSG levels in such cells, tissues, or bodily fluid with levels of BSG in preferably the same cells, tissues, or bodily fluid t:ype of a normal human control sample, wherein a change in BSG levels in the p,:~~ient: versus the normal human control i~~ associat:.ed with a c<~ncer which is progressing or regressing or in remission.
Further provided is a method of monitoring breast cancer in a human having sur_h cancer for the onset of metastasis.
The method comprises identifying a human patient having such cancer that is not known to have metastasized; periodically analyzing a sample of: cells, tissues, or bodily fluid from such patient for BSG; compai_-ing the BSG levels in such cells, tissue, or bodily fluid with levels of BSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, whewein a change in BSG levels in the patient versus the normal human control is associated with a cancer which has metastasized.
Further provided is a method of monitoring the change in stage c>f breast cancer in a human having such cancer by looking at levels of BSG in a human having such cancer. The method comprises identifying a human patient having such cancer; periodically analyzing a sample of cells, tissues, or bodily fluid from r~wc:h patient for BSG; comparing the BSG
levels in .such cell;, tissue, or bodily fluid with levels of BSG in preferably t:he same cells, tissues, or bodily fluid type of a normal human control sample, wherein a change in BSG
levels in the patient versus the normal human control is associated with a cancer which is progressing or regressing or in remission.
Furvher provided are antibodies against the BSGs or fragments of such ant:ibodies which can be used to detect or image loc<~lization of the BSGs in a patient for the purpose of detecting or diagnosing a disease or condition. Such antibodie:~ can be polyclonal c>r monoclonal, or prepared by molecular biology t:ec:hniques . The term "antibody" , as used herein and throughout. the instant specification is also meant to include aptamers and single-stranded oligonucleotides such as those derived from an in vitro evolution protocol referred to as SEL~EX and we_L1 known tc those skilled in the art.
Antibodies can be labeled with <~ variety of detectable labels including, but not l.~.mited to, radioisotopes and paramagnetic metals. 'these antibodies or fragments thereof can also be used as therapeutic agents in the treatment of diseases characterized by expression of a BSG. In therapeutic applications, the antibody can be used without or with derivatiza.tion to a cytotoxic agent such as a radioisotope, enzyme, toxin, drug or a prodrug.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the iol.lowing description. It should be understood., however, that t:he following description and the specific examples, Wh=Lle indicating preferred embodiments of the invention, are given by way of illustration only. various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.
DESCRIPTION OF THE IPdVENTION
The present invention relates to diagnostic assays and methods, both quant~_tative and qualitative for detecting, diagnosing, monitoring, staging, prognosticating and imaging cancers by comparing levels of BSG with those of BSG in a normal human contrcil. What is meant by levels of BSCi as used herein, means level: of the native protein expressed by the genes comprising the pc~lynuclectide sequence of any of SEQ ID
NO: 1-9. In the alternative, what is meant by levels of BSG
as used herein, means level: of the native mRNA encoded by any of the genes comprising any of the polynucleotide sequences of SEQ ID NO: 1-9 or levels of the gene comprising any of the polynucleotide sequence of SEQ ID NO: 1-9. Such levels are preferably measured :in at least one of, cells, tissues and/or bodily fluids, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention fo:r measuring changes in levels of any one of the BSCi proteins compared to normal control bodily fluids, cells, or tissue samples may be used to diagnose the presence of cancer~~, including breast cancer. By "change" it is meant either an increase or decrease in levels of the BSG. For example, :Eor BSGs such as Mam001 (SEQ ID N0:2), Mam004 (SEQ
ID N0:4) ;end Mam00~:~ (SEQ ID N0:3), an increase in levels as compared 1~o normal human controls is associated with breast cancer, metastasis and progression of the cancer, while a decrease in levels is as;~ociation with regression and/or remission. For thE: BSG Mam002 (SEQ ID NO:1), a decrease in levels as compared t:o normal. human controls is associated with breast cancer, metastasis and progression while an increase is associated with regression and/or remission. Any of the 9 BSGs may be measured alone in the methods of the invention, or all together or a:ny combination of the nine.
All the methods of the present invention may optionally include measuring the levels of other cancer markers as well as BSG. Other cancer markers, in addition to BSG, such as BRCAl are known to t.'.~ose of skill in the art.

Diagnostic' Assays The present invention provides methods for diagnosing the presence of bre<~st cancer by analyzing for changes in levels of :BSG in cells, tissues or bodily fluids compared with levels of :BSG in cells, tissues or bodily fluids of preferably the same type from a norma7_ human control. As demonstrated herein an increase i:n level.s of= BSGs such as Mam001 (SEQ ID
N0:2), Mam004 (SEQ ID N0:4) or Mam005 (SEQ ID N0:3) in the patient versus the normal human control is associated with the presence of breast c=ancer, while a decrease in levels of BSGs such as Mam002 (SEQ TD NO:1) in the patient versus the normal human cont=rol is a~;sociatc=_d with the presence of breast cancer.
Without limiting the instant invention, typically, for a quantitative diagnostic a:~say a positive result indicating the patient being trested has cancer is one in which cells, tissues, o:r bodily fluid levels of the cancer marker, such as BSG, are at least two times higher or lower, and most preferably are at least five times higher or lower, than in preferably the same cells, tissues, or bodily fluid of a normal human contro:L.
The present invention also provides a method of diagnosing metastatic breast cancer in a patient having breast cancer which has not. yet metastasized for the onset of metastasis. In the method of the present invention, a human cancer patient suspected of having breast cancer which may have metastasized (but which was not previously known to have metastasized) is identified. This is accomplished by a variety of means known to those of skill in the art . For example, in the case o:f breast cancer, patients are typically diagnosed with breast cancer following traditional detection methods.
In the present :invention, determining the presence of BSG level in cells, tissues, or bodily fluid, is particularly useful for discriminat::ing between breast cancer which has not _ g _ metastasi~:ed and breast cancer which has metastasized.
Existing technique.:; have difficulty discriminating between breast cancer which has metastasized and breast cancer which has not metastasized and proper treatment selection is often dependent upon such knowledge.
In the present invention, the cancer marker levels measured in such cel:Ls, tissues, or bodily fluid is BSG, and are compared with levels of BSG in preferably the same cells, tissue, or bodily f:LL;id type of a normal human control. That is, if the cancer m,~rke:r being observed is just BSG in serum, this level- is preferably compared with the level of BSG in serum of a normal han:an pat-gent. An increase in BSGs such as Mam001 (SEQ ID N0:2), Mam004 (SEQ ID N0:4) or Mam00S (SEQ ID
N0:3) in the patient: versus the normal human control is associated with brea:~t cancer which has metastasized while a decrease in BSGs such as Mam002 (SEQ ID NO: l) in the patient versus the normal human control is associated with breast cancer which has metastasized.
Without limiting the instant invention, typically, for a quantitative diagnostic assay a positive result indicating the cancer in the ~,~atient being tested or monitored has metastasized is one in which cells, tissues, or bodily fluid levels of the cancer marker, sucr: as BSC:~, are at least two times higher or lower, and most preferably are at least five times higher or lowe r, than in preferably the same cells, tissues, or bodily f7_uid of a normal patient.
Normal human control. as used herein includes a human patient without cancer and/or non cancerous samples from the patient; ~~n the methods for diagnosing or monitoring for metastasis, normal human contro~_ preferably comprises samples from a human patient that i.s determined by reliable methods to have breast cancer- which has not metastasized, such as earlier samples of the same patient.

