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Method and composition for detection and treatment of breast cancer

Abstrict

The present invention provides a method for the detection of breast cancer using breast by measuring expression levels of breast cancer specific marker (BCSM) genes, and in particular the level of polynucleotides transcribed from and polypeptides encoded by the BCSM genes. The present invention also provide a method for the treatment and/or prevention of breast cancer by modulating the activity of BCSM genes or the products of BCSM genes.

Claims

We claim:

1. A method for detecting breast cancer in a subject, said method comprising the steps of: (a) contacting a biological sample from the subject with an agent that binds to a polypeptide comprising an amino acid sequence recited in any one of SEQ ID NOS:20-38; (b) determining a level of binding of said agent to said polypeptide; (c) comparing the level of binding of said agent to said polypeptide to a control level of binding; and (d) producing a diagnosis based on a result from step (c).

2. The method of claim 1, wherein said agent is an antibody directed against said polypeptide.

3. The method of claim 2, wherein the antibody is selected from the group consisting of Fab fragment, Fab.sub.2 fragment, single chain antibody, chimeric antibody, monoclonal antibody and polyclonal antibody.

4. The method of claim 1, wherein the level of binding of said agent to said polypeptide in said biological sample is determined using a technology selected from the group consisting of ELISA, microarray technology, and biochip technology.

5. The method of claim 1, wherein said agent binds to a polypeptide comprising an amino acid sequence recited in SEQ ID NO:29.

6. A method for detecting breast cancer in a subject, said method comprising the steps of: (a) determining a level of a transcribed polynucleotide in a biological sample from said subject, wherein said transcribed polynucleotide comprises a nucleic acid sequence recited in any one of SEQ ID NOS:1-19, or a complement of any of the foregoing nucleic acid sequences; (b) comparing the level of said transcribed polynucleotide in said biological sample to a control level of said transcribed polynucleotide; and (c) producing a diagnosis based on a result from step (b).

7. The method of claim 6, wherein said transcribed polynucleotide is an mRNA, and wherein the level of mRNA in said biological sample is determined using a method selected from the group consisting of Northern hybridization, RT-PCR, microarray technology, and biochip technology.

8. The method of claim 6, wherein the transcribed polynucleotide comprises a nucleic acid sequence recited in SEQ ID NO:10, or a complement thereof.

9. A method for detecting breast cancer in a subject, said method comprising the steps of: (a) determining an expression pattern of two or more breast cancer-specific markers in a biological sample from said subject; (b) comparing the expression pattern of the two or more breast cancer-specific markers in said biological sample to a control expression pattern; and (c) producing a diagnosis based on a result from step (b), wherein said breast cancer-specific marker is a polynucleotide comprising a nucleic acid sequence recited in any one of SEQ ID NOS:1-19 or a polypeptide comprising an amino acid sequence recited in any one of SEQ ID NOS:20-38.

10. The method of claim 9, wherein the expression pattern of transcribed polynucleotides in the biological sample is determined using a method selected from the group consisting of Northern hybridization and RT-PCR.

11. The method of claim 9, wherein the expression pattern of polypeptides in the biological sample is determined using antibodies directed against the polypeptides.

12. The method of claim 9, wherein the expression pattern of two or more breast cancer-specific markers is determined using microarray or biochip technology.

13. A pharmaceutical composition for preventing or treating breast cancer, comprising pharmaceutically acceptable carrier and an agent capable of modulating an activity of a breast cancer-specific marker or an expression level of a breast cancer-specific gene, wherein said breast cancer-specific marker is a polynucleotide comprising a nucleic acid sequence recited in any one of SEQ ID NOS:1-19 or a polypeptide comprising an amino acid sequence recited in any one of SEQ ID NOS:20-38, and wherein said breast cancer-specific gene is any one of the genes listed in Tables 4 and 5.

14. A method for preventing or treating breast cancer in a subject, said method comprising the step of: introducing into the subject an effective amount of the pharmaceutical composition of claim 13.

15. A method of identifying an agent capable of binding to a breast cancer-specific marker, said method comprising: contacting a breast cancer-specific marker with a candidate agent; and determining a binding affinity of said candidate agent to said breast cancer-specific marker, wherein said breast cancer-specific marker is a polynucleotide comprising a nucleic acid sequence recited in any one of SEQ ID NOS:1-19 or a polypeptide comprising an amino acid sequence recited in any one of SEQ ID NOS:20-38.

16. The method of claim 15, wherein the breast cancer-specific marker or the candidate agent contains a label.

17. A method of identifying an agent capable of modulating an activity of a breast cancer-specific marker, comprising: contacting a breast cancer-specific marker with a candidate agent; determining an activity of said breast cancer-specific marker in the presence of said candidate agent; determining the activity of said breast cancer-specific marker in the absence of said candidate agent; and determining whether said candidate agent affects the activity of said breast cancer-specific marker, wherein said breast cancer-specific marker is a polynucleotide comprising a nucleic acid sequence recited in any one of SEQ ID NOS:1-19 or a polypeptide comprising an amino acid sequence recited in any one of SEQ ID NOS:20-38.

18. A biochip comprising any one of: (a) a polynucleotide comprising a nucleic acid sequence recited in any one of SEQ ID NOS:1-19; (b) a variant of the polynucleotides of (a); (c) a polypeptide comprising an amino acid sequence recited in any one of SEQ ID NOS:20-38; and (d) a variant of the polypeptide of (c), wherein the biochip is utilized for diagnosing breast cancer or screening agents that inhibit breast cancer.

19. A kit for diagnosing breast cancer, said kit comprising a polynucleotide probe or an antibody, wherein said polynucleotides probe specifically binds to a transcribed polynucleotide comprising a nucleic acid sequence recited in any one of SEQ ID NOS:1-19, and wherein said antibody is capable of immunospecific binding to a polypeptide comprising an amino acid sequence recited in any one of SEQ ID NOS:20-38.

20. The kit of claim 19, wherein the polynucleotides probe specifically binds to a transcribed polynucleotide comprising a nucleic acid sequence recited in SEQ ID NO:10, and wherein the antibody is capable of immunospecific binding to a polypeptide comprising an amino acid sequence recited in SEQ ID NO:29.

Description

RELATED APPLICATION

[0001] This application is related to U.S. Provisional Application Serial No. 60/359,999, filed Feb. 28, 2002.

TECHNICAL FIELD

[0002] The present invention relates generally to the detection and treatment of cancer, and in particular breast cancer. The invention specifically relates to breast cancer-specific genes (BCSG), and to polynucleotides transcribed from and polypeptides encoded by the BCSGs. Such polynucleotides and polypeptides may be used for the detection and treatment of breast cancer.

BACKGROUND

[0003] Breast cancer is the second leading cause of cancer-related deaths of women in North America. Although advances have been made in detection and treatment of the disease, breast cancer remains the second leading cause of cancer-related deaths in women, affecting more than 180,000 women in the United States each year.

[0004] Approximately 10% of all breast cancers are currently classified as strongly familial with many of these appearing to be caused by mutations in the hereditary breast cancer genes BRCA1 or BRCA2. However, at least one-third of breast cancers that seem to run in families are not linked to BRCA1 or BRCA2, suggesting the existence of an additional hereditary breast cancer gene or genes. Recently, structural and functional studies of cancer cell lines and tissues have demonstrated the involvement of many genetic loci and genes in the development of human breast cancer. Cytogenesis and loss of heterozygocity (LOH) studies have led to the discoveries of alterations in human chromosomes including 1p, 1q, 3p, 6q, 7q, 11p, 13q, 16q, 17p, 17q, and 18q, at frequencies as high as 20-60%. Thus, multiple genes are involved in the development of extensively heterogeneous breast cancers.

[0005] No vaccine or other universally successful method for the prevention or treatment of breast cancer is currently available. Management of the disease currently relies on a combination of early diagnosis (through routine breast screening procedures) and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. The course of treatment for a particular breast cancer is often selected based on a variety of prognostic parameters, including an analysis of specific tumor markers. (See, e.g., Porter-Jordan and Lippman, Breast Cancer 8:73-100, 1994). However, the use of established markers often leads to a result that is difficult to interpret, and the high mortality observed in breast cancer patients indicates that improvements are needed in the treatment, diagnosis and prevention of the disease.

[0006] Accordingly, there is a need in the art for improved methods for therapy and diagnosis of breast cancer. The identification of expression profiles and differentially expressed genes in the genomic scale would greatly facilitates the molecular classification of tumors and discovery of genes that are causally related to breast cancer development.

SUMMARY OF THE INVENTION

[0007] The present invention provides compositions and methods for the diagnosis and treatment of breast cancer. Specifically, the present invention discloses genes that are differentially expressed in breast cancer cell lines and breast cancer tissue samples as compared to control cell lines and normal tissue samples, the polynucleotides transcribed from these genes (SEQ ID NOS:1-19), and the polypeptides encoded by these polynucleotides (SEQ ID NOS:20-38). The differentially expressed genes are designated as breast cancer specific genes (BCSG). The polynucleotides transcribed from and the polypeptides encoded by the BCSGs are designated as breast cancer specific markers (BCSM).

[0008] In one aspect, the present invention provides a method for diagnosing and monitoring breast cancer by comparing the expression levels of one or more BCSM in biological samples from a subject to control samples.

[0009] In a related aspect, the present invention provides a kit for diagnosing breast cancer. The kit comprises at least one of the following (1) polynucleotide probe that specifically hybridizes to a polynucleotide transcribed from a BCSG, and (2) an antibody capable of immunospecific binding to a BCSM.

[0010] In another aspect, the present invention provides a pharmaceutical composition for the treatment of breast cancer. The pharmaceutical composition comprises a pharmaceutically acceptable carrier and at least one of the following: (1) a BCSM or a functional variant of a BCSM, (2) an antibody directed against a BCSM or its functional variant, (3) a vaccine generated using a BCSM or its variant, (4) an agent that modulate an expression level of a BCSG or an activity of a BCSM.

[0011] In a related aspect, the present invention provides a method for treating breast cancer in a patient with the pharmaceutical composition described above. The patient may be afflicted with breast cancer, in which case the methods provide treatment for the disease. The patient may also be considered at risk for breast cancer, in which case the methods provide prevention for cancer development.

[0012] In another embodiment, the present invention provides methods for screening anti-breast cancer agents based on the agents interaction with the BCSMs, or the agents' effect on the expression of the BCSGs.

[0013] In another embodiment, the present invention provides animals transgenic for one or more of the BCSGs, or a knockout animal in which one or more of the BCSGs is disrupted. These animals may be used to study the relevance of BCSGs to the development of breast cancer.

[0014] In another embodiment, the present invention provides host cells harboring a transfected BCSG. These cells may be used for the treatment of breast cancer.

[0015] Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention.

BRIEF DESCRIPTION OF FIGURES

[0016] The inventions of this application are better understood in conjunction with the following drawings, in which:

[0017] FIG. 1 shows patterns of gene expression in MDA-MB-231 (breast cancer) and MDA/H6 (non-tumorigenic) cell lines. (A) Phosphor images of gene filters. Five gene filters (gf200, gf201, gf202, gf203, gf211) were hybridized first with radioactively labeled cDNA from MDA-MB-231 cells and then with that from MDA/H6 cells. (B) Color images derived from the alignment of radioactive images. (C) A scatter plot of expression intensities of 25,985 genes in MDA-MB-231 and MDA/H6. Each dot represents a gene plotted at the coordinate of its two expression intensities on a log-scale. The genes with the equal intensities are condensed along a diagonal line. (D) The original and color images of 30 genes up-regulated in MDA/H6 with low, medium, and high levels of the expression. Three equally expressed genes were indicated. Red: up-regulated in MDA/H6; green: down regulated in MDA/H6; yellow: no changes.

[0018] FIG. 2 shows analysis of images and expression data on the customized microarrays. (A and B): The images of two sets of 768 genes on the same glass slide. The image A shows the identical patterns with the image B. (i and i'): the gene encoding for prostaglandin endoperoxide synthase 2; (ii and ii') the gene for 3-hydroxymethyl-3-methylglutaryl-Co- enzyme A lyase; (iii and iii') the gene for ribosomal protein L10. (C and D) Statistical analysis of the expression ratios of 202 informative genes between MDA-MB-231 and MDA/H6 were detected by two sets of genes (images A and B) on Slide 1 (C) and on Slide 2 (D). (E) The average ratios of the gene expression from Slide 1A and 1B were plotted against the average ratios from Slide 2A and 2B. The linear regression and Pearson coefficient of correlation were computed from the scatter plots that are on log-scale. The strong linear relations and high values of Pearson coefficient of correlation (r) are indicated in each comparison. "x": an gene expression ratio between MDA-MB-231 and MDA/H6 on x-axis; "y": the ratio between these two samples on the y-axis corresponding to a given "x".

[0019] FIG. 3 depicts clustering of the gene expression data. (A) Multidimensional scaling analysis. 3-dimentional plot of all 15 cancer samples showing two identical MDA-MB-231 samples (MB231 1 and 2, green), the most dissimilar melanoma sample (MelTis in yellow), three most similar breast cancer samples (BT20, ZR75-1, and BT474 in red) and others in blue. (B and C) Gene and sample dendrograms from the hierarchical clustering analysis reveal co-regulated genes and relationship among the samples. Two MDA-MB-231 samples are essentially identical (r=0.982). Human melanoma specimen (MelTis) is the most dissimilar to MDA-MB-231 (r=0.325). Twelve breast cancer samples are clustered in the center. Three most similar samples were BT20, BT474 and ZR-75-1 (r=0.796). The numbers on the nodes indicate the values of Pearson coefficient of correlation. (D) Nine genes with significantly up-regulated expression (.gtoreq.2 folds) in at least 10 of 13 breast cancer samples. These nine genes were also over-expressed in the metastatic melanoma. (E) Ten genes with significantly down-regulated expression (.gtoreq.0.5 folds) in at least 10 of 13 breast cancer samples. The clone ID and the gene names are listed on the left and the right of the panels, respectively.

[0020] FIG. 4 shows the correlation of thrombomodulin (THBD) RNA expression to THBD protein expression as measured by cDNA microarrays and Western blots, respectively. (A) The THBD RNA levels in 13 breast cancer cell lines measured by cDNA microarrays using MDA/H6 as the reference. The values of the intensity means (I.M.), the intensity standard deviations (I.D.), and the calibrated (Cal.) ratios for the test samples and the reference are the averages derived from the cDNA microarray images A and B on each slide (see FIG. 2). The green filled box and Cal. ratio indicate the decrease of the TH gene in a test sample relative to the corresponding MDA/H6 reference. (B) Western blot of the whole cell lysates from the breast cancer cell lines: MDA/H6 (lane 1), MB231 (lane 2), MB436 (lane 3), MB453 (lane 4) and BT549 (lane5), using the antibody against THBD (top panel) and the antibody against actin (bottom panel) as a control for loading error. Ninety-eight kilodaltons (kD) and 43 kD indicate the THBD protein and actin protein, respectively. The protein intensities in the lanes 2, 3, 4, and 5 approximate the RNA levels in the corresponding breast cancer cells: MB231, MB231, MB436, MB453 and BT549. The lane 1 shows the THBD protein intensity in the non-tumorigenic breast cancer cell line MDA/H6 that displays the highest RNA level in all the cell lines.

[0021] FIG. 5 show representative images of the pathological sections of normal and cancerous breast tissues from Case 1 (A) and Case 6 (B) in Table 6. (A1) A section shows normal breast tissue, of which the mammary epithelial cells were stained to brown (positive) by the TH antibody (A2). (A3) A tissue section shows infiltrating ductal carcinoma, of which the cancer cells were not stained by the TH antibody (A4). (B1) A section shows normal mammary epithelial tissue (indicated by the horizontal arrowheads) and infiltrating ductal carcinoma (indicated by the vertical arrowheads); (B2) Normal mammary epithelial cells were stained to brown (positive) by the TH antibody; in contrast, the cancer cells were not. Magnification: (A1 and A2), 100-fold; (A3 and A4), 200-fold; (B1 and B2), 40 fold.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The following detailed description is presented to enable any person skilled in the art to make and use the invention. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the specific nomenclature is not required to practice the invention. Descriptions of specific applications are provided only as representative examples. Various modifications to the preferred embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the scope of the invention. The present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0023] The present invention is generally directed to compositions and methods for the diagnosis, treatment, and prevention of breast cancer. The present invention is based on the discovery of transcribed polynucleotides that are either over-expressed or under-expressed in human breast cancer cell line MDA-MB-231 as related to the non-tumorigenic derivative cell line MDA/H6.