g _ Staging The invention also provides a method of staging breast cancer in a human patient.
The method comprises identifying a human patient having such cancer; analyzing a sample of cells, tissues, or bodily fluid from such patient for BSG. Then, the method compares BSG levels in such cells, tissue~~, or bodily fluid with levels of BSG in preferably t:he same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in levels of BSGs such Gm~ Mam001 (SEQ ID N0:2), Mam004 (SEQ ID
N0:4) or M~.m005 (SEQ ..D N0:3) or a decrease in levels of BSGs such as Mam002 (SEQ TD NO:1) in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in levels of BSGs such as Mam001 (SEQ ID N0:2), Mam004 (SEQ ID N0:4) c>r Mam005 (SEQ ID N0:3) or an increase in levels of BSGs such as Mam002 (SEQ ID NO: l) is associated with a cancer which is regressing or in remission.
Monitoring Further provided is a method of monitoring breast cancer in a human having such cancer for the onset of metastasis.
The method comprises .identifying a human patient having such cancer that: is not h:nown to have metastasized; periodically analyzing ~i sample ~:~f cells, tissues, or bodily fluid from such patient fo:r BSG; comparing the BSG levels in such cells, tissue, or bodily fluid with levels of BSG in preferably the same cells, tissues, or bodily Fluid type of a normal human control sample, wherein an increase in levels of BSGs such as Mam001 (SEQ ID N0:2), Mam004 (SEQ ID N0:4) or Mam005 (SEQ ID
N0:3) or a decrease in levels of BSGs such as Mam002 (SEQ ID
NO:l} in the patient versus the normal human control is associated with a cancer which has metastasized.
Further provided by this invention is a method of monitoring the change in stage of breast cancer in a human having such cancer. The method comprises identifying a human - io -patient having such cancer; periodically analyzing a sample of cells, tissues, oz- bodily fluid from such patient for BSG;
comparing the BSG 1.=vets in such cells, tissue, or bodily fluid with levels of BSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in levels of BSGs such as Mam001 (SEQ ID NO:2) , MamC)04 (SEQ TD N0:4) or Mam005 (SEQ ID N0:3) or a decrease in leve_Ls of BSGs such as Mam002 (SEQ :ID NO:1) in the patient versu:~ the normal human control is associated with a cancer which is progressing in stage and a decrease in the levels of BSGs such as Mam001 (SEQ ID N0:2), Mam004 (SEQ
ID N0:4) or Mam005 (SEQ ID N0:3) or an increase in levels of BSGs such as Mam002 {SEQ ID NO:1) is associated with a cancer which is regressing -_.n stage or in remission.
Monitoring such patient for onset of metastasis is periodic and preferab-.~y done on a quarterly basis. However, this may be=_ more or less frequent depending on the cancer, the particular patient, and the stage of the cancer.
Assay Techniques Assay techniques that can be used to determine levels of gene ex:oression, such as BSG of the present invention, in a sample df:rived frc:>m a host are well-known to those of skill in the art:. Such a:~say methods include radioimmunoassays, reverse transcri.ptase PCR (RT-PCR) assays, immunohist~achemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA
assays and proteomic: approaches. Among these, ELISAs are frequently preferred to diagnose a gene's expressed protein in biologi~~al fluids.
An ELISA assay initially comprises preparing an antibody, _Lf not readily available from a commercial source, specific t:o BSG, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds specifical=Ly to BSG. The reporter antibody is attached to a detectable reagent:: such as radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme or alkalize phosphatase.
To carry out:. the ELISA, antibody specific to BSG is incubated on a solid support, e.g. a polystyrene dish, that binds the antibody . Any f ree protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the sample to be analyzed is incubated in the dish, during which time BSG binds to the specific antibody attached to the polystyrene dish.
Unbound sample is wa~,hed outs with buffer. A reporter antibody specifica7_ly directed to BSG and linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody tc> any monoclonal antibody bound to BSG.
Unattached reporter antibod~~ is then washed out. Reagents for peroxidase activity, including a colorimetric substrate are then added to the dash. Immobilized peroxidase, linked to BSG
antibodie~~, produces a colored reaction product. The amount of color c.eveloped in a given time period is proportional to the amount of BSG protein present. in the sample. Quantitative results tarpically ar_e obtained by reference to a standard curve.
A competitiozx essay may be employed wherein antibodies specific t:o BSG attached to a solid support and labeled BSG
and a sample derived :From the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of BSG in the sample.
Nuc7.eic acid methods may be used to detect BSG mRNA as a marker for breast cancer. Polymerase chain reaction (PCR) and other nucleic ac~id methods, such as ligase chain :reaction (LCR) and nucleic acid sequence based amplification (NASABA), can be u~~ed to detect malignant cells for diagnosis and monitoring' of various malignancies. For example, reverse-transcriptase PCR (R7:--PCR) is a powerful technique which can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species. In RT-PCR, an mRNA species is first reverse transcribed to complementary DNA (cDNA) with use of the enzyme reverse transcriptase; the c:DNA is then amplified as in a standard PCR
reaction. RT-PCR can thus reveal by amplification the presence of a single' species of mRNA. Accordingly, if the mRNA is highly spec:ifi.c for the cell that produces it, RT-PCR
can be usE~d to identify the presence of a specific type of cell.
Hybridization to clones or oligonucleotides arrayed on a solid support (i.e., griddinc~) can be used to both detect the expre~;sion of and quant:itat:.e the level of expression of that gene. In th~.s approach, a cDNA encoding the BSG gene is fixed to a subst~:-ate. The substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon or plastic. At least a portion of the DNA encoding the BSG
gene is attached to the subs>trate and then incubated with the analyte, which may be RNA or a complementary DNA (cDNA) copy of the ;RNA, isolated from the tissue of interest.
Hybridization between the substrate bound DNA and the analyte can be detE~cted and qi~antitated by several means including but not limited to radi«ac:tive :Labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid. c2uantitation of the level of gene expression can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards.
The standards can be obtained by in vitro transcription of the target gene, quant:itating the yield, and then using that material to generate a standard curve.
Of the proteomic approaches, 2D electrophoresis is a technique well known t.o those in the art. Isolation of individual proteins from a sample such as serum is accomplished using sequential separation of proteins by different characteristics 'usually on polyacrylamide gels.
First, proteins are separated by size using an electric WO 00/08210 PCT/iJS99/1681 t current. The current acts uniformly on all proteins, so smaller proteins move farther on the gel than larger proteins.
The second dimensi<:7n applies a current perpendicular to the first and separates proteins not on the basis of size but on the specific electric) charge carried by each protein. Since no two proteins with different sequences are identical on the basis of both size and charge, the result of a 2D separation is a square gel in ~.Nhich each protein occupies a unique spot.
Analysis of the spots with chemical or antibody probes, or subsequent: protein microsequencing can reveal the :relative abundance of a given protein and the identity of the proteins in the sample.
The above tests can be carried out. on samples derived from a variety of patients' cells, bodily fluids and/or tissue extracts (homogenates or solubilized tissue) such as from tissue biopsy and out=opsy material. Bodily fluids useful in the present inventi.en include blood, urine, saliva, or any other bodily secretion or derivative thereof. Blood can include whole blood, plasma, serum, or any derivative of blood.
In Vivo Antibody Use Antibodies ac.~ai.nst BSGs can also be used in vivo in patients with disease of the breast. Specifically, antibodies against a BSG can be injected into a patient suspected of having a disease of the breast for diagnostic and/or therapeutic purposes. The use of antibodies for in vivo diagnosis is well known in the art. For' example, antibody-chelators labeled with :Lndium-11_L have been described for use in the radioimmunoscintographic imaging of carcinoembryonic antigen expressing tumors 1 Sumerdon et al . Nucl . Med. Biol .
1990 17:2~E7-254) . ~_n particular, these antibody-chelators have been used in detecting tumors in patients suspected of having recurrent colorectal cancer (Griffin et al. J. Clin.
Onc. 1991 9:631-640). Anti_bod~es with paramagnetic ions as labels fox- use in magnetic resonance imaging have also been described (Lauffer, R.B. Magnetic Resonance in Medicine 1991 22:339-342). Antibod.i.es directed against BSGs can be used in a similar manner. Labeled antibodies against a BSG can be injected into patients suspected of having a disease of the breast such as brea:~t cancer- for the purpose of diagnosing or staging of the disease status of= the patient. The label used will be selected in accordance with the imaging modality to be used. For examp:l.e, radioactive labels such as Indium-111, Technetium-99m or Iodine-131 can be used for planar scans or single photon emission computed tomography (SPELT). Positron emitting labels such as Fluorine-19 can be used in positron emission t:omography. Paramagnetic ions such as Gadlinium (III) or Manganese (I:II can used in magnetic resonance imaging (MRI). hocalization of the 1_abel within the breast or external to the breast permits determination of the spread of the disease. The amount of label within the breast also allows determination of the presence or absence of cancer in the breast.
For patients diagnosed with breast cancer, injection of an antibod~r against a BSG can also have a therapeutic benefit.
The antibody may exert its therapeutic effect alone.
Alternatively, the ant::ibody is conjugated to a cytotoxic agent such as a drug, t~~.xin or radionuclide to enhance its therapeutic effect. Drug monoclonal antibodies have been described in the arty for example by Garnett and Baldwin, Cancer Research 1986 46:2407--2412. The use of toxins conjugated to monoclonal antibodies for the therapy of various cancers has also been described by Pastan et al. Cell 1986 47:641-648). Yttrium-90 labeled monoclonal antibodies have been described for maximization of dose delivered to the tumor while limiting toxicity to normal tissues (Goodwin and Meares Cancer Supplement 1997 80:2675-2680). Other cytotoxic radionuclides including, but not limited to Copper-67, Iodine-131 and Rhenium-186 can also be used for labeling of antibodies; against BSGs.
Antibodies which can be used in these in vivo methods include both polyclonal and monoclonal antibodies and antibodies; prepared via molecular biology techniques.
Antibody fragments and aptamers and single-stranded oligonucleotides such as those derived from an in vitro evolution protocol referred to as SELEX and well known to those skilled in the art can also be used.
EXAMPLES
The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by reference to specific embodiments.
These exemplificati.o:ns, while illustrating certain specific aspects of: the invf:ant:ion, do not portray the limitations or circumscribe the sco~.~e of the disclosed invention.
Example 1 Identification of BSGs were carried out by a systematic analysis of data in the LIFESEQ database available from Incyte Pharmaceuticals, Pa:Lo Alto, CA, using the data mining Cancer Leads Automatic Search Package (CLASP) developed by diaDexus LLC, Santa Clara, CA.
The CLASP performs the fo7_lowing steps:
Selection of highly expressed organ specific genes based on the abundance level of the corresponding EST in the targeted organ versu;~ all the other organs.
Analysis of the expression level of each highly expressed organ specific genes in normal, tumor tissue, disease tissue and tissue libraries associated with tumor or disease.
Selection of the candidates demonstrating component ESTs were exclusively or more frequently found in tumor libraries.

CLAS P allows the identification of highly expressed organ and cancer spE~cific genes useful in the diagnosis of breast cancer.
Table 1: BSGs Sequences SEQ ID NO: LS Clone ID LSA Gene ID

1 2740238(Mam002) 242151 2 1730886(Mam001) 238469 3 y155b03(Mam005) 348845 4 2613064(Mam004) 27052 The following example was carried out using standard techniques, which are well known and routine to those of skill in the art, except:. where otherwise described in detail.
Routine molecular biology techniques of the following example can be c~irried out as described in standard laboratory manuals, :such as .~ambrook et al., MOLECULAR CLONING: A
LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Example 2: Relative Quantitation of Gene Expression Real-time quantitative PCR with fluorescent Taqman probes is a quantitat=.ive detection system utilizing the 5'-3' nuclease activity of Taq DNA polymerase. The method uses an interna7_ fluorescent oligonuc~Leotide probe (Taqman) labeled with a 5' reporter dye and a downstream, 3' quencher dye.
During PCR,, the 5'-~' nuclease activity of Taq DNA polymerase releases tree reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosysterns, Foster City, CA, USA).

_ 17 _ Amplification of an endogenous control was used to standardize= the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency. Either cyclophilin, glyceraldehyde--3-phosphate dehydrogenase (GAPDH) or 18S ribosomal RNA (rRNA) was used as this endogenous control. To calculate relative Quantitation between all the samples studied, the target RNA levels for one sample were used as t:he basis f:or comparative results (calibrator).
Quantitation relative to the "calibrator" can be obtained using the standard c:llrVe method or the comparative method (User Bull.°tin #2: ABI PRISM 7700 Sequence Detection System).
To evaluate the tissLe distribution, and the level of. breast specific markers (BSM) Mam001 (SEQ ID N0:2), Mam002 (SEQ ID
NO: l), Mam004 (SEQ ID N0:4) and Mam005 (SEQ ID N0:3) in normal and cancer tissue, total RNA was extracted from cancer and matched normal adjacent tissues (NAT) and from unmatched cancer and normal. ti;~sues. Subsequently, first strand cDNA
was prepared with reverse transcriptase and the pol.ymerase chain reaction carried out using primers and Taqman probes specific t.o each of Mam001 (SEQ ID N0:2), Mam002 (SEQ ID
N0 : 1 ) , MamO 04 ( SEQ ID N0 : 4 ) and MamO 0 5 ( SEQ II) N0 : 3 ) respectively. The results are obtained using the ABI PRISM
7700 Sequence Detector. The numbers are relative levels of expression of Mam001 (SEQ ID DJ0:2), Mam002 (SEQ ID NO: l), Mam004 (SEQ ID N0:4) and Mam005 (SEQ ID N0:3) compared to their respective ca:l.ibrator:~.
Measurement: of SEQ ID N0:2; Clone ID:1730886; Gene ID: 238469 (Mam001) The numbers deb>i.cted :in Table 2 are relative levels of expression in 12 normal tissues of Mam001 (SEQ ID N0:2) compared t.o testis (~~alibrator). These RNA samples were obtained commercially and were generated by pooling samples from a parvicular tissue from different individuals.

Table 2: Relative levels of Mam001 (SEQ ID N0:2) Expression in Pooled Samples Tissue NORMAL

Brai.n_ 0 Heart 0 Kidney 0 Liver 0 Lunc:f _ 0 Mammary 6 Prostate 0 Muscl~_ 0 Small Intestine 0 Test.i;s 1 Thymus 0 Utazu~ 0 The relative levels of expression in Table 2 show that Mam001 (S~~Q ID N0:2) mRNA expression is detected in the pool of normal mammary and in testis but not in the other 1.0 normal tissue pools analyzed. These results demonstrate that Mam001 (SBQ ID N0:2) mRNA expression is highly specific for mammary tissue and is als~_:~ found in testis . Expression in a male specific tissue is not relevant in detecting cancer in female specific tissues The tissues shown in Table 2 are pooled samples from different individua~.s. The tissues shown in Table 3 were obtained from individuals and are not pooled. Hence the values fo=r mRNA expression leve:Ls shown in Table 2 cannot be directly compared to the values shown in Table 3.
The numbers depicted in Table 3 are relative levels of expression of Mam001 (SEQ ID N0:2) compared tc> testis (calibrator), in 24 lairs of matching samples. Each matching pair contains the cancer sample for a particular tissue and the norma:L adjacent tissue (NAT) sample for that same tissue from the ~~ame individual.