[0024] Definitions and Terms

[0025] To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

[0026] As used herein, the term "breast cancer specific gene (BCSG)" refers to a gene that is over-expressed by at least two-fold (i.e. .gtoreq.200% of normal) or under-expressed by at least two-fold (i.e., .ltoreq.50% of normal) in breast cancer tissue or cell lines relative to normal tissue or cell lines. Specifically, BCSG refers to the genes listed in Table 1 and the alleles of these genes.

[0027] As used herein, "a breast cancer-specific marker (BCSM)" refers to a polynucleotide transcribed from a BCSG or a polypeptide translated from such a polynucleotide. BCSM and "BCSG product" are used interchangeably.

[0028] As used herein, "a BCSM and its variants" refers to variants of a polynucleotide transcribed from a BCSG and variants of a polypepetide encoded by a BCSG.

[0029] As used herein, the terms "polynucleotide" "nucleic acid" and "oligonucleotide" are used interchangeably, and include polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, DNA, cDNA, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0030] As used herein, the terms "variants of a polynucleotide" refers to polynucleotides that, as a result of the degeneracy of the genetic code, encode the same polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. A variant may contain one or more substitutions, additions, deletions and/or insertions such that the activity or immunogenicity of the encoded polypeptide is not substantially enhanced or diminished, relative to a native polypeptide.

[0031] Variants of a polynucleotide may also be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native breast tumor protein (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5.times.SSC, 0.5% SDS. 1.0 mM EDTA (pH 8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC, overnight; followed by washing twice at 65.degree. C. for 20 minutes with each of 2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS. Standard hybridization techniques are described in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989.

[0032] As used herein, a "variant of a polypeptide" is a polypeptide that differs from a native polypeptide in one or more substitutions, deletions, additions and/or insertions, such that the functionality of the polypeptide is not substantially enhanced or diminished. In other words, a variant retains the biological activities of the native peptide. The biological activities of the variant may be enhanced or diminished by less than 50%, preferably less than 20%, relative to the native polypeptide. Similarly, the ability of a variant to react with antigen-specific antisera may be enhanced or diminished by less than 50%, preferably less than 20%, relative to the native polypeptide. Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein.

[0033] Preferably, a variant polypeptide contains conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Variants may also be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.

[0034] Polypeptide variants preferably exhibit at least about 70%, more preferably at least about 90% and most preferably at least about 95% homology to the original polypeptide.

[0035] A polypeptide variant also include a polypeptides that is modified from the original polypeptides by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0036] As used herein, a "biologically active portion" of a polypeptide encoded by a BCSG includes a fragment of the polypeptide comprising amino acid sequences derived from the original polypeptide, which include fewer amino acids than the full length polypeptide, and exhibit at least one activity of the full length polypeptide. Typically, biologically active portions comprise a domain or motif with at least one activity of the full length polypeptide. A biologically active portion of a polypeptide encoded by a BCSG can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length.

[0037] As used herein, an "immunologenic portion" or "epitope" of a polypeptide encoded by a BCSG includes a fragment of the original polypeptide comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the original polypeptide, which include fewer amino acids than the full length polypeptide and can be used as an antigen to stimulate anti-BCSG peptide immune response.

[0038] As used herein, the term "modulation" includes, in its various grammatical forms (e.g., "modulated", "modulation", "modulating", etc.), up-regulation, induction, stimulation, potentiation, inhibition, down-regulation, or suppression.

[0039] As used herein, the term "control sequences" or "regulatory sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The term "control/regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Control/regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

[0040] A nucleic acid sequence is "operably linked" to another nucleic acid sequence when it is placed into a functional relationship with another nucleic acid sequence. For example, coding sequences of a BCSG can be operably linked to the regulatory sequences in a manner which allows for expression of the BCSG (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0041] As used herein, the term "immunospecific binding" refers to the specific binding of an antibody to an antigen at an affinity that is at least 10.sup.5M.sup.-1.

[0042] As used herein, the term "biomolecules" refers to molecules having a bioactivity in a mammal. Examples of biomolecules include, but are not limited to, amino acids, nucleic acids, lipids, carbohydrates, polypeptides, polynucleotides, and polysaccsharides.

[0043] Breast Cancer Specific Genes

[0044] Breast cancer consists of extensively heterogeneous tumors and individual tumor cells may have specific genetic defects that determine gene expression patterns. Identification of expression profiles of multiple cancer samples may reveal genes and their expression patterns that consist of portions specific to the individual samples and common to most, if not all, samples studied. The common expression patterns might represent a common "passage" through which the cells evolve from one status to another. Although the high throughput technology DNA microarray is very useful to reveal genome-wide gene expression profiles, high density microarrays of thousands of genes are currently too expensive for routine research activities in majority laboratories.

[0045] The present invention uses an alternative approach to combine high density gene filters and low-cost high quality microarrays to study genome-wide gene expression. Gene expression profiles between the parental metastatic breast cancer cell line MDA-MB-231 and the chromosome 6-mediated suppressed non-tumorigenic derivative cell line MDA/H6 were initially compared using gene filters with 19,592 unique human genes/6,393 controls and radioactive detection technique. Six hundred and fifty-one genes were found to have more than 800 radioactive signal intensities and more than 2-fold changes in expression between the parental breast cancer cell line MDA-MB-231 and the non-tumorigenic cell line MDA/H6.

[0046] The 651 differentially expressed genes were further examined using customized DNA microarrays and fluorescence detection techniques. Since gene expression levelsin the same cells detected by microarrays can be affected by many factors including cell culture conditions, RNA purification, cDNA labeling methods and the quality of microarrays, high quality microarrays were used in the present invention to reduce the variance that could otherwise be introduced by different microarray slides. Strong positive linear relations with high values of Pearson coefficient of correlation were obtained between 2 sets of genes on the same slides and between the genes on the different slides, demonstrating the consistency of the microarrays and reproducibility of the experiments. The microarray analysis revealed 202 genes that were expressed differentially in breast cancer cell lines (n=10) and clinical breast cancer specimens (n=3) as related to normal tissues. The genes identified by the microarray and their expression profiles are listed in Tables 1 and 2, respectively.

1TABLE 1 Genes with informative expression profiles in breast cancer cell lines Clone ID Gene Name Title Plate Position 23185 TNC hexabrachion (tenascin C, cytotactin) LCC9d11 23831 ALDOC aldolase C, fructose-bisphosphate LCC1e11 26617 ALCAM activated leucocyte cell adhesion molecule LCC2b1 26711 NCBP2 nuclear cap binding protein subunit 2, 20 kD LCC1g10 28098 LOC57862 clones 23667 and 23775 zinc finger protein LCC1e5 28116 karyopherin a2 karyopherin alpha 2 (RAG cohort 1, importin alpha 1) LCC9e1 30476 ESTs ESTs LCC9d8 32517 FLJ10509 hypothetical protein FLJ10509 LCC8e12 33949 PRPSAP1 phosphoribosyl pyrophosphate synthetase-associated protein 1 LCC1d8 36191 Fibronectin 1 fibronectin 1 LCC9d10 39884 IMPDH1 IMP (inosine monophosphate) dehydrogenase 1 LCC8a8 40026 SLC25A4 solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4 LCC8g5 44178 TEGT testis enhanced gene transcript LCC9d7 44255 RPML3 ribosomal protein, mitochondrial, L3 LCC8a7 45641 MAP2K3 mitogen-activated protein kinase kinase 3 LCC3a10 45801 ESTs ESTs LCC5b5 49496 AMID programmed cell death 8 (apoptosis-inducing factor) LCC9a12 49553 ARF4L ADP-ribosylation factor 4-like LCC8g6 49987 GRIA2 glutamate receptor, ionotropic, AMPA 2 LCC8e6 51718 ESTs ESTs LCC9b3 66686 RPL10 ribosomal protein L10 LCC1a11 71101 PROCR protein C receptor, endothelial (EPCR) LCC2h2 79710 KIAA0174 KIAA0174 gene product LCC2e10 80910 SLC1A5 solute carrier family 1 (neutral amino acid transporter), member 5 LCC2e8 108667 SF3A1 splicing factor 3a, subunit 1, 120 kD LCC8b6 112576 ESTs ESTs LCC3e1 114101 ESTs ESTs LCC9c8 127519 POH1 26S proteasome-associated pad1 homolog LCC1f11 127821 ACP5 acid phosphatase 5, tartrate resistant LCC2a8 128243 ADK adenosine kinase LCC2b5 129585 EST(Metallothionein2) EST, Moderately similar to Cd-7 Metallothionein-2 [H. sapiens] LCC3d9 131563 FLJ13443 Homo sapiens cDNA FLJ13443 fis, clone PLACE1002853 LCC4a1 134495 FLJ10976 Homo sapiens cDNA FLJ10976 fis, clone PLACE1001399 LCC4a10 135083 GRP58 glucose regulated protein, 58 kD LCC8c10 136798 Fibronectin 1 fibronectin 1 LCC9a5 138345 PTP IVA protein tyrosine phosphatase type IVA, member 1 LCC9a6 139883 ESTs ESTs LCC4b2 142586 MCT-1 MCT-1 protein LCC4f6 144926 ESTs ESTs, Weakly similar to B0495.6 [C. elegans] LCC3e5 147050 PTGS2 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) LCC9d4 147338 ESTs ESTs LCC9a7 163097 MAM melanoma adhesion molecule LCC9d1 173554 SFRS3 splicing factor, arginine/serine-rich 3 LCC6d8 191603 TUBB tubulin, beta polypeptide LCC8a4 198871 ESTs ESTs LCC9b6 201436 LCC9c4 205185 THBD thrombomodulin LCC1a7 207358 SLC2A1 solute carrier family 2 (facilitated glucose transporter), member 1 LCC3b3 208001 CD59 CD59 antigen p18-20 (antigen identified by monoclonal antibodies 16.3A5, EJ16, EJ30, EL32 LCC2b4 and G344) 208699 ESTs ESTs LCC4f7 212165 PRDX2 peroxiredoxin 2 LCC2h7 220376 ESTs ESTs LCC9b8 221632 EIF2B2 eukaryotic translation initiation factor 2B, subunit 2 (beta, 39 kD) LCC6e1 223141 ESTs ESTs LCC9d9 232772 EST(Metallothionein-1B ESTs, Highly similar to MT1B_HUMAN METALLOTHIONEIN-1B [H. sapiens] LCC1c12 233581 HIP2 huntingtin interacting protein 2 LCC3b10 234398 TCCCIA00427 Homo sapiens clone TCCCIA00427 mRNA sequence LCC3g11 236305 HARS histidyl-tRNA synthetase LCC8c12 239877 HDAC3 histone deacetylase 3 LCC3b9 244147 ZFP92 zinc finger protein homologous to Zfp92 in mouse LCC3e6 245547 KIAA0700 KIAA0700 protein LCC6c4 251753 ESTs ESTs LCC5c9 257197 NRBF-2 nuclear receptor binding factor-2 LCC4h8 271478 MAX-interacting protein MAX-interacting protein 1 LCC9b10 276547 DNMT1 DNA (cytosine-5-)-methyltransferase 1 LCC8b11 284592 PRO1659 PRO1659 protein LCC4f8 292213 PERQ1 PERQ amino acid rich, with GYF domain 1 LCC1c7 295140 FLJ0330 hypothetical protein FLJ10330 LCC4d2 295410 ESTs ESTs LCC3f6 296998 ART4 ADP-ribosyltransferase 4 LCC1h9 298155 ACADM acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain LCC2b2 298965 COX6B cytochrome c oxidase subunit VIb LCC5g11 307532 EIF4A2 eukaryotic translation initiation factor 4A, isoform 2 LCC8d7 310493 FACL3 fatty-acid-Coenzyme A ligase, long-chain 3 LCC3c10 321189 RAP1B RAP1B, member of RAS oncogene family LCC3d12 321661 PPP2R5C protein phosphatase 2, regulatory subunit B (B56), gamma isoform LCC1b4 321859 ESTs ESTs LCC4h10 322759 SNAPC5 small nuclear RNA activating complex, polypeptide 5, 19 kD LCC4b8 323474 ARF1 ADP-ribosylation factor 1 LCC8d11 325062 SLC20A1 solute carrier family 20 (phosphate transporter), member 1 LCC1e3 325102 EST(CTB2) ESTs, Moderately similar to CTB2_HUMAN C-TERMINAL BINDING PROTEIN 2.quadrature. LCC3d10 [H. sapiens] 327304 H326 H326 LCC1f9 340840 FLJ20263 (AKAP450) Homo sapiens cDNA FLJ20263 fis, clone COLF7804, highly similar to AJ131693 LCC3f3 Homo sapiens mRNA for AKAP450 protein 342378 DUSP5 dual specificity phosphatase 5 LCC1d5 346009 PFKL phosphofructokinase, liver LCC8f9 358531 JUN v-jun avian sarcoma virus 17 oncogene homolog LCC3b4 359835 SAT spermidine/spermine N1-acetyltransferase LCC8a5 359933 GNAS1 guanine nucleotide binding protein (G protein), alpha stimulating activity polypeptide 1 LCC8d9 361565 GLUD1 glutamate dehydrogenase 1 LCC8a11 365930 TAF2F TATA box binding protein (TBP)-associated factor, RNA polymerase II, F, 55 kD LCC1e12 399562 NUP54 nucleoporin p54 LCC6h2 430318 PVALB parvalbumin LCC2c1 436051 ESTs ESTs, Weakly similar to putative p150 [H. sapiens] LCC6h10 449112 EST(G3PDH) ESTs, Highly similar to G3P2_HUMAN GLYCERALDEHYDE 3-PHOSPHATE LCC6h9 DEHYDROGENASE, LIVER.quadrature. [H. sapiens] 454970 DKFZP434G032 DKFZP434G032 protein LCC9g12 469151 EIF2S2 eukaryotic translation initiation factor 2, subunit 2 (beta, 38 kD) LCC8f10 471863 DKFZp586C1817 Homo sapiens mRNA; cDNA DKFZp586C1817 (from clone DKFZp586C1817) LCC9h9 509516 LOC56966 hypothetical protein from EUROIMAGE 1034327 LCC5c5 511521 CANX calnexin LCC2a6 511586 HNRPA1 heterogeneous nuclear ribonucleoprotein A1 LCC8c11 564803 FOXM1 forkhead box M1 LCC2h3 628357 ACTN3 actinin, alpha 3 LCC2a11 665774 EIF4E eukaryotic translation initiation factor 4E LCC1h7 711959 RPC62 polymerase (RNA) III (DNA directed) (62 kD) LCC2f12 712840 STAT5B signal transducer and activator of transcription 5B LCC2d4 712848 MADD MAP-kinase activating death domain LCC2h4 713647 TSPAN-3 tetraspan 3 LCC2f6 714210 RY1 putative nucleic acid binding protein RY-1 LCC3a4 725274 TTC1 tetratricopeptide repeat domain 1 LCC2d3 730149 TCEA2 transcription elongation factor A (SII), 2 LCC1d4 739183 CD68 CD68 antigen LCC3a12 739625 KIAA0973 KIAA0973 protein LCC2h10 739993 BRE brain and reproductive organ-expressed (TNFRSF1A modulator) LCC2g11 740914 CTBP1 C-terminal binding protein 1 LCC2h5 741067 SMARCD2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, LCC1f6 member 2 741988 ACY1 aminoacylase 1 LCC8f6 745604 BCAR1 breast cancer anti-estrogen resistance 1 LCC8g8 753313 LAPTM5 Lysosomal-associated multispanning membrane protein-5 LCC1e2 753457 NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1 (75 kD) (NADH-coenzyme Q reductase) LCC1b11 753897 AMFR autocrine motility factor receptor LCC2a10 755444 TMSB4X thymosin, beta 4, X chromosome LCC6e12 756490 BCAT2 branched chain aminotransferase 2, mitochondrial LCC5a12 756600 PPIB peptidylprolyl isomerase B (cyclophilin B) LCC8d8 756769 CHAF1B chromarin assembly factor 1, subunit B (p60) LCC8b3 756968 EFNB1 ephrin-B1 LCC2g7 758365 OS4 conserved gene amplified in osteosarcoma LCC3b7 758662 PSMD9 proteasome (prosome, macropain) 26S subunit, non-ATPase, 9 LCC2e1 759200 DHPS deoxyhypusine synthase LCC8e9 760298 PRSC1 protease, cysteine, 1 (legumain) LCC2e7 770080 PXN paxillin LCC1d2 770388 CLDN4 claudin 4 LCC5b1 773147 FLJ10491 Homo sapiens cDNA FLJ10491 fis, clone NT2RP2000239 LCC5e3 773367 COMT catechol-O-methyltransferase LCC8f4 774071 CLTH Clathrin assembly lymphoid-myeloid leukemia gene LCC2g2 781704 TRIP7 thyroid hormone receptor interactor 7 LCC2g12 783698 KIAA0188 KIAA0188 protein LCC2e12

784278 SF100 nuclear antigen Sp100 LCC2c9 784841 EIF2S3 eukaryotic translation initiation factor 2, subunit 3 (gamma, 52 kD) LCC2b10 786048 E2F4 E2F transcription factor 4, p107/p130-binding LCC3a3 788574 GCN5L2 GCN5 (general control of amino-acid synthesis, yeast, homolog)-like 2 LCC2g8 789232 PSMD4 proteasome (prosome, macropain) 26S subunit, non-ATPase, 4 LCC2g4 795282 HSPC126 HSPC126 protein LCC4h3 795330 NR1D1 nuclear receptor subfamily 1, group D, member 1 LCC2b11 795888 RBBP2 retinoblastoma-binding protein 2 LCC2b12 809517 PRO2605 hypothetical protein PRO2605 LCC4g7 809648 ZNF162 zinc finger protein 162 LCC2g10 809835 HNRPC heterogeneous nuclear ribonucleoprotein C (C1/C2) LCC8d12 809992 PSMD2 proteasome (prosome, macropain) 26S subunit, non-ATPase, 2 LCC1g9 809992 PSMD2 proteasome (prosome, macropain) 26S subunit, non-ATPase, 2 LCC8b8 810019 HNRPD heterogeneous nuclear ribonucleoprotein D (AU-rich element RNA-binding protein 1, 37 kD) LCC8a10 810791 MNAT1 menage a trois 1 (CAK assembly factor) LCC8b7 810873 SCNN1A sodium channel, nonvoltage-gated 1 alpha LCC1a8 811792 GSS glutathione synthetase LCC1h2 813158 DRG2 developmentally regulated GTP-binding protein 2 LCC1g11 813280 ADSL adenylosuccinate lyase LCC2a12 813426 G53955 GS3955 protein LCC1f4 813648 DLD dihydrolipoamide dehydrogenase (E3 component of pyruvate dehydrogenase complex, LCC8b10 2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex) 813742 PTK7 PTK7 protein tyrosine kinase 7 LCC1b2 814508 PPP1R7 protein phosphatase 1, regulatory subunit 7 LCC2h9 814595 PRKCBP1 protein kinase C binding protein 1 LCC2d5 814636 SMARCA2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, LCC2e3 member 2 815542 MX1 myxovirus (influenza) resistance 1, homolog of marine (interferon-inducible protein p78) LCC2c10 815575 ACTR1A ARP1 (actin-related protein 1, yeast) homolog A (centractin alpha) LCC8f3 823930 ARPC1A actin related protein 2/3 complex, subunit 1A (41 kD) LCC1g7 824024 NQO2 NAD(P)H menadione oxidoreductase 2, dioxin-inducible LCC2c3 824031 HSJ2 heat shock protein, DNAJ-like 2 LCC3a7 824602 IFI16 interferon, gamma-inducible protein 16 LCC2f7 825470 TOP2A topoisomerase (DNA) II alpha (170 kD) LCC2b7 838366 HMGCL 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria) LCC8g4 840404 MGAT2 mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase LCC2g6 840940 PABPC1 poly(A)-binding protein, cytoplasmic 1 LCC2c8 841691 MNPEP methionine aminopeptidase; eIF-2-associated p67 LCC8c9 843016 P130 nucleolar phosphoprotein p130 LCC2f5 843328 DUSP12 dual specificity phosphatase 12 LCC5c2 852520 UQCRC2 ubiquinol-cytochrome c reductase core protein II LCC8e2 853570 SLC25A6 solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 LCC8f5 855910 LGALS3 lectin, galactoside-binding, soluble, 3 (galectin 3) LCC5a9 866882 FDFT1 farnesyl-diphosphate farnesyltransferase 1 LCC8e8 868368 TMSB4X thymosin, beta 4, X chromosome LCC5a11 877613 DCTN1 dynactin 1 (p150, Glued (Drosophila) homolog) LCC2h8 877832 DXS1357E accessory proteins BAP31/BAP29 LCC8e5 878545 RPL18 ribosomal protein L18 LCC6c9 884644 HBG1 hemoglobin, gamma A LCC5a10 897164 CTNNA1 catenin (cadherin-associated protein), alpha 1 (102 kD) LCC8e7 897177 PGAM1 phosphoglycerate mutase 1 (brain) LCC8e3 897626 PRO2706 hypothetical protein PRO2706 LCC2h11 897880 CCT4 chaperonin containing TCP1, subunit 4 (delta) LCC8d6 897983 KIAA0106 anti-oxidant protein 2 (non-selenium glutathione peroxidase, acidic calcium-independent LCC2f9 phospholipase A2) 898262 UBE1 ubiquitin-activating enzyme E1 (A1S9T and BN75 temperature sensitivity complementing) LCC8c3 949928 ZNF220 zinc finger protein 220 LCC2e2 950489 SOD1 superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult)) LCC8b12 950682 PFKP phosphofructokinase, platelet LCC8c5 951117 SHMT2 serine hydroxymethyltransferase 2 (mitochondrial) LCC3b6 951313 GP1 glucose phosphate isomerase LCC5c6 969854 CALM3 calmodulin 3 (phosphorylase kinase, delta) LCC8e4 971367 RPS8 ribosomal protein S8 LCC6c10 1160558 PTS 6-pyruvoyltetrahydropterin synthase LCC6c3 1340595 HNRPL heterogeneous nuclear ribonucleoprotein L LCC6b12 1416782 CKB creatine kinase, brain LCC8f7 1473300 HADHA hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A LCC8f11 hydratase (trifunctional protein), alpha subunit 1475028 RPS27 ribosomal protein S27 (metallopanstimulin 1) LCC6c8 1475730 CCT6A chaperonin containing TCP1, subunit 6A (zeta 1) LCC8f12

[0047]

2TABLE 2 Gene expression profiles in breast cancer cell lines Gene Name Clone ID Plate Pos. MB231-1A MB231-1B MB231-2A MB231-2B MB231-1 TNC 23185 LCC9d11 0.744 0.811 0.773 0.611 0.7775 ALDOC 23831 LCC1e11 0.877 0.944 1.038 1.043 0.9105 ALCAM 26617 LCC2b1 0.563 0.595 0.582 0.526 0.579 NCBP2 26711 LCC1g10 0.55 0.562 0.603 0.615 0.556 LOC57862 28098 LCC1e5 0.846 0.849 0.713 0.688 0.8475 karyopherin a2 28116 LCC9e1 1.167 1.23 1.169 1.204 1.1985 ESTs 30476 LCC9d8 2.033 2.229 2.179 1.895 2.131 FLJ10509 32517 LCC8e12 1.039 1.09 1.14 1.028 1.0645 PRPSAP1 33949 LCC1d8 1.38 1.575 1.347 1.296 1.4775 Fibronectin 1 36191 LCC9d10 0.559 0.59 0.639 0.542 0.5745 IMPDH1 39884 LCC8a8 0.541 0.572 0.547 0.53 0.5565 SLC25A4 40026 LCC8g5 1.716 1.829 1.465 1.704 1.7725 TEGT 44178 LCC9d7 1.4 1.458 1.511 1.291 1.429 RPML3 44255 LCC8a7 0.594 0.627 0.552 0.639 0.6105 MAP2K3 45641 LCC3a10 0.605 0.627 0.602 0.67 0.616 ESTs 45801 LCC5b5 0.62 0.66 0.661 0.579 0.64 AMID 49496 LCC9a12 1.494 1.709 1.686 1.712 1.6015 ARF4L 49553 LCC8g6 1.379 1.483 1.443 1.321 1.431 GRIA2 49987 LCC8e6 0.803 0.919 0.796 0.74 0.861 ESTs 51718 LCC9b3 1.88 1.979 2.247 1.845 1.9295 RPL10 66686 LCC1a11 2.54 2.746 2.748 2.784 2.643 PROCR 71101 LCC2h2 0.905 0.765 0.77 0.769 0.835 KIAA0174 79710 LCC2e10 0.963 1.036 1.137 1.114 0.9995 SLC1A5 80910 LCC2e8 3.853 4.213 3.84 4.022 4.033 SF3A1 108667 LCC8b6 0.574 0.585 0.695 0.498 0.5795 ESTs 112576 LCC3e1 0.684 0.75 0.973 0.699 0.717 ESTs 114101 LCC9c8 0.685 0.716 0.682 0.733 0.7005 POH1 127519 LCC1f11 1.143 1.191 1.155 1.102 1.167 ACP5 127821 LCC2a8 0.723 0.775 0.866 0.785 0.749 ADK 128243 LCC2b5 0.506 0.801 0.854 0.803 0.6535 EST 129585 LCC3d9 0.858 0.879 0.868 0.863 0.8685 FLJ13443 131563 LCC4a1 0.844 0.87 0.83 0.825 0.857 FLJ10976 134495 LCC4a10 0.851 0.878 0.947 0.896 0.8645 GRP58 135083 LCC8c10 0.651 0.67 0.612 0.602 0.6605 Fibronectin 1 136798 LCC9a5 0.548 0.574 0.559 0.557 0.561 PTP IVA 138345 LCC9a6 0.405 0.406 0.418 0.426 0.4055 ESTs 139883 LCC4b2 0.679 0.762 0.742 0.691 0.7205 MCT-1 142586 LCC4f6 1.36 1.416 1.389 1.488 1.388 ESTs 144926 LCC3e5 0.859 0.988 0.923 1.047 0.9235 PTGS2 147050 LCC9d4 0.066 0.088 0.06 0.065 0.077 ESTs 147338 LCC9a7 0.67 0.737 0.71 0.769 0.7035 MAM 163097 LCC9d1 0.652 0.839 0.681 0.634 0.7455 SFRS3 173554 LCC6d8 1.796 2.076 1.532 1.884 1.936 TUBB 191603 LCC8a4 0.892 0.96 0.871 0.951 0.926 ESTs 198871 LCC9b6 0.99 1.022 1.053 0.904 1.006 201436 LCC9c4 1.138 1.28 1.219 1.231 1.209 THBD 205185 LCC1a7 0.173 0.185 0.114 0.108 0.179 SLC2A1 207358 LCC3b3 0.593 0.592 0.649 0.696 0.5925 CD59 208001 LCC2b4 0.818 0.914 0.832 0.898 0.866 ESTs 208699 LCC4f7 0.574 0.641 0.671 0.596 0.6075 PRDX2 212165 LCC2h7 0.735 0.782 0.721 0.721 0.7585 ESTs 220376 LCC9b8 1.517 1.46 1.46 1.283 1.4885 EIF2B2 221632 LCC6e1 0.443 0.474 0.43 0.499 0.4585 ESTs 223141 LCC9d9 1.125 1.116 1.18 0.962 1.1205 EST(MTT-1B) 232772 LCC1c12 0.84 0.848 1.174 0.92 0.844 HIP2 233581 LCC3b10 0.615 0.643 0.623 0.59 0.629 TCCCIA00427 234398 LCC3g11 1.221 1.317 1.198 1.333 1.269 HARS 236305 LCC8c12 1.073 1.131 1.239 1.175 1.102 HDAC3 239877 LCC3b9 1.046 1.013 1.062 1.175 1.0295 ZFP92 244147 LCC3e6 0.829 0.865 0.784 0.89 0.847 KIAA0700 245547 LCC6c4 0.646 0.713 0.668 0.769 0.6795 ESTs 251753 LCC5c9 0.852 0.901 0.833 0.878 0.8765 NRBF-2 257197 LCC4h8 0.981 0.963 0.908 0.967 0.972 MAX-IP1 271478 LCC9b10 0.758 0.789 0.772 0.687 0.7735 DNMT1 276547 LCC8b11 0.923 1.099 1.13 1.059 1.011 PRO1659 284592 LCC4f8 1.06 1.145 1.012 1.042 1.1025 PERQ1 292213 LCC1c7 1.213 1.347 1.097 1.096 1.28 FLJ10330 295140 LCC4d2 1.248 1.565 1.34 1.17 1.4065 ESTs 295410 LCC3f6 1.362 1.364 1.409 1.355 1.363 ART4 296998 LCC1h9 0.697 0.762 0.602 0.606 0.7295 ACADM 298155 LCC2b2 1.56 1.724 1.612 1.633 1.642 COX6B 298965 LCC5g11 1.604 1.678 1.565 1.799 1.641 EIF4A2 307532 LCC8d7 1.494 2.159 1.418 1.37 1.8265 FACL3 310493 LCC3c10 0.884 1.289 1.328 1.478 1.0865 RAP1B 321189 LCC3d12 1.156 1.294 1.227 1.057 1.225 PPP2R5C 321661 LCC1b4 0.696 0.734 0.768 0.645 0.715 ESTs 321859 LCC4h10 0.471 0.535 0.569 0.503 0.503 SNAPC5 322759 LCC4b8 0.94 0.994 0.952 0.978 0.967 ARF1 323474 LCC8d11 1.149 1.114 1.09 0.95 1.1315 SLC20A1 325062 LCC1e3 1.269 1.438 1.257 1.385 1.3535 EST(CTB2) 325102 LCC3d10 1.258 1.287 1.149 1.201 1.2725 H326 327304 LCC1f9 0.833 1.007 0.865 0.929 0.92 FLJ20263(AKAP450) 340840 LCC3f3 0.883 1.089 0.803 0.673 0.986 DUSP5 342378 LCC1d5 0.45 0.488 0.429 0.478 0.469 PFKL 346009 LCC8f9 1.132 1.188 1.099 1.027 1.16 JUN 358531 LCC3b4 0.527 0.565 0.523 0.472 0.546 SAT 359835 LCC8a5 0.469 0.517 0.467 0.515 0.493 GNAS1 359933 LCC8d9 0.929 1.131 0.959 0.825 1.03 GLUD1 361565 LCC8a1 1.114 1.173 1.182 1.244 1.1435 TAF2F 365930 LCC1e12 0.888 0.918 0.941 0.992 0.903 NUP54 399562 LCC6h2 1.101 1.05 1.225 1.062 1.0755 PVALB 430318 LCC2c1 0.866 0.676 0.796 0.81 0.771 ESTs 436051 LCC6h10 0.712 0.931 0.921 0.919 0.8215 EST(G3PDH) 449112 LCC6h9 0.935 0.925 1.034 0.89 0.93 DKFZP434G032 454970 LCC9g12 0.724 0.681 0.727 0.67 0.7025 EIF2S2 469151 LCC8f10 1.07 1.08 1.209 0.934 1.075 DKFZp586C1817 471863 LCC9h9 1.486 1.689 1.51 1.086 1.5875 LOC56966 509516 LCC5c5 1.035 1.219 0.908 0.93 1.127 CANX 511521 LCC2a6 1.473 1.601 1.737 1.655 1.537 HNRPA1 511586 LCC8c11 1.492 1.527 1.535 1.566 1.5095 FOXM1 564803 LCC2h3 0.813 0.884 0.893 0.815 0.8485 ACTN3 628357 LCC2a11 0.782 0.756 0.821 0.855 0.769 EIF4E 665774 LCC1h7 0.705 0.752 0.672 0.699 0.7285 RPC62 711959 LCC2f12 1.993 2.185 1.827 2.177 2.089 STAT5B 712840 LCC2d4 0.867 0.91 0.837 0.914 0.8885 MADD 712848 LCC2h4 0.775 0.832 0.893 0.854 0.8035 TSPAN-3 713647 LCC2f6 0.715 0.749 0.747 0.784 0.732 RY1 714210 LCC3a4 0.838 0.917 0.832 0.906 0.8775 TTC1 725274 LCC2d3 0.96 0.965 0.976 0.955 0.9625 TCEA2 730149 LCC1d4 0.796 1.113 1.051 1.03 0.9545 CD68 739183 LCC3a12 0.88 0.92 0.974 0.952 0.9 KIAA0973 739625 LCC2h10 1.495 1.761 1.496 1.612 1.628 BRE 739993 LCC2g11 0.926 0.966 0.963 1.082 0.946 CTBP1 740914 LCC2h5 1.039 1.061 1.103 1.064 1.05 SMARCD2 741067 LCC1f6 1.212 1.206 1.103 1.037 1.209 ACY1 741988 LCC8f6 1.556 1.691 1.71 1.413 1.6235 BCAR1 745604 LCC8g8 0.698 0.78 0.729 0.718 0.739 LAPTM5 753313 LCC1e2 0.845 0.75 0.762 0.815 0.7975 NDUFS1 753457 LCC1b11 0.997 1.149 1.107 1.057 1.073 AMFR 753897 LCC2a10 1.516 1.667 1.592 1.535 1.5915 TMSB4X 755444 LCC6e12 1.373 1.534 1.192 1.265 1.4535 BCAT2 756490 LCC5a12 1.264 1.367 1.235 1.102 1.3155 PPIB 756600 LCC8d8 1.434 1.485 1.461 1.31 1.4595 CHAF1B 756769 LCC8b3 0.972 0.902 0.891 0.769 0.937 EFNB1 756068 LCC2g7 1.258 1.292 1.17 1.287 1.275 OS4 758365 LCC3b7 1.23 1.278 1.202 1.323 1.254 PSMD9 758662 LCC2e1 0.822 0.842 0.826 0.931 0.832 DHPS 759200 LCC8e9 1.048 1.228 1.05 1.161 1.138 PRSC1 760298 LCC2e7 0.405 0.422 0.375 0.422 0.4135 PXN 770080 LCC1d2 0.844 0.852 0.832 0.868 0.848 CLDN4 770388 LCC5b1 5.1407 5.7961 6.276 5.436 5.4684 FLJ10491 773147 LCC5e3 1.42 1.466 1.521 1.42 1.443 COMT 773367 LCC8f4 1.074 1.195 1.173 0.939 1.1345 CLTH 774071 LCC2g2 0.773 0.811 0.731 0.729 0.792 TRIP7 781704 LCC2g12 0.846 0.937 1.027 0.992 0.8915 KIAA0188 783698 LCC2e12 1.417 1.489 1.562 1.435 1.453 SP100 784278 LCC2c9 0.773 0.811 0.803 0.851 0.792 EIF2S3 784841 LCC2b10 2.165 2.741 2.062 2.676 2.453 E2F4 786048 LCC3a3 1.269 1.422 1.103 1.283 1.3455 GCN5L2 788574 LCC2g8 0.601 0.571 0.682 0.634 0.586 PSMD4 789232 LCC2g4 0.996 1.072 1.143 1.174 1.034 HSPC126 795282 LCC4h3 1.172 1.343 1.487 1.315 1.2575 NR1D1 795330 LCC2b11 0.301 0.363 0.315 0.282 0.332 RBBP2 795888 LCC2b12 1.416 1.68 1.423 1.535 1.548 PRO2605 809517 LCC4g7 0.937 0.976 0.894 0.908 0.9565 ZNF162 809648 LCC2g10 1.518 1.725 1.648 1.797 1.6215 HNRPC 809835 LCC8d12 1.04 1.115 1.319 1.011 1.0775 PSMD2 809992 LCC1g9 0.564 0.581 0.634 0.587 0.5725 PSMD2 809992 LCC8b8 0.542 0.573 0.597 0.507 0.5575 HNRPD 810019 LCC8a10 0.665 0.705 0.73 0.684 0.685 MNAT1 810791 LCC8b7 0.578 0.607 0.683 0.591 0.5925 SCNN1A 810873 LCC1a8 1.945 1.789 1.741