_ 19 _ Table 3: Relative levels of Mam001 (SEQ ID N0:2) Expression in Individual Sample;
Sample ID Tissue Cancer Matching Normal Mam 47XP Mammary Gland 0 0 Mam A06X Mammary Gland 23 1 Mam BOllX Mammary Gland 0 5 Mam 603X/C034 Mammary Gland 0 2.10 Mam 16a?X Mammary Gland 1.96 0.15 Mam 42DN Mammary Gland 0.38 1.27 Mam 5079 Mammary Gland 0.34 0.36 Mam Sla?3 Mammary Gland 0.03 0.87 Mam S5u6 Mammary Gland 0.43 0.53 Mam 5639 Mammary Gland 0.40 0.66 Mam S9~)7 Mammary Gland 0.41 0.51 Sto AC~64 ~>tomach 0 0 TST 39X Testis 0 () Cln SG~65 Colon 0 0 Cln TX01 Colon 0 0 Cvx NK.?3 Cervix 0 0 Cvx NKa?4 Cervix 0 0 Endo 3~~X Endometrium 0 0 Endo 4XA Endometrium 0 0 Endo 5XA Endometrium 0 0 Kid 11XD Kidney 0 0 Kid 5XD Kidney 0 0 Lng C20X Lung 0 0 Lng SQ56 Lung 0 0 Among 48 samples in Table 3 representing 8 different tissues expression is seen only in mammary tissues. These results confirm the tissue specificity results obtained with normal sarlples shown in Table 2. Table 2 and Table 3 represent a combined total of 60 samples in 16 human tissue types. Thirty-six samples representing 14 different tissue types excluding breast and testis had no detected Mam001 (SEQ
ID N0:2) rciRNA {Tabl.e 2 and 3) . Other than breast tissue, Mam001 (SEC) ID N0:2) i:~ detected only in one other tissue type (Testis) and then or~l.y in the pooled tissue sample (Table 2) but not in the matched testis cancer samples (Table 3).
Comparisons of t=he level of mRNA expression in breast cancer samples and the normal adjacent tissue from the same individual: are shown in Table 3. Mam001 (SEQ ID N0:2) is expressed ~~t higher levels in 2 of 11 breast cancer tissues (Mam A06X a.nd Mam 16:.'X) compared with the corresponding normal adjacent tissue. The level of Mam001 (SEQ ID N0:2) expression is lower in breast cancer compared to normal adjacent tissue in four matched samples (Mam BO11X, Mam 603X/C034, Mam 42DN
and Mam S123). No Expression was detected in one set of matched samples (Mam 47XP). Equivalent levels or very similar levels of expression were detected in four other matched samples (Mam S079, Mam S516, Mam 5699 and Mam S997). However increasing cancer mass might in these cases result in an overall increase in the total amount of expression.
The high level. of tissue specificity and increased or equivalent expression in 6 of 11 individuals is demonstrative of Mam00=L (SEQ Ii:) NO:2) being a diagnostic marker for detection of mammary cancer cells using mRNA.
Measurement of SEQ ID NO:1; Clone ID: 2740238; Gene ID 242151 (Mam002) The numbers depicted in Table 5 are relative levels of expression in 12 normal tissues of Mam002 (SEQ ID NO:1) compared to Thymus (calibratorj. These RNA samples were obtained commercially and were generated by pooling samples from a particular ti.s;~ue from different individuals.

Table 4: Relative levels of Mam002 (SEQ ID NO:1) Expression in Pooled Samples Tissue NORMAL

Brain 0.03 Heart O.Ol Kidney 0 Liver 0 ~

Lung 0.06 Mammary 289.01 Muscle 0 Prostate 0.31 Smal l :Int: 0 .

Testis 0.08 Thymus 1.00 Uterus 0 The relative levels of: expression in Table 4 show that Mam002 (SEQ ID NO:1) mRNA expression is detected at a high level in the pool of normal mammary but at very low levels in the other 11 normal tissue pools analyzed. These results demonstrate that Mam002 (SEQ ID NO:1) mRNA expression is highly specific for mammary tissue.
The tissues :shown in Table 4 are pooled samples from different individuals. The ti.:>sues shown in Table 5 were obtained i_rom individuals and are not pooled. Hence the values for mRNA expression levels shown in Table 4 cannot be directly compared to t=he values shown in Table 5.
The numbers depicted in Table 5 are relative levels of expression of Mam002 (SEQ ID NO: l) compared to thymus (calibrator) in 27 pairs of matching samples. Each matching pair contains the cancer sample for a particular tissue and the normal adjacent tissue (NAT) sample for that same tissue from the ;game indiv=_dual. In addition 2 unmatched mammary samples from normal tissues and one unmatched ovarian cancer and one normal (non--cancerous) ovary were also tested.

PCT/US99l168I 1 Table 5: Relative levels of Mam002 (SEQ ID NO:1) Expression in Individual Samples Sample ID Tissue Cancer Matching Normal Mam 12X Mammary 7.2 69 Gland Mam 42DrJ Mammary 1051 2075 Glanci Mam 59X Mammary 7.0 15.5 Gland Mam A06?F Mamm;:~z~y 1655 1781 G 1 axlCi Mam B011X Mamm<~ry 32.1 2311 Gland Mam S12',~ Mamm~x~y 1 . 7 3 0 Glanc;

Mam S51F> Mammary 9.72 69.95 Gland Mam S 6 9 Mamm,::~ 8 3 . 4 6 7 5 . 6 5 ~3 zwy Gland Mam S85~6 Mammary 133.23 836.56 Gland Mam S967 Mammary 59.77 188.28 Glarncl Mam S99'' Mammary 94.14 73.64 GlancL

Mam 162; Mammary 674.0 31.1 Gland Mam Cola; Mammaz~y N/A N/A 11379.3 Gland Mam C039: Mammary N/A N/A 3502.6 Gland Mam 507~~ Mammary 11772.5 903.5 GlancL

Mam S 12 Mamma~~y 3 . 4 17 0 . 5 _;

Gland Ovr 103; Ovarv 0 0 Ovr 1118 Ovary 0.13 ~N/A

- z~ -Ovr 35GA Ovary N/A N/A 0.13 Utr 23XU Uteru:~ 5.6 0 Utr 135X0 Uteru:> 0 0 Cvx NK24 Cervix: 0.9 1.4 End 4XA Endometz~iu 32.2 0 m Cln AS43 Colon 2.3 0 Cln AS45 Colon 0 0 Cln RCO1 Colc:>n 0.2 0 Lng AC90 Lung 0 2.0 Lng LC10 Lun:~ 0 0 . 6 ~~