1.749 1.867 GSS 811792 LCC1h2 0.366 0.368 0.392 0.366 0.367 DRG2 813158 LCC1g11 0.602 0.631 0.615 0.631 0.6165 ADSL 813280 LCC2a12 1.341 1.449 1.439 1.556 1.395 GS3955 813426 LCC1f4 1.088 1.165 1.162 1.005 1.1265 DLD 813648 LCC8b10 0.703 0.762 0.78 0.599 0.7325 PTK7 813742 LCC1b2 0.509 0.576 0.425 0.481 0.5425 PPP1R7 814508 LCC2h9 1.66 1.609 1.458 1.501 1.6345 PRKCBP1 814595 LCC2d5 0.754 0.834 0.762 0.686 0.794 SMARCA2 814636 LCC2e3 1.96 2.11 2.035 2.137 2.035 MX1 815542 LCC2c10 1.237 1.526 1.168 1.083 1.3815 ACTR1A 815575 LCC8f3 1.318 1.384 1.193 1.145 1.351 ARPC1A 823930 LCC1g7 0.876 0.88 0.902 0.818 0.878 NQO2 824024 LCC2c3 0.92 0.984 0.952 1.186 0.952 HSJ2 824031 LCC3a7 1.182 1.255 1.108 1.176 1.2185 IFI16 824602 LCC2f7 0.607 0.613 0.596 0.527 0.61 TOP2A 825470 LCC2b7 0.68 0.69 0.671 0.65 0.685 HMGCL 838366 LCC8g4 0.934 0.932 0.897 0.951 0.933 MGAT2 840404 LCC2g6 1.537 1.567 1.492 1.521 1.552 PABPC1 840940 LCC2c8 0.56 0.664 0.633 0.586 0.612 MNPEP 841691 LCC8c9 1.081 1.135 1.186 1.297 1.108 P130 843016 LCC2f5 1.017 1.067 1.09 0.996 1.042 DUSP12 843328 LCC5c2 1.156 1.218 1.183 1.208 1.187 UQCRC2 852520 LCC8e2 1.073 1.134 1.116 1.082 1.1035 SLC25A6 853570 LCC8f5 1.724 2.027 1.775 1.649 1.8735 LGALS3 855910 LCC5a9 0.741 0.805 0.706 0.772 0.773 FSFT1 866882 LCC8e8 0.749 0.779 0.834 0.783 0.764 TMSB4X 868368 LCC5a11 1.407 1.484 1.239 1.237 1.4433 DCTN1 877613 LCC2h8 0.878 0.944 0.877 0.956 0.911 DXS1357E 877832 LCC8e5 1.342 1.426 1.156 1.283 1.384 RPL18 878545 LCC6c9 1.91 2.026 2.03 2.26 1.968 HBG1 884644 LCC5a10 1.237 1.444 1.376 1.44 1.3405 CTNNA1 897164 LCC8e7 1.061 1.128 0.948 1.098 1.0945 PGAM1 897177 LCC8e3 0.993 1.113 0.97 1.1 1.053 PRO2706 897626 LCC2h11 1.06 1.123 1.119 1.013 1.0915 CCT4 897880 LCC8d6 1.012 0.997 1.109 0.906 1.0045 KIAA0106 897983 LCC2f9 1.302 1.27 1.26 1.226 1.296 UBE1 898262 LCC8c3 0.859 1.01 0.783 0.913 0.9345 ZNF220 949928 LCC2e2 1.124 1.114 1.246 1.285 1.119 SOD1 950489 LCC8b12 1.219 1.255 1.434 1.124 1.237 PFKP 950682 LCC8c5 1.325 1.398 1.271 1.454 1.3615 SHMT2 951117 LCC3b6 2.854 3.233 2.71 2.87 3.0435 GPI 951313 LCC5c6 1.078 1.16 1.134 1.186 1.119 CALM3 969854 LCC8e4 1.075 1.153 1.031 1.042 1.114 RPS8 971367 LCC6c10 1.691 1.806 1.775 1.977 1.7485 PTS 1160558 LCC6c3 1.199 1.259 1.163 1.337 1.229 HNRPL 1340595 LCC6b12 0.87 0.943 0.978 0.948 0.9065 CKB 1416782 LCC8f7 0.23 0.225 0.257 0.247 0.2275 HADHA 1473300 LCC8f11 1.113 1.151 1.234 1.041 1.132 RPS27 1475028 LCC6c8 1.125 1.225 1.17 1.208 1.175 CCT6A 1475730 LCC8f12 1.472 1.531 1.582 1.287 1.5015 Gene Name MB231-2 MelTis BCTis-1 BCTis-2 MB468 ZR75-1 BT549 TNC 0.692 3.4165 0.322 0.401 0.083 0.019 0.225 ALDOC 1.0405 3.5685 0.7555 2.9265 0.19 0.8445 0.75 ALCAM 0.554 2.2295 0.3495 1.2215 0.258 0.394 3.323 NCBP2 0.609 1.118 0.498 0.7145 0.412 0.535 0.3855 LOC57862 0.7005 2.5825 0.899 2.1045 0.9105 1.349 1.0285 karyopherin a2 1.1865 0.562 0.182 0.4275 1.1675 1.706 0.737 ESTs 2.037 2.8725 0.845 2.0715 1.45 0.577 0.634 FLJ10509 1.084 1.567 0.5135 1.509 1.2675 1.106 0.9395 PRPSAP1 1.3215 3.6375 1.1135 2.146 1.6545 2.5855 0.9365 Fibronectin 1 0.5905 1.2415 0.3365 3.1255 0.3715 0.0335 2.6545 IMPDH1 0.5385 0.8995 0.4395 1.2965 0.331 0.5735 0.387 SLC25A4 1.5845 9.0595 1.2235 2.1625 1.1405 1.867 1.2185 TEGT 1.401 3.427 0.884 1.7315 0.8495 1.1535 0.6225 RPML3 0.5955 0.7495 0.296 0.7785 0.289 0.637 0.462 MAP2K3 0.636 1.1385 0.431 0.741 0.7065 0.906 0.591 ESTs 0.62 3.725 0.2185 0.5475 0.438 0.1825 1.017 AMID 1.699 2.6895 1.2005 2.6595 3.6495 5.6195 3.925 ARF4L 1.382 8.4535 1.1145 1.645 1.01 1.508 0.885 GRIA2 0.768 1.3815 1.826 0.724 0.3935 0.8915 0.487 ESTs 2.046 3.758 1.7975 2.428 2.9085 2.009 0.7815 RPL10 2.766 8.6115 0.9505 1.3005 2.265 2.556 2.6805 PROCR 0.7695 3.6755 0.4425 0.6025 1.0405 0.852 0.9815 KIAA0174 1.1255 1.211 0.881 1.5945 0.631 1.416 0.583 SLC1A5 3.931 3.868 5.366 2.341 3.6915 2.947 2.5335 SF3A1 0.5965 0.733 0.406 0.8295 0.2405 0.635 0.4675 ESTs 0.836 0.8115 0.387 0.6965 0.65 0.98 0.5765 ESTs 0.7075 1.7875 1.388 0.974 1.4545 0.329 0.992 POH1 1.1285 2.8295 0.441 0.937 0.9295 1.2075 0.7855 ACP5 0.8255 1.307 0.4865 0.521 1.0315 0.817 0.535 ADK 0.8285 1.209 0.3015 0.435 0.993 0.797 0.53 EST 0.8655 0.179 0.1535 0.2685 0.639 0.0795 0.645 FLJ13443 0.8275 0.5205 0.1785 0.266 0.8715 0.1775 0.303 FLJ10976 0.9215 2.0805 0.85 1.592 1.117 0.9175 0.6435 GRP58 0.607 0.9855 3.1965 2.688 0.8735 0.544 1.415 Fibronectin 1 0.558 6.0425 0.8805 3.9185 0.3745 0.0895 3.237 PTP IVA 0.422 1.3295 0.422 1.0725 0.594 2.0775 1.0525 ESTs 0.7165 0.904 0.1415 0.3345 0.367 0.115 0.5965 MCT-1 1.4385 1.292 0.546 1.4685 1.293 1.1955 1.31 ESTs 0.985 1.779 0.5135 1.1165 0.5125 0.7995 0.7305 PTGS2 0.0625 0.1975 0.024 0.0715 0.021 0.0145 0.193 ESTs 0.7395 1.495 0.371 0.813 0.6285 0.218 0.873 MAM 0.6575 5.7525 0.42 1.218 1.116 0.698 1.0755 SFRS3 1.708 3.2255 1.4135 1.2645 1.758 2.104 1.325 TUBB 0.911 1.3455 0.226 0.506 1.0425 0.9855 0.9375 ESTs 0.9785 0.986 1.0615 1.086 0.883 1.6875 1.4255 1.225 3.9395 2.966 1.942 3.1845 1.92 1.091 THBD 0.111 2.3235 0.3525 0.2235 0.244 0.2945 0.1125 SLC2A1 0.6725 0.3475 0.198 0.531 0.7935 1.893 0.2185 CD59 0.865 2.3895 0.82 1.177 0.961 0.8495 1.922 ESTs 0.6335 1.144 0.088 0.2355 0.3225 0.115 0.481 PRDX2 0.721 1.848 2.8795 2.5795 1.1035 2.311 0.767 ESTs 1.3715 15.3045 3.606 1.223 1.013 1.4025 2.688 EIF2B2 0.4645 0.9435 0.233 0.387 0.359 0.49 0.4835 ESTs 1.071 3.8935 0.7155 0.9165 1.4355 0.8805 0.906 EST(MTT-1B) 1.047 0.391 0.162 0.2235 0.7335 0.084 0.6565 HIP2 0.6065 1.079 0.5295 0.761 0.634 0.8495 0.545 TCCCIA00427 1.2655 1.59 1.501 3.332 0.783 0.7435 0.5815 HARS 1.207 1.187 0.634 1.1255 0.9515 1.273 1.015 HDAC3 1.1185 1.8585 0.8905 1.0185 0.6555 1.1155 0.618 ZFP92 0.837 1.59 0.5205 1.409 0.559 1.411 0.572 KIAA0700 0.7185 1.694 0.5715 0.666 1.2795 0.5085 0.4635 ESTs 0.8555 0.961 0.2585 0.756 0.7315 0.389 0.717 NRBF-2 0.9375 2.587 0.2265 0.5405 0.959 0.526 0.683 MAX-IP1 0.7295 3.3305 0.597 1.407 0.577 0.762 0.366 DNMT1 1.0945 0.8405 2.0275 2.1585 0.8315 0.954 1.284 PRO1659 1.027 3.599 0.187 0.275 0.842 0.021 1.444 PERQ1 1.0965 4.796 1.0245 3.2815 1.568 1.1255 1.252 FLJ10330 1.255 1.5485 0.486 0.833 1.04 1.6045 1.177 ESTs 1.382 2.779 2.1015 2.0485 1.25 1.8075 1.2765 ART4 0.604 3.014 0.7945 1.3 0.393 0.6045 0.686 ACADM 1.6325 1.4885 0.3485 0.8055 0.612 0.477 1.179 COX6B 1.682 6.4865 3.065 2.678 1.67 1.9215 1.21 EIF4A2 1.394 6.512 0.4635 2.4105 0.792 0.9455 0.911 FACL3 1.403 1.8865 0.5105 1.947 2.4115 1 1.1605 RAP1B 1.142 0.7935 0.3005 0.6745 0.535 1.029 1.0795 PPP2R5C 0.7065 1.481 0.565 1.0015 1.1035 1.545 0.7925 ESTs 0.536 1.083 0.442 3.847 0.349 0.2975 0.538 SNAPC5 0.965 3.2825 0.449 1.2875 0.7585 1.5055 0.3945 ARF1 1.02 4.0365 0.4755 1.469 0.586 1.2805 0.985 SLC20A1 1.321 3.6525 0.414 1.1745 0.278 0.818 0.5445 EST(CTB2) 1.175 2.5405 2.2555 2.0525 1.285 2.805 1.4345 H326 0.897 3.9665 1.95 1.2305 0.819 0.6035 0.484 FLJ20263(AKAP450) 0.738 7.9685 1.9345 2.2725 0.603 0.8755 1.638 DUSP5 0.4535 0.484 0.51 1.2275 0.156 0.062 0.061 PFKL 1.063 3.658 0.286 0.419 1.109 0.743 0.74 JUN 0.4975 1.1825 0.8815 0.365 0.6585 0.823 0.8815 SAT 0.491 0.642 0.1795 1.3215 3.9775 0.111 0.469 GNAS1 0.892 3.6785 0.6385 0.7105 0.6795 1.2895 1.128 GLUD1 1.213 0.9235 2.0105 2.4145 0.8475 0.81 1.265 TAF2F 0.9665 1.076 0.3775 0.575 0.443 0.998 0.5435 NUP54 1.1435 1.0595 0.253 0.5955 0.93 1.2285 1.196 PVALB 0.803 3034.015 29.026 34162.963 4.5445 2.8805 3.5565 ESTs 0.92 0.999 0.289 0.4215 0.414 0.854 0.685 EST(G3PDH) 0.962 3.451 0.259 0.421 0.3485 0.8045 0.54 DKFZP434G032 0.6985 6.512 89.957 22.3825 1263.422 1.834 5.4505 EIF2S2 1.0715 0.6975 0.2215 0.4095 1.34 0.5935 0.661 DKFZp586C1817 1.298 4.086 0.332 0.778 0.5305 0.317 1.083 LOC56966 0.919 3.756 2.7275 4.3095 1.323 1.782

0.8035 CANX 1.696 0.7885 0.5045 1.3235 1.6535 1.408 1.1875 HNRPA1 1.5505 0.478 0.589 1.0015 0.882 1.5625 1.062 FOXM1 0.854 2.216 0.4285 0.5505 0.76 0.9105 1.1685 ACTN3 0.838 2.366 0.6445 0.709 0.4335 0.541 0.671 EIF4E 0.6855 1.0055 0.1965 0.5985 0.5445 1.118 0.568 RPC62 2.002 3.1745 1.7305 1.367 4.4425 2.506 1.769 STAT5B 0.8755 3.293 1.177 0.9845 0.6425 0.5175 1.269 MADD 0.8735 3.8565 1.0775 2.284 0.6735 1.2 0.606 TSPAN-3 0.7655 1.165 1.198 1.5835 0.921 0.929 0.6765 RY1 0.869 1.419 0.557 2.081 0.9705 1.567 0.6545 TTC1 0.9655 4.09 1.0065 0.7975 0.664 0.5235 0.673 TCEA2 1.0405 3.398 1.9575 2.3155 1.239 0.6775 0.8565 CD68 0.963 2.552 0.444 1.186 0.204 0.27 0.2555 KIAA0973 1.554 4.203 1.791 1.516 1.752 1.1825 1.133 BRE 1.0225 3.466 1.002 1.482 0.8155 0.6545 0.6075 CTBP1 1.0835 4.6745 3.0685 1.571 1.2955 1.6935 1.3785 SMARCD2 1.07 6.044 3.235 2.036 2.4705 1.552 1.0145 ACY1 1.5615 3.971 1.0215 1.689 0.9565 0.9915 0.654 BCAR1 0.7235 1.059 0.587 0.633 0.801 1.277 0.56 LAPTM5 0.7885 2.657 1.379 0.8495 0.2375 0.2335 0.229 NDUFS1 1.082 3.4775 0.5085 1.333 1.171 1.4045 0.8045 AMFR 1.5635 4.083 1.4875 3.179 1.9635 2.4385 1.5355 TMSB4X 1.2285 0.476 0.165 0.2335 0.872 0.559 1.877 BCAT2 1.1685 0.12 0.1615 0.2255 1.442 0.6745 1.9365 PPIB 1.3855 1.404 2.69 1.053 0.6775 1.278 2.1405 CHAF1B 0.83 4.714 1.1595 3.514 2.2375 2.71 1.473 EFNB1 1.2285 2.168 0.9755 1.162 1.6805 1.826 3.9375 OS4 1.2625 2.4505 1.0495 2.309 1.038 1.5465 1.335 PSMD9 0.8785 2.708 0.539 1.236 0.73 0.8435 0.401 DHPS 1.1055 2.053 1.2455 1.435 1.2985 1.235 0.924 PRSC1 0.3985 2.1255 0.341 1.0805 0.522 0.499 0.9825 PXN 0.85 1.794 0.271 0.324 0.4075 0.3385 0.321 CLDN4 5.856 6.512 7.0405 3211.802 56.151 30.974 1.379 FLJ10491 1.4705 6.512 0.536 3.4925 1.51 1.7855 3.243 COMT 1.056 2.4605 0.505 0.905 0.709 0.6915 0.962 CLTH 0.73 1.967 0.4775 0.69 1.009 0.864 0.7125 TRIP7 1.0095 2.8065 1.123 1.442 2.3765 1.3995 0.797 KIAA0188 1.4985 1.3005 0.601 1.195 0.5375 0.298 0.75 SP100 0.827 3.287 0.855 1.373 0.6015 0.5465 0.72 EIF2S3 2.369 14.7625 2.0405 3.6135 4.507 3.2215 3.625 E2F4 1.193 2.0005 0.7935 0.955 0.729 1.0715 0.6285 GCN5L2 0.658 1.792 0.5175 0.575 0.298 0.3075 0.4975 PSMD4 1.1585 5.0575 3.592 1.7545 1.3555 1.7 1.2155 HSPC126 1.401 2.18 0.63 1.098 0.836 1.199 1.346 NR1D1 0.2985 4.848 0.84 3.061 0.375 0.206 0.4425 RBBP2 1.479 6.402 2.1615 3.8545 1.5485 1.958 2.908 PRO2605 0.901 1.724 0.658 1.3111 2.235 1.0535 0.5415 ZNF162 1.7225 4.0705 1.985 1.5815 1.8715 1.17 1.0205 HNRPC 1.165 1.2015 0.351 0.949 0.739 0.8545 0.9 PSMD2 0.6105 1.1135 0.3255 0.6645 0.313 0.5505 0.3975 PSMD2 0.552 0.7245 0.332 0.6755 0.354 0.565 0.389 HNRPD 0.707 1.3535 2.669 3.918 0.8065 0.8555 1.2305 MNAT1 0.637 3.608 0.3295 0.721 0.326 0.6645 0.419 SCNN1A 1.745 20.372 3.2285 4.603 30.8785 1.8085 2.378 GSS 0.379 1.2795 1.039 0.4315 0.1675 0.2085 0.475 DRG2 0.623 1.545 0.4875 0.797 0.3885 0.562 0.431 ADSL 1.4975 2.099 0.483 0.8595 0.7255 0.7545 0.7115 GS3955 1.0835 11.633 3.84 3.052 1.99 0.3595 0.907 DLD 0.6895 0.941 3.2445 3.756 0.871 1.072 1.3605 PTK7 0.453 4.8115 1.469 0.784 1.893 0.6335 1.8735 PPP1R7 1.4795 4.766 2.2 3.2585 2.2935 1.287 1.175 PRKCBP1 0.724 3.5355 0.701 1.0465 0.7785 1.212 1.093 SMARCA2 2.086 5.0805 3.333 2.851 2.124 1.235 0.3525 MX1 1.1255 13.161 11.482 7.6555 11.108 1.24 38.2705 ACTR1A 1.169 4.803 0.9565 1.267 0.7945 0.91 0.8815 ARPC1A 0.86 8.3555 1.001 1.348 0.7215 0.8435 0.616 NQO2 1.069 0.7825 0.1415 0.126 0.2615 0.055 0.049 HSJ2 1.142 2.51 0.9115 1.985 0.7255 1.4695 1.8275 IFI16 0.5615 1.5035 0.275 0.204 0.019 0.016 0.3175 TOP2A 0.6605 0.3715 0.181 0.4205 0.82 0.844 0.5655 HMGCL 0.924 3.8995 1.1475 1.1965 0.8875 0.858 0.721 MGAT2 1.5065 1.1425 0.547 0.6215 0.3465 0.611 0.993 PABPC1 0.6095 2.278 0.5095 0.3195 0.469 0.2935 0.7905 MNPEP 1.2415 0.7725 1.6045 1.8895 0.7735 1.037 1.3945 P130 1.043 1.3645 0.2815 0.681 0.625 0.6075 1.689 DUSP12 1.1955 1.7505 1.0515 0.9915 2.0275 1.0155 0.7885 UQCRC2 1.099 2.431 0.5465 1.148 0.94 2.072 0.858 SLC25A6 1.712 12.1205 1.356 2.082 1.161 2.0135 1.3713 LGALS3 0.739 5.6375 0.461 0.1555 0.602 0.151 1.371 FSFT1 0.8085 0.697 0.876 0.704 0.749 0.935 1.0365 TMSB4X 1.238 0.1725 0.19 0.244 1.331 0.6875 2.1755 DCTN1 0.9165 3.3475 0.7175 1.7875 0.6715 0.99 0.573 DXS1357E 1.2195 4.5985 1.069 1.6945 0.933 1.2605 1.012 RPL18 2.145 6.2765 2.634 1.9115 1.002 1.9055 1.202 HBG1 1.408 1.973 0.463 0.779 0.976 1.5835 0.947 CTNNA1 1.023 0.8295 0.295 0.57 0.1075 0.5995 0.521 PGAM1 1.035 1.873 0.657 0.7745 0.3095 0.6495 0.927 PRO2706 1.066 1.6325 0.5065 1.412 0.9995 1.1125 0.818 CCT4 1.0075 0.2765 0.0505 0.4785 0.8575 1.2675 0.393 KIAA0106 1.243 0.6715 0.3915 0.845 1.1175 0.754 0.6045 UBE1 0.848 3.496 1.2705 3.717 2.1655 2.7045 1.523 ZNF220 1.2655 2.1565 1.451 6.1565 0.855 0.9455 1.333 SOD1 1.279 2.449 1.3525 2.821 2.0195 1.3995 0.7495 PFKP 1.3625 4.3135 0.763 0.7515 1.092 1.319 0.9995 SHMT2 2.79 2.6455 0.6015 0.4655 1.561 1.2105 0.9965 GPI 1.16 1.4485 1.2495 1.3795 1.229 1.0365 1.1715 CALM3 1.0365 3.228 0.779 1.0685 0.8625 0.8105 0.733 RPS8 1.876 3.672 0.974 1.876 1.696 1.4415 1.407 PTS 1.25 1.9595 1.036 1.4485 2.795 0.9325 0.9975 HNRPL 0.963 1.693 1.4435 1.281 1.511 1.8605 1.2505 CKB 0.252 3.5425 1.5175 0.653 0.1605 1.278 0.2115 HADHA 1.1375 3.8725 0.326 0.424 1.185 0.687 0.747 RPS27 1.189 5.5065 1.9225 1.4805 1.096 1.156 0.771 CCT6A 1.4345 1.3275 0.6925 1.0815 3.487 1.0275 0.9125 Gene Name MB134 MB157 MB436 MB453 BT20 BT474 BCTis-3 TNC 0.083 0.229 0.2915 1.322 0.0275 0.31 0.862 ALDOC 0.6725 0.084 0.477 0.846 0.5965 0.3565 1.0645 ALCAM 1.2535 0.193 0.299 2.2875 0.0715 0.607 0.248 NCBP2 0.823 0.3445 0.394 0.3165 0.6855 0.709 0.9885 LOC57862 1.4175 1.389 0.753 1.8475 1.212 1.746 0.953 karyopherin a2 0.536 1.156 0.672 1.1825 0.913 1.327 0.9935 ESTs 5.3745 3.409 0.5135 2.5625 1.2645 2.3425 1.269 FLJ10509 1.4435 0.6365 0.9125 1.3555 1.6975 1.211 1.487 PRPSAP1 2.1275 2.588 0.828 2.205 1.626 1.487 3.9915 Fibronectin 1 0.052 7.429 0.6615 0.047 0.2645 0.0825 14.9675 IMPDH1 0.746 0.294 0.3385 0.3705 0.7115 0.739 0.788 SLC25A4 1.1555 3.7145 1.3925 1.3215 0.5115 2.456 1.3635 TEGT 2.5555 0.7165 0.3945 1.3355 1.243 1.5195 1.5285 RPML3 0.75 0.2245 0.3375 0.3575 0.733 0.7445 0.7515 MAP2K3 0.8695 1.1805 0.8895 0.533 1.0785 0.7475 0.6815 ESTs 0.205 0.711 0.376 0.1465 0.1745 0.143 0.437 AMID 4.7245 4.1005 6.505 2.0425 6.565 3.195 1.4385 ARF4L 0.9695 2.686 1.1445 1.018 0.604 1.83 1.2565 GRIA2 1.084 0.488 0.365 1.245 0.7055 0.7945 0.8765 ESTs 1.709 1.3445 1.0245 1.998 2.459 3.2003 1.6245 RPL10 1.3545 1.761 3.2775 5.325 1.172 1.668 2.157 PROCR 0.6095 1.3195 0.9655 0.6315 1.338 0.534 0.9085 KIAA0174 1.2295 0.7765 0.6725 0.502 2.6715 1.483 0.993 SLC1A5 6.2575 2.794 2.0725 3.821 2.165 2.8855 1.453 SF3A1 0.7375 0.293 0.3325 0.298 0.716 0.6905 0.714 ESTs 0.53 0.0445 0.563 0.291 0.677 0.671 0.8965 ESTs 0.98 1.7145 1.764 0.3215 0.346 0.8265 2.1375 POH1 1.282 0.707 1.1035 0.821 1.447 1.0855 0.924 ACP5 0.5365 0.4775 1.424 0.4095 0.7035 0.872 0.5625 ADK 0.639 0.4465 0.3545 0.424 0.7015 0.992 0.4065 EST 0.3 0.147 0.2525 0.2465 0.165 0.11 0.122 FLJ13443 0.224 0.584 0.1705 0.653 0.2 0.098 0.344 FLJ10976 1.1 0.851 0.705 0.921 2.1265 1.29 2.2155 GRP58 2.3025 1.113 1.101 2.3115 1.8155 0.762 1.4895 Fibronectin 1 0.211 10.624 0.691 0.114 0.2675 0.076 32.204 PTP IVA 1.931 1.3455 0.6125 0.8825 0.2185 1.107 1.0365 ESTs 0.539 0.7465 0.4645 0.1635 0.2185 0.1845 0.817 MCT-1 1.5805 5.669 1.3235 0.516 1.963 1.1625 0.958 ESTs 0.758 0.96 0.8875 0.584 0.456 0.523 0.8465 PTGS2 0.044 0.0095 0.0815 0.0495 0.0125 0.028 0.013 ESTs 0.359 1.08 0.7935 0.175 1.193 0.5335 1.8195 MAM 0.7305 4.4885 0.443 0.3025 0.23 0.5795 2.241 SFRS3 1.511 1.7895 1.3305 1.3975 1.632 2.5915 2.3715 TUBB 0.4865 0.578 0.4805 0.3135 0.5895 0.867 0.935 ESTs 1.476 0.8335 0.935 1.194 1.0705 3.561 2.8205 1.843 1.4565 0.554 1.357 2.8505 5.027 1.082 THBD 0.1645 0.0075 0.151 0.3495 0.0405 0.089 0.1725 SLC2A1 3.522 0.817 0.502 0.344 0.5585 0.8135 0.855 CD59 0.7925 2.476 0.7755 0.734 0.735 0.8725 7.412 ESTs 0.114 1.0425 0.3505 0.0955 0.1055 0.158 0.7315