Lng SC32 Lung 0.8 0 Sto AC93 Stomach 0 0 Tst 39X Testi~> 1.97 0 Among 58 sam~:~les in Table 5 representing 9 different tissues, the highest expression is seen in mammary tissues.
Amongst the non-breasr_ tissues which show expression, only one sample (End 4XA) has expression comparable to that seen in the majority of the breast samples tested. This sample is endometrial tissue, which is a female specific tissue. These results confirm the tissue specificity results obtained with normal samples shown in Table 4. Table 4 and Table 5 represent a combined total of 7C) samples in 17 human tissue types. Twenty-two samples representing 11 different. tissue types excluding brea:~t had no detected Mam002 (SEQ ID NO:1) mRNA (Table 4 and Table 5).
Comparisons of the level of mRNA expression in breast cancer samples and the normal adjacent tissue from the same individuals are shown in Table 5. Mam002 (SEQ ID NO:1) is expressed at higher Levels in ?'. of 13 matched breast cancer tissues (Samples Mam 5127, Mam 162X and Mam 5079) compared with the correspondin.c~ normal adjacent tissue. The level of Mam002 (SE;Q ID NO:'1) expression is lower in breast cancer compared to normal adjacent tissue in eight individuals (Mam 12X, Mam ~~2DN, Mam 59X, Mam BOllX, Mam S516, Mam 5854, Mam S967, and Mam 5123). Equivalent levels or very similar levels of expres:~ion were detected in three other matched samples (Samples Nfam A06X, M<~m 5699 and Mam 5997).
The high level of tissue specificity is demonstrative of Mam002 (SEQ ID NO:l) being a diagnostic marker for detection of mammary cancer cells using mRNA. Breast tissue is the on7_y signif a.cant source of this gene' s expression so far detected. Eight:. of 13 matched samples have lower levels of express>ion in cancer than normal adjacent tissue. Thus, decreased expression of this gene appears to be diagnostic of cancer presence.
Measurement of SEQ ID N0:4; Clone ID: 2613064; Gene ID: 27052 (Mam004) The numbers depicted in Table 6 are relative levels of expression in 12 :uc%rmal tissues of Mam004 (SEQ ID N0:4) compared t:o mammary (calibrator). These RNA samples were obtained commerciall~~ and were generated by pooling samples from a particular ti;~sue from different individuals.
Table 6: Relative levels of Mam004 (SEQ ID N0:4) Expression in Pooled Samples Tissue NORMAL

Brain 0.059 Heart 0.131 Kidney 0.018 Liver 0 ~

Lung 0.478 Mammary 1.000 Prostate' 0.459 Muscle 0.003 Small Intestine 0.048 Testis 0.130 Thymus 0.030 Uterus 0.071 The relative levels of expression in Table 6 show that Mam004 (SEQ ID N0:4) mRNA expression is detected in the pool of normal mammary and also in other tissues including lung, prostate, testis and heart. These results demonstrate that although more highly expressed in normal breast tissue Mam004(SEQ ID N0:4) mRNA expression is not specific for mammary gl<~nd.
The tissues shown. in Table 6 are pooled samples from different individual:. The tissues shown in Table 7 were obtained from individuals and are not pooled. Hence the values for mRNA expression levels shown in Table 6 cannot be directly compared to the values shown in Table 7.
The :numbers depicted in Table 7 are relative levels of expression of Mam004 (SEQ ID N0:4) compared to mammary (calibrator), in 23 pairs of matching samples. Each matching pair contains the cancer sample for a particular tissue and the normal adjacent tissue (NAT) sample for that same tissue from the s<~me indiv:i.dual.
Table 7: Relative levels of Mam004 (SEQ ID N0:4) Expression in Individual Samples Sample :ID T:Lssue Cancer Matching Mam 1213 Mammary Gland 0 0 Mam 12:~ Mammary Gland 13.454 0 Mam 603X Mammary Gland 30.484 0 Mam 59:~ Mammary Gland 1.306 0 Mam 162X Mammary Gland 0.71 0.04 Mam 42DN Mammary Gland 0.25 2.17 Mam S079 Mammary Gland 42.18 0.47 Mam S123 Mammary Gland 0.01 0 Mam S516 Mammary Gland 1.17 0.41 Mam S699 Mammary Gland 0.11 0.55 Mam 5997 Mammary Gland 10.43 1.29 Sto AC44 '.:>tomach 0.61 0 Cln SG45 Colon 0.04 0 Cln TXO1 Colon 0 0 Cvx NK23 Cervix 0 0 Cvx NK24 Cervix 0 0 Endo 3F.X Enc3ometriurn 0 0 Endo 4xA En<~ometrium 0 0 Endo 5xA Enciometrium 0 2.73 Kid 11X:D K_~dney 0 0 Kid 5X1 Kidney 0 2.63 Lng C20X Dung 0 0 Lng SQ56 Lung 10.37 0 Among 46 samples in Table 7 representing 7 different tissues expression i.s highest in breast tissues particularly cancers. Expression comparable to that seen in breast samples is also seen in 1 of 4 lung camp-les (Sample 23), 1 of 4 kidney samples (Sample 21) <~:nd 1 of 6 endometrial samples (Sample 19). Table E~ and Table 7 represent a combined total of 58 samples in 16 human ti.~~sue types. Twenty samples representing 7 different tissue types excluding breast had no detected Mam004 (SE(~ ID N0:4 j mRNA (''able 6 and Table 7) .
Comparisons o:f the level of mRNA expression in breast cancer samples and the normal adjacent tissue from the same individual; are shown in Table 7. Mam004 (SEQ ID N0:4) is expressed ~~t higher 1_evels in 8 of 11 breast cancer tissues (Mam 12X, N(am 603X, Mam 59X, Mam 162X, Mam 5079, Mam S123, Mam S516 and hlam 5997) compared with the corresponding normal adjacent tissue. The level of Mam004 (SEQ ID N0:4) expression is lower in breast <:ancer compared to normal adjacent tissue in two matched samples (Mam 42DN and Mam 5699). No expression was detected in one matched sample (Mam 12B).
Elevated expre:~sion in the majority of matched cancer samples compared to normal adjacent tissue is indicative of Mam004 (SEQ ID N0:4) being a diagnostic marker for detection of mammary cancer cells using mRNA.
Measurement. of SEQ ID N0:3; Clone ID:y155b03; Gene ID: 348845 (Mam005) The numbers depicted in Table 8 are relative levels of expression in 12 normal tissues of Mam005 (SEQ ID N0:3) compared to testis (calibrator). These RNA samples were obtained commercially and were generated by pooling samples from a part:icul.ar ta.ssue from d~.fferent individuals.
Table 8: Relative levels of Mam005 (SEQ ID N0:3) Expression in Pooled Samples Tissue NORMAL

Brain 0 Heart ~ 0.0002 Kidney 0.0001 Liver ~ 0 Lung 0 Mammary 5.4076 Muscle 0 Prostate 0 Small )intestine0 Testis 1 ~

Thymus 0 Uterus 0 The relati~le levels o:E expression in Table 8 show that Mam005 (SEQ ID NO:3) mRNA E=xpression is detected in the pool of normal mammary and in testis but is not present at significant levels in the other 7..0 normal tissue pools analyzed. These results demonstrate that Mam005 (SEQ ID N0:3) mRNA expression is highly specific for mammary tissue and is also found in testis. Expression in a male specific tissue is not relevant in detecting cancer in female specific tissues.
The tissues shown in Table 8 are pooled samples from different individua:l:~. The tissues shown in Table 9 were obtained from individuals and are not pooled. Hence the values for mRNA expression leve:Ls shown in Table 8 cannot be directly c~smpared to t:he va:Lues shown in Table 9.