PRDX2 0.501 0.1875 1.208 1.525 0.8105 2.3125 0.787 ESTs 3.729 0.3425 1.6685 0.993 4.8455 5.5175 1.9915 EIF2B2 0.5015 1.21 0.295 0.364 0.2235 0.435 0.328 ESTs 0.9505 1.981 1.319 0.246 1.01 0.764 0.9785 EST(MTT-1B) 0.2805 0.296 0.3035 0.197 0.2125 0.1045 0.1745 HIP2 0.8555 1.167 0.8825 0.506 0.9325 0.7075 0.6435 TCCCIA00427 1.7045 1.3355 1.066 1.752 1.272 2.3445 1.254 HARS 0.834 1.173 1.0685 1.2255 1.1475 1.0605 1.3 HDAC3 1.1245 1.085 0.8495 0.571 0.898 1.044 0.7 ZFP92 0.9415 1.1055 0.603 1.1 0.6825 5.3125 2.365 KIAA0700 0.512 1.3745 0.7875 0.3855 1.117 0.7535 0.457 ESTs 0.9295 1.2825 0.733 0.403 0.5775 0.5245 0.4285 NRBF-2 0.7695 0.819 0.95 0.6775 0.644 0.599 0.53 MAX-IP1 1.0845 0.8425 0.397 0.4715 0.4095 0.3235 1.5375 DNMT1 1.7225 0.8565 1.044 2.0605 1.5975 0.754 1.1775 PRO1659 0.099 0.921 0.0595 0.5335 0.0485 0.07 1.159 PERQ1 1.5895 5.2495 1.145 1.556 1.03 3.25 2.3 FLJ10330 0.7975 1.808 0.559 1.5015 1.8595 1.081 1.4795 ESTs 1.3085 1.2165 1.022 0.744 0.9165 1.873 1.553 ART4 1.066 0.4065 0.518 0.391 0.8325 0.902 1.1305 ACADM 2.2965 0.821 2.154 0.4005 1.266 0.8335 2.1475 COX6B 2.76 5.2755 1.4165 1.1105 2.1125 3.3935 2.335 EIF4A2 0.64 0.002 0.3925 2.472 1.831 1.081 1.3555 FACL3 4.1225 3.1055 0.893 3.682 1.4815 3.4015 1.081 RAP1B 1.0365 0.9895 1.3155 0.5945 0.9845 0.7125 1.6955 PPP2R5C 0.949 1.0355 0.7945 0.906 1.0525 1.3081 1.4955 ESTs 0.7285 0.393 0.254 0.279 0.3775 0.5245 0.3985 SNAPC5 1.091 0.945 1.3685 0.5625 1.384 1.0605 0.662 ARF1 0.5135 0.194 0.804 1.633 1.467 0.4765 0.8595 SLC20A1 0.533 0.0015 0.851 0.5355 0.3375 0.2725 0.489 EST(CTB2) 3.8435 2.0365 0.8045 2.281 0.934 1.801 3.129 H326 1.026 0.7975 0.616 0.5505 0.666 1.2165 1.6595 FLJ20263(AKAP450) 2.2365 2.7495 0.735 0.67 0.8525 1.4945 2.4845 DUSP5 0.218 0.096 0.1545 0.2435 0.037 0.0425 0.304 PFKL 0.955 1.388 0.859 0.8835 0.918 0.4175 0.447 JUN 0.1985 0.767 0.773 1.0665 0.507 0.6835 1.2975 SAT 0.6935 0.1755 0.3135 0.2115 0.225 0.627 1.149 GNAS1 0.8 1.0245 0.9545 1.253 2.394 1.083 0.9915 GLUD1 1.7325 0.777 0.947 2.33 1.265 0.795 1.0165 TAF2F 0.602 1.6335 0.4445 0.539 0.8575 0.8705 1.049 NUP54 0.8125 0.826 0.9855 1.041 1.152 1.7865 0.837 PVALB 5.18 9.3575 2.5555 2.3745 2.8045 6.814 4.736 ESTs 0.4155 0.1175 0.7225 0.4055 1.0255 0.6755 0.5645 EST(G3PDH) 0.31 0.117 0.657 0.3365 0.9385 0.5835 0.5265 DKFZP434G032 4.919 9.817 1.4585 2.224 2.4305 2.6 10279.539 EIF2S2 0.9275 1.254 0.7515 0.7625 0.847 0.293 0.373 DKFZp586C1817 1.0435 0.683 0.4245 0.145 1.73 0.443 1.008 LOC56966 2.044 2.3915 0.757 0.7455 2.525 2.741 1.254 CANX 2.065 2.773 2.262 1.528 1.2645 1.365 0.4385 HNRPA1 0.697 0.5545 0.882 1.1935 0.788 0.8645 0.956 FOXM1 0.4075 0.2225 1.927 0.506 1.124 0.949 1.111 ACTN3 0.7415 0.4775 0.5715 0.619 0.3935 0.5585 0.9855 EIF4E 0.7465 0.5455 0.701 1.369 0.937 0.9685 0.553 RPC62 1.2155 1.8925 2.1285 0.976 2.1585 1.261 3.415 STAT5B 0.9355 1.9855 0.6505 0.4215 0.5355 0.756 0.6965 MADD 0.9985 0.829 0.4595 0.606 0.6385 0.781 1.221 TSPAN-3 2.062 1.3325 0.468 0.0535 0.278 0.1785 0.7485 RY1 1.142 1.3275 1.419 0.9495 0.936 0.8085 0.9415 TTC1 1.0205 1.882 0.7765 0.6485 0.4945 0.7855 0.6875 TCEA2 0.9595 2.5575 0.375 0.5555 1.7475 1.577 0.7495 CD68 0.246 1.02 0.4565 0.19 0.325 0.248 1.716 KIAA0973 2.847 1.851 1.41 1.1345 1.404 3.31 1.978 BRE 1.341 1.467 0.736 0.62 0.737 0.878 1.261 CTBP1 1.6185 1.4985 2.015 1.4565 1.1645 1.4155 2.496 SMARCD2 2.005 1.2945 0.28 1.42 2.3675 4.6585 0.3875 ACY1 0.622 0.526 0.4715 0.855 1.084 1.0755 0.925 BCAR1 1.102 1.231 0.6615 1.2765 0.6835 0.7295 0.7375 LAPTM5 0.459 0.1235 0.2265 0.3665 0.1855 0.146 1.895 NDUFS1 1.2935 1.4475 1.171 0.83 1.1985 1.193 1.0725 AMFR 2.039 1.5275 1.252 1.993 2.527 1.3955 1.577 TMSB4X 0.359 1.0855 0.86 0.1465 0.1915 1.476 1.1765 BCAT2 0.467 2.32 0.816 0.1215 0.168 1.5805 1.7565 PPIB 2.4055 2.753 1.6725 2.5865 1.3085 0.533 1.289 CHAF1B 2.8405 2.8915 1.8245 1.4385 1.4295 2.78 2.123 EFNB1 2.488 3.235 1.4295 0.8675 0.6145 1.9215 2.2775 OS4 1.8455 0.979 0.5125 2.106 1.167 1.5885 3.1285 PSMD9 0.905 0.8305 0.851 0.5325 0.5155 0.9055 0.988 DHPS 0.6635 1.165 0.923 0.868 0.761 1.175 0.6 PRSC1 0.9015 1.2235 0.287 0.308 0.673 0.331 2.384 PXN 0.543 0.39 0.6675 0.39 0.678 0.529 0.5175 CLDN4 5.72155 5.215 1.543 9.817 28.0275 136.3065 4175.7835 FLJ10491 1.4965 0.006 2.1085 0.45 2.634 2.1025 1.371 COMT 0.5755 0.7285 0.6055 0.683 0.7345 0.777 0.916 CLTH 0.495 0.7515 0.9225 0.669 1.188 0.5015 0.905 TRIP7 2.5205 1.942 1.8265 1.3735 0.5195 2.927 1.4265 KIAA0188 0.9945 2.7635 0.596 0.904 0.6005 0.736 1.779 SP100 1.148 2.4075 0.502 0.3085 0.6435 0.291 1.857 EIF2S3 3.7685 4.4755 4.35 2.4815 3.083 5.41 5.58 E2F4 1.0375 0.4285 0.7435 0.7525 1.846 1.1245 1.037 GCN5L2 0.627 2.0175 3.049 0.206 0.214 0.288 0.9845 PSMD4 1.5415 1.667 2.1205 1.345 1.2155 1.497 2.6205 HSPC126 1.463 0.493 1.4065 1.563 1.875 1.4075 1.557 NR1D1 0.613 0.983 0.45 0.243 0.2725 0.1655 2.318 RBBP2 2.299 1.3615 4.235 2.2035 3.2825 2.618 3.394 PRO2605 15.176 1.2685 0.4715 0.6135 0.5905 1.4295 0.666 ZNF162 1.9525 2.015 1.765 1.14 1.2475 3.7065 2.343 HNRPC 0.7345 0.553 1.094 1.247 0.9495 0.879 0.8185 PSMD2 0.7405 0.2575 0.361 0.3135 0.6915 0.6895 0.758 PSMD2 0.7315 0.2665 0.311 0.3305 0.704 0.6935 0.649 HNRPD 2.118 1.0625 1.042 2.2275 1.763 0.8475 1.3195 MNAT1 0.7715 0.2935 0.3705 0.417 0.784 0.7525 0.7295 SCNN1A 71.293 2.253 2.0385 1.5535 30.312 19.2445 4.658 GSS 0.205 0.2935 0.3145 0.3595 0.269 0.203 0.3285 DRG2 0.7805 0.2655 0.421 0.3535 0.6865 0.72 1.04 ADSL 0.9785 1.2665 0.7895 0.6425 0.553 0.988 0.946 GS3955 1.6365 1.6395 1.1165 0.62 1.1645 0.356 1.472 DLD 2.229 1.1315 1.05 2.6815 1.805 0.965 1.273 PTK7 0.8635 1.899 0.368 0.542 2.02 1.1355 3.0105 PPP1R7 2.95 2.09 1.1385 1.368 1.981 1.947 2.1225 PRKCBP1 1.304 1.3 0.713 1.4 1.7865 1.563 1.526 SMARCA2 3.287 1.0865 1.5145 1.4175 2.0375 0.5805 3.353 MX1 9.4925 177.459 5.1435 3.2365 6.8535 2.7345 17.302 ACTR1A 0.602 0.662 1.055 0.561 0.762 0.776 0.908 ARPC1A 1.1655 0.5525 0.712 0.771 1.0795 0.9775 1.057 NQO2 0.073 0.0275 0.145 0.064 0.139 0.031 0.6335 HSJ2 1.268 1.8435 1.872 1.81 0.719 1.747 2.3705 IFI16 0.082 2.3755 0.631 0.0455 0.0685 0.0295 1.175 TOP2A 0.2155 0.1445 0.4195 0.1865 0.61 0.6195 0.769 HMGCL 0.648 1.1385 1.1415 0.5735 0.839 0.9715 1.1035 MGAT2 0.5085 0.629 0.3385 1.029 0.5015 0.438 0.532 PABPC1 0.4085 0.957 2.0415 0.3885 0.5955 0.33 0.69 MNPEP 1.697 0.9545 0.9245 1.6125 1.3275 0.757 1.0515 P130 0.4415 0.716 0.8015 0.451 0.267 0.667 1.3695 DUSP12 1.057 1.1505 1.004 0.92 1.0685 1.9875 1.592 UQCRC2 3.0075 1.551 1.834 1.178 1.097 1.759 1.0965 SLC25A6 1.2885 4.3495 1.6095 1.4065 0.472 2.906 1.522 LGALS3 1.6405 2.215 0.5115 0.27 0.75 0.8815 1.233 FSFT1 1.2785 0.9515 0.608 1.719 0.767 0.6835 0.7845 TMSB4X 0.514 2.523 0.8575 0.1475 0.1925 1.693 1.629 DCTN1 1.0465 0.9455 0.494 0.467 1.0355 0.443 1.3535 DXS1357E 0.8015 1.354 1.111 1.005 0.8185 1.3365 0.9385 RPL18 0.91 0.6875 0.914 0.859 1.5585 1.5095 1.271 HBG1 0.4 0.8015 0.9945 0.7335 0.645 0.8825 0.615 CTNNA1 1.4065 0.0005 0.309 0.6765 0.795 1.2165 0.7585 PGAM1 0.5585 0.507 0.4985 0.414 0.328 0.587 0.8925 PRO2706 2.172 1.4275 0.7875 1.065 1.4985 1.67 1.056 CCT4 0.4345 0.608 0.9985 0.4555 1.1 0.549 0.8025 KIAA0106 0.8625 0.7125 0.8905 0.51 1.583 1.272 0.868 UBE1 2.7625 2.7385 1.754 1.391 1.324 2.75 2.168 ZNF220 1.349 1.5735 0.6875 1.0335 0.969 1.2055 2.333 SOD1 1.633 2.692 2.051 1.0645 2.5225 3.224 2.2375 PFKP 1.37 1.374 0.6555 0.7935 1.5745 1.422 1.168 SHMT2 1.106 1.502 0.717 1.0935 0.6265 0.969 1.5105 GPI 1.981 0.804 0.368 1.126 1.1215 1.0235 0.988 CALM3 0.5515 0.9525 0.8255 0.621 0.7405 0.889 1.0095 RPS8 0.9335 2.07 2.18 1.119 0.867 1.7025 1.418 PTS 1.498 1.5405 1.3865 0.893 1.5505 1.363 3.1175 HNRPL 1.26 1.499 0.771 0.9405 1.6075

2.3 1.939 CKB 1.374 0.8095 0.1075 2.6755 0.111 0.0265 1.051 HADHA 1.1565 1.4865 0.8275 0.7885 0.8945 0.358 0.615 RPS27 0.585 1.8435 1.2445 1.0555 0.6795 1.972 2 CCT6A 1.2785 2.0385 1.201 1.0285 3.4195 2.9105 1.096

[0048] The high quality cDNA microarrays were then used to measure expression of 768 arrayed elements in malignant breast cancer cell lines (n=10) and tissue samples (n=3) using the non-tumorigenic cell line MDA/H6 as a common reference. The name and origin of the breast cancer cell lines and tissues are listed in Table 3. Pearson coefficient of correlation was used to compute the matrix of similarities and dissimilarities between samples and genes. The complex matrix relationships between 15 cancer samples and between 202 genes were simplified and visualized by multidimensional scaling and hierarchical dendrogram clustering analyses. First, the expression profiles of 202 genes in two MDA-MB-231 samples were essentially identical (r=0.982) and the expression pattern of the melanoma sample was the most dissimilar to that of MDA-MB-231 (r=0.325), as expected. Secondly, the expression profiles of other 12 breast cancers were distributed between the identity and the dissimilarity and had their own expression patterns, demonstrating the extensive heterogeneous nature of these breast cancer cells. Thirdly, the expression profiles of BT20, BT474 and ZR75-1 cell lines were more similar to each other (r=0.796) than to others.

3TABLE 3 Human Cancer Cell Line and Tissue Original Name Symbol Clinical Diagnosis Source MDA-MB-231 MB231 Adenocarcinoma ATCC.sup.1 MDA/H6 MDA/H6 Non-tumorigenic Dr. Negrini.sup.3 MDA-MB-134 MB134 Carcinoma ATCC MDA-MB-157 MB157 Carcinoma ATCC MDA-MB-436 MB436 Adenocarcinoma ATCC MDA-MB-453 MB453 Carcinoma ATCC MDA-MB-468 MB468 Adenocarcinoma ATCC BT-20 BT20 Adenocarcinoma ATCC BT-474 BT474 Ductal carcinoma ATCC BT-549 BT549 Ductal carcinoma ATCC ZR75-1 ZR751 Ductal carcinoma ATCC Breast cancer tissue 1 BCTis-1 Poorly differentiated LCC.sup.2 invasive ductal carcinoma Breast cancer tissue 2 BCTis-2 Poorly differentiated LCC infiltrating ductal carcinoma Breast cancer tissue 3 BCTis-3 Poorly differentiated LCC infiltrating carcinoma Melanoma tissue MelTis Metastatic malignant LCC melanoma .sup.1American Type Culture Collection; .sup.2Lombardi Cancer Center Histology Facility; .sup.3Department of Experimental and Diagnostic Medicine, Section of Microbiology, University of Ferrera, Via Luigi Borsari 46, 44100 Ferrara, Italy.

[0049] The microarray gene expression analysis revealed 19 genes with high frequent alterations in their expression in human breast cancers. Out of the 19 genes, 9 genes were over-expressed (Table 4) and 10 genes were under-expressed (Table 5) in breast cancers at the frequencies greater than 77% (n=13).

4TABLE 4 Over-expressed BCSGs in breast cancer cell line and tissue samples Symbol Locus ID Nucleotide Seq. Amino acid Seq. MX1 4599 SEQ ID NO:1 SEQ ID NO:20 PVALB 5816 SEQ ID NO:2 SEQ ID NO:21 RBBP2 5927 SEQ ID NO:3 SEQ ID NO:22 AIF 84883 SEQ ID NO:4 SEQ ID NO:23 CLDN4 1364 SEQ ID NO:5 SEQ ID NO:24 KRT23 25984 SEQ ID NO:6 SEQ ID NO:25 SLC1A5 6510 SEQ ID NO:7 SEQ ID NO:26 EIF2S3 1968 SEQ ID NO:8 SEQ ID NO:27 SCNN1A 6337 SEQ ID NO:9 SEQ ID NO:28

[0050]

5TABLE 5 Under-expressed BCSGs in breast cancer cell line and tissue samples Symbol Locus ID Nucleotide Seq. Amino acid Seq. THBD 7056 SEQ ID NO:10 SEQ ID NO:29 PTGS2 5743 SEQ ID NO:11 SEQ ID NO:30 GSS 2937 SEQ ID NO:12 SEQ ID NO:31 DUSP5 1847 SEQ ID NO:13 SEQ ID NO:32 NQO2 4835 SEQ ID NO:14 SEQ ID NO:33 TNC 3371 SEQ ID NO:15 SEQ ID NO:34 LAPTM5 7805 SEQ ID NO:16 SEQ ID NO:35 IFI16 3428 SEQ ID NO:17 SEQ ID NO:36 CD68 968 SEQ ID NO:18 SEQ ID NO:37 EIF2B2 8892 SEQ ID NO:19 SEQ ID NO:38

[0051] Six of the nine over-expressed genes were known to be involved in human cancers. The interferon-inducible protein p78 (MX1) is over-expressed in human prostate cancer cell line LNCaP (Vaarala et al., Lab. Invest., 80:1259-1268, 2000). Parvalbumin (PVALB) is a Ca.sup.2+ binding protein and was highly expressed in human carcinoma, mouse neuroblastoma and rat glioma (Pfyffer et al., 412:135-144, 1987). The retinoblastoma binding protein 2 (RBBP2) can bind to the tumor suppressor gene RB and reverse RB-mediated suppression of the activity of the E2F transcription factor (Kim et al., Mol. Cell Biol., 14:7256-7264, 1994). The apoptosis inducible factor (AMID) is a flavoprotein that is normally confined to mitochondria and is sufficient to induce apoptosis of isolated nuclei (Susin et al., Nature, 397:441-446, 1999). Claudin 4 (CLDN4) is a member of the family of tight junction proteins and was shown to up-regulated in ovarian cancer (Hough et al., Cancer Res., 60:6281-6287, 2000). An expression of keratin 23 (KRT23) was highly inducible by pro-apoptotic agent sodium butyrate in different pancreatic cancer cells and this induction was blocked by expression of p21 (WAF1/CIP1) antisense RNA (Zhang et al., 30:123-135, 2001). In addition, soluble carrier family 1 member 5 (SLC1A5) is a neutral amino acid transport-like protein and was up-regulated in 12 of the 13 breast cancer cell lines/tissue samples. Eukaryotic translation initiation factor 2B gamma (EIF2S3) and sodium channel nonvoltage-gated 1.alpha. (SCNN1A) were up-regulated in 12 and 10 of the 13 breast cancer cell lines/tissue samples, respectively.

[0052] Among the under-expressed genes listed in Table 5, thrombomodulin (THBD), a negative regulator of coagulation, was reported to involve in vascular diseases and cancers. (Kim et al., Anticancer Res., 17:2319-2323, 1997; Hosaka et al., Cancer Lett., 161:231-240,2000). Prostaglandin-endoperoxide synthase 2 (PTGS2) was reported to be undetectable in mammary invasive carcinomas and was more likely detected in ductal carcinomas in situ (Soslow et al., Cancer, 89:2637-2645, 2000). PTGS2 was down-regulated in all 13 breast cancer cell lines/tissue samples. Up-regulation of glutathione synthetase (GSS) is known to increase the cellular levels of glutathione that in turn facilitates growth of certain cells (Huang et al., FASEB J, 15:19-21, 2001). The GSS expression was decreased in 12 of 13 breast cancer cell lines/tissue samples, suggesting that the advanced cancer cells do not require high levels of glutathione for their growth. Dual specificity protein tyrosine phosphatase 5 (DUSP5) is inducible by serum stimulation of fibroblasts and by heat shock, and the DUSP5 induction may lead to the deactivation of mitogen- or stress-activated protein kinase 3 (MAPK3) that participates in cell cycle progression (Ishibashi et al., J. Biol. Chem., 269:29897-29902, 1994). NAD(P)H menadione oxidoreductase 2 (NQO2) is expressed in human heart, brain, lung, liver, and skeletal muscle but is not expressed in placenta, implying its decrease in fast growth tissue. The expression of NQO2 is inducible by antioxidants and its role in cancer remains unknown. Interestingly, the expression of the eukaryotic translation initiation factor 2 beta subunit (EIF2B2) was decreased more than 2 fold in 11 of 13 breast cancer cell lines/tissue samples, whereas the gamma subunit (EIF2S3) was up-regulated in all 13 breast cancer cell lines/tissue samples. The discovery that the inverse levels of EIF2B2 and EIF2S3 were associated with breast cancer progression has not been reported before. Hexabrachion (TNC) is an extracellular matrix glycoprotein that modulates cellular organization (Talts et al. J. Cell Sci., 112:1855-1864, 1999) and the TNC expression was down-regulated in 10 of the 13 breast cancer cell lines/tissue samples.

[0053] Further analysis demonstrated that the THBD RNA levels decreased in all 13 breast cancer cell lines/tissue samples (FIG. 4, panel (B)). In addition, Western blot analysis correlated the THBD protein expression to its RNA levels in all five cell lines tested. Furthermore, the THBD protein levels were negative in all 18 cases of the advanced breast cancer cells in contrast to normal mammary epithelial cells, measured by in situ immunohistochemical staining (Table 6). It thus appears that THBD expression is inversely correlated to the development of breast cancer.

6TABLE 6 Results of in situ immunohistochemical staining of THBD antibody on 20 cases of breast normal and cancer specimens Pathological diagnosis THBD staining Case Malignancy Metastasis to RLN MEC BCC 1 Infiltrating ductal To 19 of 20 RLN + + + - carcinoma 2 Infiltrating ductal To 2 of 2 RLN + + + - carcinoma 3 Infiltrating lobular and To 5 of 15 RLN + + - ductal carcinoma 4 Infiltrating ductal To 1 of 9 RLN + + + - carcinoma 5 Infiltrating ductal To 1 of 12 RLN + + - carcinoma 6 Infiltrating ductal NE + + + - carcinoma 7 Infiltrating carcinoma, NE + + + - poorly differentiated 8 Infiltrating ductal NE + + + - carcinoma 9 Infiltrating ductal NE + + - carcinoma 10 Infiltrating ductal NE + + + - carcinoma 11 Infiltrating ductal NE + + - carcinoma 12 Infiltrating ductal NE + + - carcinoma 13 Infiltrating ductal NE + + + - carcinoma 14 Infiltrating ductal NE + + + - carcinoma 15 Infiltrating ductal NE + + + - carcinoma 16 Infiltrating ductal NE + + - carcinoma 17 Infiltrating ductal NE + + + - carcinoma 18 Infiltrating ductal NE + + - carcinoma 19 Infiltrating NE + + + + + adenocarcinoma, moderately well differentiated 20 Infiltrating ductal NE + + + + carcinoma with intramammary lymphatic invasion

[0054] Each case of the normal and breast cancer specimens was from the same patients. All the sections were purchased from Lombardi Cancer Center (LCC) Histology Facility. The malignant diagnosis was derived from LCC pathological reports and further verified using HE stained sections. The Metastatic diagnosis was from the LCC pathological reports. The criteria for scoring the positive and negative results are follows: the intensities of immunohistochemical staining: 0 (none), 1 (weak), 2 (moderate), and 3 (strong); distribution of the intensities: 0 (none), 1 (1-15%), 2 (26-50%), 3 (51-75%), and 4 (76-100%); sum=an intensity number+distribution number, sum 0 is score 0, sums 1, 2, and 3 were grouped as score 1, sums 4 and 5 were grouped as score 2, sums 6 and 7 were grouped to score 3; negative (-): means score 0 or score 1, positive (++) means score 2 and positive (+++) means score 3. THBD: thrombomodulin. MEC: normal mammary epithelial cells; BCC: breast cancer cells; RLN: regional lymph nodes. NE: non evidence.

[0055] BCSG Products as Markers for Breast Cancer

[0056] Most of the BCSGs listed in Tables 4 and 5 have not been previously associated with breast cancer. BCSG homologs from other organisms may also be useful in the use of animal models for the study of breast cancer and for drug evaluation. BCSG homologs from other organisms may be obtained using the techniques outlined below.

[0057] In one aspect, the present invention is based on the identification of a number of genes, designated breast-cancer specific genes (BCSGs) set forth in Tables 4 and 5, which are differentially expressed between the breast cancer tissues/cell lines and the non-tumorigenic control tissues/cell lines. The proteins encoded by these genes may in turn be components of disease pathways and thus may serve as markers of breast cancer development or as novel therapeutic targets for treatment and prevention of breast cancer.

[0058] Accordingly, the present invention pertains to the use of polynucleotides transcribed from and polypeptides encoded by the BCSGs of Table 4 and 5 as markers for breast cancer. Moreover, the use of expression profiles of these genes can indicate the presence of or a risk of breast cancer. These markers are further useful to correlate differences in levels of expression with a poor or favorable prognosis of breast cancer. For example, panels of the BCSGs can be conveniently arrayed on solid supports for use in kits. The BCSGs can be used to assess the efficacy of a treatment or therapy of breast cancer, or as a target for a treatment or therapeutic agent. The BCSGs can also be used to generate gene therapy vectors that inhibit breast cancer.

[0059] Therefore, without limitation as to mechanism, the invention is based in part on the principle that modulation of the expression of the BCSGs of the invention may ameliorate breast cancer, when they are expressed at levels similar or substantially similar to normal (non-diseased) tissue.

[0060] As an example, the expression of THBD, one of the BCSGs listed in Table 5, is dowregulated in the parental metastatic breast cancer cell line MDA-MB-231 comparing to the non-tumorigenic derivative MDA/H6. Accordingly, modulation of the down-regulated THBD gene to normal levels (e.g., levels similar or substantially similar to tissue substantially free of breast cancer) may allow for amelioration of breast cancer.

[0061] In another embodiment of the invention, a BCSG product (including polynucleotides transcribed from a BCSG and polypeptide translated from such polynucleotides) can be used as a therapeutic compound of the invention. In yet other embodiments, a modulator of an BCSG product of the invention may be used as a therapeutic compound of the invention, or may be used in combination with one or more other therapeutic compositions of the invention. Formulation of such compounds into pharmaceutical compositions is described in subsections below.

[0062] In another aspect of the invention, the levels of BCSMs are determined in a particular subject sample for which either diagnosis or prognosis information is desired. The level of a number of BCSMs simultaneously provides an expression profile, which is essentially a "fingerprint" of the presence or activity of a BCSG or plurality of BCSGs that is unique to the state of the cell. In certain embodiments, comparison of relative levels of expression is indicative of the severity of breast cancer, and as such permits for diagnostic and prognostic analysis. Moreover, by comparing relative expression profiles of BCSGs from tissue samples taken at different points in time, e.g., pre- and post-therapy and/or at different time points within a course of therapy, information regarding which genes are important in each of these stages is obtained. The identification of genes that are abnormally expressed in breast cancer tissue versus normal tissue, as well as differentially expressed genes during breast cancer development, allows the use of this invention in a number of ways. For example, comparison of expression profiles of BCSGs at different stages of the disease progression provides a method for long-term prognosing, including survival.

[0063] The discovery of the differential gene expression patterns for individual or panels of BCSMs allows for screening of test compounds with the goal of modulating a particular expression pattern. For example, screening can be done for compounds that will convert an expression profile for a poor prognosis to one for a better prognosis. In certain embodiments, this may be done by making biochips comprising sets of BCSMs, which can then be used in these screens. These methods can also be done on the protein level. For example, protein expression levels of the BCSGs can be evaluated for diagnostic and prognostic purposes or to screen test compounds. Furthermore, the modulation of the activity or expression of a BCSM may be correlated with the diagnosis or prognosis of breast cancer.

[0064] BCSG-Related Polynucleotides

[0065] BCSG-related polynucleotides can be prepared using any of a variety of techniques. For example, a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs for tumor-associated expression (i.e., expression that is at least two fold greater in a breast tumor than in normal tissue, as described in the present invention. Alternatively, polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as breast cancer cells. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesized.

[0066] An amplified portion may be used to isolate a full length gene from a suitable library (e.g., a breast cancer cDNA library) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequences.

[0067] Alternatively, there are numerous amplification techniques for obtaining a full length coding sequence from a partial cDNA sequence. Within such techniques, amplification is generally performed via PCR. Any of a variety of commercially available kits may be used to perform the amplification step. Primers may be designed using, for example, software well known in the art. Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68.degree. C. to 72.degree. C. The amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.

[0068] One such amplification technique is inverse PCR, which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR and walking PCR. Other methods employing amplification may also be employed to obtain a full length cDNA sequence.

[0069] In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA sequences may also be obtained by analysis of genomic fragments.

[0070] Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a breast tumor protein, or portion thereof, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein. In addition a portion may be administered to a patient such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding a breast tumor polypeptide, and administering the transfected cells to the patient).

[0071] A portion of a sequence complementary to a coding sequence (i.e., an antisense polynucleotide) may also be used as a probe or to modulate gene expression. cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells or tissues to facilitate the production of antisense RNA. An antisense polynucleotide may be used, as described herein, to inhibit expression of a BCSG protein. Antisense technology can be used to control gene expression through triple-helix formation, which compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Alternatively, an antisense molecule may be designed to hybridize with a control region of a gene (e.g., promoter, enhancer or transcription initiation site), and block transcription of the gene; or to block translation by inhibiting binding of a transcript to ribosomes.

[0072] A portion of a coding sequence, or of a complementary sequence, may also be designed as a probe or primer to detect gene expression. Probes may be labeled with a variety of reporter groups, such as radionuclides and enzymes, and are preferably at least 10 nucleotides in length, more preferably at least 20 nucleotides in length and still more preferably at least 30 nucleotides in length. Primers, as noted above, are preferably 22-30 nucleotides in length.

[0073] Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2-O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

[0074] Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). The polynucleotides may also be administered as naked plasmid vectors. Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art.

[0075] Other formulations for therapeutic purposes include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

[0076] BCSG-Related Polypeptides

[0077] Within the context of the present invention, BCSG-related polypeptides comprise at least a biologically active portion or an immunogenic portion of a BCSG encoded polypeptide or a variant thereof.

[0078] Immunogenic portions may generally be identified using well known techniques. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are "antigen-specific" if they show immunospecific binding to an antigen (i.e., binding to the antigen with an affinity that is at least 10.sup.5M.sup.-1). Such antisera and antibodies may be prepared as described herein, and using well known techniques. An immunogenic portion of a native breast cancer protein is a portion that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, .sup.125I-labeled Protein A.

[0079] BCSG related polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

[0080] BCSG related polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by polynucleotides as described above may be readily prepared from the polynucleotides using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.

[0081] Portions and other variants having less than about 100 amino acids, and generally less than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, Calif.), and may be operated according to the manufacturer's instructions.

[0082] Within certain specific embodiments, a polypeptide may be a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and a fusion partner. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.

[0083] Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

[0084] A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

[0085] The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.

[0086] Antibodies

[0087] The present invention further provides antibodies and antigen-binding fragments thereof, that specifically bind to a BCSM (BCSM-specific antibodies). As used herein, an antibody, or antigen-binding fragment thereof, is said to "specifically bind" to a BCSM if it binds to an antigen with an affinity that is at least 10.sup.5M.sup.-1. As used herein, "binding" refers to a noncovalent association between two separate molecules such that a complex is formed.