The numbers depicted in Table 9 are relative levels of expression of Mam00!~ (SEQ ID N0:3) compared to testis (calibrator), in 46 pairs of matching samples. Each matching pair contains the canee:r sample for a particular tissue and the normal adjacent tissue sample for that same tissue from the same individual. In additioru 2 unmatched mammary samples from norma7_ tissues and one unmatched ovarian cancer and one normal (non-cancerous) ovary were also tested.
Table 9: Relative levels of Mam005 (SEQ ID N0:3) Expression in Individual Samples Sample ID Tissue Cancer Matching Normal Mam 12X Mamma:r~r 0 . 3 0 . 71 Gland Mam 42DN Mammar~r 0.22 0.63 Gland Mam 59X Mammary 0.03 0.23 Gland Mam A06X Mammary 70.77 0.56 Gland Mam BO11X Mammary 0.0:~ 1.52 Gland Mam 162X Mammary 0.43 0.09 Glanca Mam C012 Mammary N/A N/A 1..6 Gland Mam C034 Mammary N/A N/A 2.9 Gland 2 Mam S 0 7 Mamma z~y 0 . 2 2 0 . 13 0 9 ) Gland Mam 5123 Mammary 0.01. 0.23 Gland Mam 5127 Mammary 0 0.28 Gland Mam 5516 Mammary 0.15 0.05 Gland Mam S699 Mammary 0.21 0.42 Gland Mam S854 Mammary 1.12 0.54 Gland Mam 5967 Mammary 30.61 0.54 Gland Mam 5997 Mammary 0.40 0.22 Gland Mam 14DN Mammary 0.07 0 Gland Mam 6 9 9 Mamma.r~T 0 . 01 0 . 0 9 F Gland Mam 5621 Mammary 1.82 0 Gl.an;~

Mam 5918 Mammary 6.89 1.06 Gland Cln CM67 Colon 0 0 Cln DC19 Colon 0 0 Cln AS43 Colon 0 0 Cln AS45 Colon 0 0 Cln RCO1 Colon 0.0012 0.0003 Lng AC90 Lung 0 0 Lng LC109 Lung 0 0 Lng SQ3 2 Lunch 0 0 Lng SQ43 Lung 0 0 Ovr 103X Ovary 0 0 Ovr 1118 Ovary 0 N/A

Ovr A084 Ovary 0 0 Ovr 6021 Ovary 0 0 Ovr 35GA Ovary N/A N/A 0 Cvx NK23 Cervix 0 0 Cvx NK24 Cervix 0 0 Endo 3AX Endomet::riu0 0 m Endo 4XA Endomet:.riu0 0 m Sto 758S Stomach 0 0 Sto AC44 Stomach 0 0 Sto AC93 Stomach 0 0 Tst 39X Testis 0.01 0.01 Utr 85XU Uterus 0 0 Utr 135X0 Uterus 0 0 Utr 23XU Uterus 0 0 Kid 124D Kidney 0 0 Lvr 15XA Liver 0 0 Pan C044 Pancreas 0 0 Skn 4485 Skirl. 0 0 SmInt 21X~~ Small 0 0 Intestines Among 96 samples in 'rar>le 9 representing 14 different tissues significant expression is seen only in breast tissues. These results confirm the tissue specificity results obtained with normal samples shown in Table 8. Table 8 and Table 9 represent ~. combined total of 108 samples in 18 human tissue types. Sixay-seven s<~mples representing 16 different tissue types excluding breast and t=estis had either no or very low levels of detected M<~m005 (SEQ ID N0:3) mRNA {Table 8 and Table 9 ) .
Comp<zrisons of. the level of mRNA expression in breast cancer samples and r~he normal adjacent tissue from the same individuals are shown in Table 9. Mam005 (SEQ ID NO:3) is expressed ;~t higher levels in 7.0 of 18 cancer and normal adjacent tissue samples (Mam A06X, Mam 162X, Mam 5079, Mam 5516, Mam 3854, Mam 5967, Mam S997, Mam 14DN, Mam 5621, and Mam 5918) compared with the corresponding normal adjacent tissue. The level oi: Mam005 (SEQ ID N0:3) expression is lower in breast cancer compared to normal adjacent tissue in eight WO 00!08210 PCT/US99/16811 cancer and normal adjacent tissue samples (Mam 12X, Mam 42DN, Mam 59X, Mam BO11X, Mam 51.23, Mam 5127, Mam S699 and Mam 699F). No expression was detected in two matching samples.
The :high leve7_ of tissue specificity and overexpression in 10 of lf3 matched cancer and normal adjacent tissue samples is indicative of Marn005 (SEQ ID N0:3) being a diagnostic marker for detection of mammary cancer cells using mRNA.

SEQUENCE LISTING
<1.10> Sun, Yongming Recipon, Herv~>_ Cafferkey, Robert DIADEXUS LLC
<1.20> A NOVEL METHOD OF DIAGNOSING, MONITORII'IG, STAGING , IMAGING AND T:ftEATING BREAST CANCER
<7.30> DEX-0040 <7.40>
<7.41>
<~_50> 60/095,232 <:_51> 1998-08-04 <=_60> 9 <:_70> PatentIn Ver. 2.0 W?10>1 W?11>544 <;?12>DNA

<;?13>Homo sapiens <:?20>
<;?21> unsure <:?22> (505) . . (506) <220>
<221> unsure <222> (510) < 220>
<221> unsure <222> (521?
<220>
<221> unsure <:022> (527)..(528) <220>
<221> unsure <222> (531>
<220>

<221> unsure <222> (534)..(535) <220>
<221> unsure <222> (540)..(541) <400> 1 ctagtctcga gtctagagcg ccttgcctt:.c tcttaggctt t.gaagcattt ttgtctgtgc 60 tccctgatct tcatgtcacc accatgaacxt.tcttagcagt c:ctggtactc ttgggagttt 120 ccatctttct ggtctctgcc cagaatcc::xa. caacagctgc t:ccagctgac acgtatccag 180 ctactggtcc tgctgatgat gaagcccct:.g ,atgctgaaac cactgctgct gc aaccactg 240 cgaccactgc tgctcctacc actgcaacaa ccg ctgcttc taccactgct cgtaaagaca 300 ttccagtttt acccaaatc~g gttgggga;-c tcccgaatgg t.agagtgtgt ccctgagatg 360 gaatcagctt gagtcttci:g caattggt::::a caactattca tccttcc~gt gatttcatcc 420 aactacttac cttgcctacg atatcccc;:t tatctctaat c:agttta-tt tctttcaaat 980 aaaaaataac tatgagcaac taaannaa~:~n aaaaaaaaaa naaaaannaa n<3annaaaan 540 naga 544 <210> 2 <211> 1066 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> (729)..(813) <400> 2 gttgaccagt ggtcatgcca ctgcctgtt:g atttgttgaa aatattgttt acacgtatgt 60 tcttgttact gattgtcac~a aagctggt?::t tgagactgca gcttggacta aattcagtca 120 tctggctgtc tggggaagc:a tgctgaccag tctggtgttc tttggca~ct actcagccat 180 ctggtccacc attctcatt:g ccccaaatat gagaggacag aagaatggta ccggtactgc 240 caatggagat ggaggaagc~a gacagaaac~a <3acagagccc agaccctagg gaccaccagc 300 atttgcagaa tggataaaca gccttctt:::c taacaaagga agcacagcaa ctgtgatcct 360 gagctgtgca cacttctgc~t tgggattat::t tctggtttct acttcctgtt tgaagatgtg 420 gcatggagag tgaacaagca gct=gcccacc <3cctggcatc acagccccag aactcagcta 480 tttccatggg accacagcat ctcatctct~g g gctgagcca gaaagacccc tactgaagtc 590 cagaggcact tttctgaaag gctctgctt~t gacctgaagt attttatcta tc ctcagtct 600 caggacactg ttgatggaat taaggccaag cacatctgca aaaaagacat tgctggagga 660 ggtgcaaaga gctggaaac:c aagtctcc<_ig tcctgggaaa agcagtggta tggaaaagca 720 atggaaagnn nnnnnnnnnn nnnnnnnnvn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780 nnnnnnnnnn nnnnnnnnnn nnnnnnnn:un nnncatagca ccaatgacct gaagagcctt 890 gttgaaggaa gactccatct gatgactc_ig agcaagtatt ttttagtgtg ttattgttat 900 tagcagaaag agggccataa aatacatg.~g c~caagctgaa tatatcttag gcaaaagaag 960 aaaatattca aattcttat:g ttatttt-_at::c taattatttt atctcttttt gtgtgtgact 1020 tataatgtgt gtattgtat:t aataaaagt:a tataaacatg tagttt 1066 <:?10>3 <:?11>649 < DNA
212>

<213>Homo Sapiens <<~00> 3 gcaatgttta atatctcata agctatacac acctcgaagc catcaatgac aaccttttct 60 tc~ctgaatag aacagtgatt gatgtcatga agacaat:ttt <stctcctttt gccttccata 120 aiatgtacca ggttatataa tagtataaca ctgccaagga g,~ggattatc tcatcttcat 180 cca gtaattc cagtgtttgt cacgtggttg ttgaataaat gaataaagaa tgagaaaacc 240 ac~aagctctg atacataatc ataatgataa ttatttc:aat :3cacaactac gggtggtgct 300 gaactagaat ctatattttc tgaaactggc_ tcctctagga ~c~tactaatg atttaaatct 360 a<~aagatgaa gttagtaaag catcagaaaa aaaaggt.aaa ,~aaattgctc ctgtggagat 920 g<3ttggcatc acatggtgtt ttgagctgat ac:accc<taca ~ttgagctca ctgcaacagt 480 ac~cagatttt caccgctatg cctcctttca ctctggc;agt .~rt:cagagg tcttgcactc 540 gc~gagagcat gctcaggttt ccccagctct acaaaat:cac ~c~agaatgcc aaagacttca 600 ac~acaagggt aaataaggtt gatct:,agaa ttgtcac:ctc aaaaaggcc 649 <2I0>9 <211>388 <212>DNA

<213>Homo Sapiens <220>
<:?21> unsure <222> (378) < 220>
<221> unsure <222> (385) <~00> 4 agctgctcaa tacggaacat attctcagtc: ctcctctggt ctacaaagcc tgtgatttct 60 t~~tctatgga cagaacgtct ggtttaat~~t acaggaaccc ataacttcct gaagctttat 120 g~~ttaacagt gacaacgtga gtcagttgaa ttttatt~gtg r_-tcagtccg tagagtatta 180 g~~taacagaa acctttccat tgccatac-tc~ agaaactggc .ag caggcagt gtgcctacag 240 gt ctacaaag aaacttcaga tcatcttctt gagggaaaga agctgaagtg ctacataaga 300 t~~cttgtgct tcataactct cagaagctg c agattct:gta r_aaatcctta gaaaagagca 360 t~~ccctgaat ccataaangt at,stngcc; 388 <.210> 5 <211> 1227 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> (327) <220>
<221> unsure <222> (352) <220>
<221> unsure <222> (369) <2 20>
<221> unsure <222> (850)..(880) <<:20>
<<:21> unsure <<:22> (122C) <900> 5 at.tttgtagt tcagcaaa~c ct~~caaatac acagcatgtt acaaggcact ggtggcacag 60 gc~cacaacag gaaatgat,~t ttatttag:::a aattcattta acaaatatta ttgggcacct 120 gt:tatgtgag acactgtc~~t aggcactgtg ggataacaac agcaaacact t~~acacaaca 180 gc:ctggcctt cctgt.gttvt acaacagcl::c ctaaagatag ctgatat ~aa gacatttgag 240 gc~acacagtt catgtaga~3t caaaatatt:a gtatttcaga at.aaggattt tttttctgaa 300 aagcatacag agaggaaaca gcttaanaat aggtcaagac caaaaaa~,ag antataatca 360 cctgaataanc tggataac~~c: agacagtc:::c: r-acagaattt cattcaggtc acagatttct 420 t~.aaactcac ccccaaaarg tg~~ctgctt~g gttgtttgaa t.cttgcataa tt aatgtcac 980 actgcgcaagc cgctgaacvt agttgagal:g cagaaaacaa acaaatgcaa tgacatatct 540 ga.gaagcatt tatgtaacvc cggttaagt:g gtgaggaggg <xtgtgtgaag acagtgtgca 600 tcfcatgagtg tgtattca~-a tatatgtgta tacatatgaa tttcactgtt attttccagg 660 gtctatggac aatgtggc<3g taagagtcta tgatgttctg aaacttttca ca gtaaatcc 720 aa.agattaca gaccttacaa ggtgcttg~:a ttctgttgct tttccatctg tc acttctca 780 gcatatttga ctgtgttcaa accttcttt~t ctttttcatt gagtttcatt ttttaagctt 840 gttaaatgcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn t.gtcatt-_rt cacattatcc 900 tcacttctct gcaacaagc~a tagtaagat:g tagatgaatg c:aaaaataat aacaacaata 960 acgaaatata ttaaagcttt aaaatatg-,:a catatgtagt t:.ctaaagagc aataacggta 1020 gtatctattt cgaacatgca ttaggcaaaa aagaaatcaa aactgaaatt ttcgtgtatt 1080 tttccccttg taagatgttc aaatgcta,ac ttcattttct cctttcctct atgtggcact 1140 ttctcaaaat atctatgaaa tactttta~Ta c:aaagattga c;ctggagaaa gagatacaaa 1200 tttccatccc cccagacagn gagacat 1227 <210>6 <<11>253 <<12>DNA

<2I3>Homo sa~iens <<:20>
<<:21> unsure <<:22> (181) n < 220>
<:?21> unsure <222> (201) <2 20>
<221> unsure <2 22> (205) < 220>
<;?21> unsure <<'?22> (238) <:?20>
<;?21> unsure <;?22> (241) .. (242) <;?20>
<<'?21> unsure <;?22> (250) «!00> 6 gaacagcctc acttgtgttg ctgtcagtgc cagtagggca <~gcaggaatg cagcagagag 60 gactcgccat cgtggccttg gctgtctgt=g cggwcctaca t_gcctcagaa gccatacttc 120 ccattgcctc cagctgtt~~c acc3gaggtt=t cacatcatat t: t:ccagaagg ctcctggaaa 180 nagtgaatat gtgtcgca~c naganagct:g atggggattg t_qacttggct gctgtcancc 240 nncatgtcan gcg 253 <:?10> 7 <<?11> 943 < a? 12 > DNA
<i?13> Homo Sapiens W?20>
W?21> unsure W?22> ( 128 ) <i?20>
<i?21> unsure <i?22> ( 130 ) W?20>
W?21> unsure W?22> (925) <400> 7 gclgggcctgg ccccggcc~~c tgtgaggacc ccgcgggtgc tggggtaaga ggctctagac 60 ccttcacctg tcagtcac~~t gagggagg<~t gaggccaagc cccatccctc agaatcaagg 120 :p cttgcaancn cccctcac:ct gcccagtctc tgtccacacc cctcgggctg aagacggccc 180 tgaccaggcc ctgggcct:ca gcgaccaccc ctccccctcc tgcctggacc cagggagcag 240 gtgcaggggg ctccgagc:cc ctggtgactg tcaccgtgca gtgcgccttc acagtggccc 300 tgagggcacg aagaggac;cc gacctgtc:-ca gcctgcgggc actgctgggc c aagccctcc 360 ctcaccaggc ccagcttc;gg caactca=:;gt gggccagaaa gcccccggtg gctgcggtgg 420 agctgggcac cgccccgact gaggcagc:a~~ ctggaagagg gggtggcaga ggtcactgcc 480 ctccctgcag gccccacc:ca ggaggcc;.:c~ t~~tgaggaat ctctttgcag ttacctagcc 540 ccaggtgagg acgggcaca g ggtcccc<atc c~~cgaggagg agtcgctgca gagggcctgg 600 caggacgcag ctgcctgc:cc cagggggc:a~3 cagctg~agt_ gcaggggagc cgggggtcgg 660 ccggtcctct accaggtc;gt ggcccagc:a~~ agctactccg cc~aggggcc agaggacctg 720 ggcttccgac agggggac:ac ggtggac~;tc ctgtgtgaag tggaccaggc atggctggag 780 ggccactgtg acggccgc:at cggcatct:t~: c~~caagtgct tcgtggtccc cgccggccct 840 cggatgtcag gagccccc:gg ccgccr_g:-:c.. cgatcc_agc agggagatca gccctaatga 900 tgctgtgtcc atgatgct:tt taatnaa<aaa aacccc~~act gca 943 <210> 8 <211> 249 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> (48) <220>
<221> unsure <222> (110) <220>
<221> unsure <222> (192) <220>
<221> unsure <222> (205) <220>
<221> unsure <222> (218) <400> 8 atcacattaa gtcattgcaa attttataaa caaaaacaat ggttttantt tgcatctccc 60 tgattggtat tgctgtacaa catatttc;ga gaagtttgtt tgtctttggn gtttatttca 120 tgaatagatt gtgtgcccat tttctctt.gc; ggtattc~agt ttr_ttattac tgatgtgagc 180 atgtgtatgg gngattattt gatgnttast<: agttttgntt agtagactgg c:aatatttag 240 tcttgctgt 249 <210> 9 <2L1> 690 <2L2> DNA
<2:L3> Homo sapiens <400> 9 gac~gcccagt gacctgccga ggtcggcagc acagagct:ct gg<sgatgaag accctgttcc 60 tgc3gtgtcac gctcggcctg gccgctgcc:c tgtccttcac c~~rggaggag gaggatatca 120 cagggacctg gtacgtgaag gccatggtc:g tcgataagga cttt:ccggag gacaggaggc 180 cc<iggaaggt gtccccagtg aaggtgacag ccctgggc:gg tgggaagttg gaagccacgt 240 tc<3ccttcat gagggaggat cggtgcatc:c a.gaagaaaat c~tgatgcgg aagacggagg 300 ago ctggcaa atacagcgcc tatgggggc:a ggaagctc:at g=acctgcag gagctgccca 360 gga gggacca ctacatcttt tactgcaaag accagca<:ca tc)ggggcr_tg ctccacatgg 420 gaa agcttgt gggtaggaat tctgatacc:a acc:gggaqgc c;tgqaagaa tttaagaaat 480 tgc)tgcagcg caagggactc tcggaggagg acattttcac g;~ccctgcag acgggaagct 540 gcc~ttcccga acactaggca gccc~ccggc~~ ctgcacctcc agagcccacc: ctaccaccag 600 ac<icagagcc cggaccacct ggacctaccc tccagccatg a--ccatc:cct gctcccaccc 660 acc~tgactcc aaataaagtc cttctcccc~= 690

Claims (14)

What is claimed is:

1. A method for diagnosing the presence of breast cancer in a patient comprising:
(a) measuring levels of BSG in cells, tissues or bodily fluids in said patient; and (b) comparing measured levels of BSG with levels of BSG
in cells, tissues or bodily fluids from a normal human control, wherein a change in measured levels of BSG in the patient versus normal human control is associated with the presence of breast cancer.

2. A method of diagnosing metastatic breast cancer in a patient having breast cancer comprising:
(a) identifying a patient having breast cancer that is not known to have metastasized;
(b) measuring levels of BSG in a sample of cells, tissues, or bodily fluid from said patient; and (c) comparing the measured BSG levels with levels of BSG
in cells, tissue, car bodily fluid type of a normal human control, wherein a change in measured BSG levels in the patient versus the normal human control is associated with a cancer which has metastasized.

3. A method of staging breast cancer in a patient comprising:
(a) identifying a patient having breast cancer;
(b) measuring levels of BSG in a sample of cells, tissues, or bodily fluid from said patient for BSG; and (c) comparing measured BSG levels with levels of BSG in cells, tissues, or bodily fluid type of a normal human control sample, wherein a change in measured BSG levels in said patient versus the normal human control is associated with a cancer which is progressing or regressing or in remission.

4. A method of monitoring breast cancer in a patient having breast cancer for the onset of metastasis comprising:
(a) identifying a patient having breast cancer that is not known to have metastasized;
(b) periodically measuring BSG levels in a sample of cells, tissues, or bodily fluid from said patient; and (c) comparing the measured BSG levels with levels of BSG
in cells, tissues, or bodily fluid type of a normal human control, wherein a change in BSG levels in the patient versus the normal. human control is associated with a cancer which has metastasized.

5. A method of monitoring the change in stage of breast cancer in a patient having breast cancer comprising:
(a) identifying a patient having breast cancer;
(b) periodically measuring BSG levels in a sample of cells, tissues, or bodily fluid from said patient; and (c) comparing the measured BSG levels with levels of BSG
in cells, tissues, or bodily fluid type of a normal human control, wherein a change in measured BSG levels in the patient versus the normal human control is associated with a cancer which is progressing in stage, which is regressing in stage, or in remission.

6. The method of claim 1, 2, 3, 4 or 5 wherein the change associated with the presence, metastasis or progression of breast cancer in said patient is an increase in measured BSG levels in the patient and the BSG comprises Mam001 (SEQ
ID NO:2), Mam004 (SEQ ID NO:4) or Mam005 (SEQ ID NO:3).

7. The method of claim 1, 2 , 3 , 4 or 5 wherein the change associated with the presence, metastasis or progression of breast cancer in said patient is a decrease in measured BSG
levels in the patient and the BSG comprises Mam002 (SEQ ID
NO: 1).

8. The method of claim 3 or 5 wherein the change associated with the regression or remission of breast cancer in said patient is a decrease i.n measured BSG levels in the patient and the BSG comprises Mam001 (SEQ ID NO:2), Mam004 (SEQ ID NO:4) or Mam005 (SEQ ID NO:3).

9. The method of claim 3 or 5 wherein the change associated with the regression or remission of breast cancer in said patient is in increase in measured BSG levels in the patient and the BSG comprises Mam002 (SEQ ID NO: 1).

10. An antibody against a BSG wherein said BSG
comprises Mam001 (SEQ ID NO:2), Mam004 (SEQ ID NO:4) or Mam005 (SEQ ID NO:3).

11. A method of imaging breast cancer in a patient comprising administering to the patient an antibody of claim 10.

12. The method of claim 11 wherein said antibody is labeled with paramagnetic ions or a radioisotope.

13. A method of treating breast cancer in a patient comprising administering to the patient an antibody of claim 10.

14. The method of claim 13 wherein the antibody is conjugated to a cytotoxic agent.