[0088] Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

[0089] Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using methods well known in the art. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, breasties of hybrids are observed. Single breasties are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

[0090] Monoclonal antibodies may be isolated from the supernatants of growing hybridoma breasties. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

[0091] Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns and digested by papain to yield Fab and Fe fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.

[0092] Additionally, recombinant anti-BCSM antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

[0093] Humanized antibodies are particularly desirable for therapeutic treatment of human subjects. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues forming a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the constant regions being those of a human immunoglobulin consensus sequence. The humanized antibody will preferably also comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin.

[0094] A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable antibody either directly or indirectly (e.g., via a linker group). A direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.

[0095] Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.

[0096] It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers that provide multiple sites for attachment can be used.

[0097] Vectors

[0098] Another aspect of the invention pertains to vectors containing a polynucleotide encoding a BCSG protein, or a portion thereof. One type of vector is a "plasmid," which includes a circular double stranded DNA loop into which additional DNA segments can be ligated. Vectors include expression vectors and gene delivery vectors.

[0099] The expression vectors of the invention comprise a polynucleotide encoding a BCSG protein or a portion thereof in a form suitable for expression of the polynucleotide in a host cell, which means that the expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the polynucleotide sequence to be expressed. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by polynucleotides as described herein (e.g., BCSG polypeptides, variants of BCSG polypeptides, fusion proteins, and the like).

[0100] The expression vectors of the invention can be designed for expression of BCSG polypeptides in prokaryotic or eukaryotic cells. For example, BCSG polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. In certain embodiments, such protein may be used, for example, as a therapeutic protein of the invention. Alternatively, the expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0101] In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1, pMFa, pJRY88, pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).

[0102] Alternatively, BCSG polypeptides of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series and the pVL series.

[0103] In yet another embodiment, a BCSG is expressed in mammalian cells using a mammalian expression vector. When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.

[0104] In another embodiment, the mammalian expression vector is capable of directing expression of the polynucleotide preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the polynucleotide). Tissue-specific regulatory elements are known in the art and may include epithelial cell-specific promoters. Other non-limiting examples of suitable tissue-specific promoters include the liver-specific albumin promoter, lymphoid-specific promoters, promoters of T cell receptors and immunoglobulins, neuron-specific promoters (e.g., the neurofilament promoter), pancreas-specific promoters, and mammary gland-specific promoters (e.g., milk whey promoter). Developmentally-regulated promoters are also encompassed, for example the marine box promoters and the .alpha.-fetoprotein promoter. In certain preferred embodiments of the invention, the tissue-specific promoter is an epithelial cell-specific promoter.

[0105] The invention provides a recombinant expression vector comprising a polynucleotide encoding a BCSG cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to mRNA corresponding to a BCSG of the invention. Regulatory sequences operatively linked to a polynucleotide cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense polynucleotides are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.

[0106] The invention further provides gene delivery vectors for delivery of polynucleotides to cells, tissue, or to a the mammal for expression. For example, a polynucleotide sequence of the invention can be administered either locally or systemically in a gene delivery vector. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constituted or regulated. The invention includes gene delivery vehicles capable of expressing the contemplated polynucleotides. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, lentiviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vectors. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, togavirus viral vector.

[0107] Delivery of the gene therapy constructs of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, liposomes, polycationic condensed DNA linked or unlinked to inactivated adenovirus, ligand linked DNA, naked DNA and eucaryotic cell delivery vehicles cells.

[0108] Another aspect of the invention pertains to the expression of BCSGs using a regulatable expression system. Systems to regulate expression of therapeutic genes have been developed and incorporated into the current viral and nonviral gene delivery vectors. Examples of regulatable systems include: the tet-on/off system, the ecdysone system, the progesterone-system, and the rapamycin system.

[0109] Methods for Detecting Breast Cancer

[0110] In general, breast cancer may be detected in a patient based on the presence of one or more BCSG products (polynucleotides or polypeptide) in a biological sample (for example, blood, sera, sputum urine and/or tumor biopsies) obtained from the patient. In other words, such BCSG products may be used as markers to indicate the presence or absence of breast cancer. In addition, such products may be useful for the detection of other cancers. The antibodies provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the levels of transcribed polynucleotides from BCSGs, which is also indicative of the presence or absence of a cancer.

[0111] There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a cancer in a patient may be determined by (a) contacting a biological sample obtained from a patient with an antibody; (b) detecting in the sample a level of polypeptide that binds to the antibody; and (c) comparing the level of polypeptide with a predetermined control value.

[0112] In a preferred embodiment, the assay involves the use of antibody immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the antibody/polypeptide complex. Such detection reagents may comprise, for example, an antibody that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the antibody, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the antibody is indicative of the reactivity of the sample with the immobilized antibody. Suitable polypeptides for use within such assays include full length breast tumor proteins and portions thereof to which the antibody binds, as described above.

[0113] The solid support may be any material known to those of ordinary skill in the art to which the tumor protein may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The antibody may be immobilized on the solid support using a variety of techniques known to those of skill in the art. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antibody and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the antibody, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of antibody ranging from about 10 ng to about 10 .mu.g, and preferably about 100 ng to about 1 .mu.g, is sufficient to immobilize an adequate amount of the antibody.

[0114] In certain embodiments, the assay is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

[0115] To determine the presence or absence of breast cancer, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined control value. In one preferred embodiment, the control value for the detection of breast cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer. A sample generating a signal that is significantly higher (e.g., .gtoreq.200%) or lower (e.g., .ltoreq.50%) than the control value determined by this method may be considered indicative of cancer.

[0116] In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the antibody is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent. Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 .mu.g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.

[0117] Numerous other assay protocols exist that are suitable for use with the BCSG products or antibodies of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use BCSG polypeptides to detect antibodies that bind to such polypeptides in a biological sample. The detection of such BCSG-specific antibodies may correlate with the presence of breast cancer.

[0118] As noted above, breast cancer may also, or alternatively, be detected based on the level of mRNA transcribed from a BCSG in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a breast tumor cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the breast tumor protein. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a breast tumor protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the tumor protein in a biological sample.

[0119] To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 70%, preferably at least about 80% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a breast tumor protein that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length. In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence recited in SEQ ID NOS:1-19. Techniques for both PCR based assays and hybridization assays are well known in the art.

[0120] One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis. Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase/decrease in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample may be considered indicative of cancer.

[0121] As noted above, to improve sensitivity, multiple BCSG markers may be assayed within a given sample. It will be apparent that antibodies specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of BCSG markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for BCSG products provided herein may be combined with assays for other known tumor antigens.

[0122] Diagnostic Kits

[0123] The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a polypeptide. Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.

[0124] Alternatively, a kit may contain at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide transcribed from a BCSG. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide transcribed from a BCSG.

[0125] Arrays and Biochips

[0126] The invention also includes an array comprising a panel of BCSMs of the present invention. The array can be used to assay expression of one or more genes in the array.

[0127] It will be appreciated by one skilled in the art that the panels of BCSMs of the invention may conveniently be provided on solid supports, as a biochip. For example, polynucleotides may be coupled to an array (e.g., a biochip using GeneChip.RTM. for hybridization analysis), to a resin (e.g., a resin which can be packed into a column for column chromatography), or a matrix (e.g., a nitrocellulose matrix for northern blot analysis). The immobilization of molecules complementary to the BCSG(s), either covalently or noncovalently, permits a discrete analysis of the presence or activity of each BCSG in a sample. In an array, for example, polynucleotides complementary to each member of a panel of BCSGs may individually be attached to different, known locations on the array. The array may be hybridized with, for example, polynucleotides extracted from a blood or colon sample from a subject. The hybridization of polynucleotides from the sample with the array at any location on the array can be detected, and thus the presence or quantity of the BCSG and BCSG transcripts in the sample can be ascertained. In a preferred embodiment, an array based on a biochip is employed. Similarly, Western analyses may be performed on immobilized antibodies specific for BCSMs hybridized to a protein sample from a subject.

[0128] It will also be apparent to one skilled in the art that the entire BCSM (protein or polynucleotide) molecule need not be conjugated to the biochip support; a portion of the BCSM or sufficient length for detection purposes (i.e., for hybridization), for example a portion of the BCSM which is 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100 or more nucleotides or amino acids in length may be sufficient for detection purposes.

[0129] Identifying Modulators of BCSM

[0130] The invention also provides methods for identifying modulators, i.e., candidate agents which (a) bind to a BCSM, or (b) have a modulatory (e.g., stimulatory or inhibitory) effect on the activity of a BCSM or, more specifically, (c) have a modulatory effect on the interactions of the BCSM with one or more of its natural substrates (e.g., peptide, protein, hormone, co-factor, or polynucleotide), or (d) have a modulatory effect on the expression of the BCSMs. Such assays typically comprise a reaction between the BCSM and one or more assay components. The other components may be either the candidate agents itself, or a combination of candidate agents and a binding partner of the BCSM.

[0131] The candidate agents of the present invention are generally either small molecules or bioactive agents. In one embodiment the test compound is a small molecule. In another embodiment, the test compound is a bioactive agent. Bioactive agents include but are not limited to naturally-occurring or synthetic compounds or biomolecules. One skilled in the art will appreciate that the nature of the candidate agents may vary depending on the nature of the protein encoded by the BCSG of the invention. For example, if the BCSG encodes an orphan receptor having an unknown ligand, the test compound may be any of a number of bioactive agents which may act as cognate ligand, including but not limited to, cytokines, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, or proteases. In another embodiment, the candidate agents can be an antisense polynucleotide molecule which is complementary to a BCSG polynucleotides.

[0132] As used herein, the term "binding partner" refers to a bioactive agent which serves as either a substrate for a BCSM, or alternatively, as a ligand having binding affinity to the BCSM.

[0133] Modulators of BCSG expression, activity or binding ability are useful as thereapeutic compositions of the invention. Such modulators (e.g., antagonists or agonists) may be formulated as pharmaceutical compositions, as described herein below. Such modulators may also be used in the methods of the invention, for example, to diagnose, treat, or prognose breast cancer.

[0134] Vaccines

[0135] Within certain aspects, BCSG products (polypeptides and polynucleotides) described herein may be used as vaccines for breast cancer. Vaccines may comprise one or more such products and an immunostimulant. An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. Examples of immunostimulants include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes. Vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be present, either incorporated into a fusion polypeptide or as a separate compound, within the composition or vaccine.

[0136] A vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be naked DNA. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a polypeptide component. Such vaccines may provide for an enhanced immune response.

[0137] It will be apparent that a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).

[0138] Any of a variety of immunostimulants may be employed in the vaccines of this invention. For example, an adjuvant may be included. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadellci pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable micro spheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

[0139] Any vaccine provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient. The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.

[0140] Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. Such carriers include microparticles of poly(lactide-co-glycolide), as well as polyacrylate, latex, starch, cellulose and dextran. Other delayed-release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

[0141] Pharmaceutical Compositions

[0142] The invention is further directed to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and at least one of the following: a BCSM, a variant of a BCSM, a BCSM modulator, a BCSM-specific antibody, a vaccine generated using a BCSM or its variant, and a vector capable of expressing a BCSM or a variant of a BCSM.

[0143] As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, lubricants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions.

[0144] The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a BCSM of the invention. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a BCSM . Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a BCSM and one or more additional bioactive agents.

[0145] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0146] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the injectable composition should be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the requited particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0147] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of a BCSM or an anti-BCSM antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active, ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0148] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0149] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0150] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the bioactive compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0151] In one embodiment, the therapeutic moieties, which may contain a bioactive compound, are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.

[0152] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0153] The BCSGs of the invention can be inserted into gene delivery vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by intravascular, intrameucular, subcutaneous, intraperitoneal injection, by direct injection into the target tissue, by inhalation, or by perfusion. The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0154] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0155] Methods for Treating Breast Cancer

[0156] In further aspects of the present invention, the pharmaceutical compositions described herein may be used for treatment of breast cancer. Within such methods, pharmaceutical compositions are typically administered to a patient. A patient may or may not be afflicted with cancer. Accordingly, the above pharmaceutical compositions may be used to prevent the development of breast cancer or to treat a patient afflicted with breast cancer. Breast cancer may be diagnosed using criteria generally accepted in the art, including the detection method described herein. Pharmaceutical compositions may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs.

[0157] Routes and frequency of administration of the pharmaceutical compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, an appropriate dosage and treatment regimen provides the pharmaceutical composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients.

EXAMPLES

[0158] The following Examples are offered by way of illustration and not by way of limitation.

Example 1

Identification of Genes Differentially Expressed Between the Metastatic Breast Cancer Cell Line MDA-MB-231 and the Non-Tumorigenic Derivative MDA/H6 Using High Density Gene Filters

[0159] Total RNA was extracted from MDA-MB-231 and MDA/H6 cells with Trizol Reagent (15596-026, Life Technologies, Rockville, Md.) following the manufacturer's instructions. Briefly, cells were lysed by adding 17.5 ml Trizol solution per 175 cm.sup.2 flask. After transferring the lysate into a tube, 0.2 ml chloroform was added per 1 ml Trizol reagent used. The samples were centrifuged at 12,000 g for 15 min at 4.degree. C. The aqueous phase was transferred to a fresh tube, and 0.9 ml isopropyl alcohol was added each ml of aqueous phase collected. The samples were incubated at room temperature for 10 min and spun at 12,000 g for 10 min at 4.degree. C. The supernatant was removed and the RNA pellet was washed once with 75% ethanol alcohol. The pellet was air-dried and then dissolved in RNase-free water (D-5758, Sigma, St. Louis, Mo.). RNA was purified using Rneasy Midi Kit 50 (75144, Qiagen, Valencia, Calif.) following manufacturer's instructions. Briefly, 500 .mu.g total RNA was purified by use of 1 mg purification column. The RNA was equalized to 1 ml Rnase-free water and then 3.8 ml Buffer RLT was added. Next, 2.8 ml 100% ethanol alcohol was added and the sample was placed on the Rneasy midi spin column. The column was centrifuged for 5 min at 5,000 g, and the flow-through was discarded. Two and a 0.5 ml Buffer RPE was added to the column that was centrifuged at 5,000 g for 5 min., and repeated once. The column was transferred to a new collection tube and 250 .mu.l RNase-free water was added to the column and spun at 5,000 g for 5 min. This elution step was repeated once. Both of the elution were transferred into a Microcon 100 column and spun at 500 g for 12 min. The column was inversely placed into a tube and spun at 3,000 g for 3 min. to collect the concentrated RNA.

[0160] High density gene filters (gf200, gf201, gf202, gf203 and gf211) consisting of 25,985 arrayed elements (19,592 unique human genes and 6,393 controls) were purchased from Research Genetics (Huntsville, Ala.). A new gene filter was first washed in boiling 0.5% SDS for 5 min. and then placed in a 35.times.150 mm roller tube (052-002, Biometra, Tampa) with the DNA side facing the center of the tube. Next, 5 ml hybridization solution (HYB125.GF, Research Genetics), 5 .mu.l Poly(dA) (POLYA.GF, Research Genetics) and 5 .mu.l Cot-1 DNA (15279-011, Life Technologies) were added to the tube, that was placed in a 42.degree. C. hybridization oven for 2 to 4 hours.

[0161] DNA for hybridization on gene filter was labeled as follows. Total RNA (0.8 .mu.g) was suspended to 8 .mu.l RNase-free water. Two .mu.l of 1 .mu.g/.mu.l 10-20 mer of Oligo-(dT) (POLYT.GF, Research Genetics) was added to the RNA solution in a tube that was then incubated in a 70.degree. C. for 10 min. Then, the tube was briefly chilled on ice. Next, 6 .mu.l 5.times. First Strand Buffer (18064-014, Life Technologies), 1 .mu.l of 0.1M DTT (18064-014, Life Technologies), 1.5 .mu.l of 100 mM dNTP (27-2035-02, Amersham Pharmacia), 1.5 .mu.l Superscript II reverse transcriptase (18064-014, Life Technologies) and 10 .mu.l .sup.33P dCTP (BF1003, Amersham Pharmacia) were added and mixed thoroughly. A count per minute for radioactivity was recorded by use of Scanner QC4000 (Bioscan Inc. Washington, D.C.). The mixture was placed in a 37.degree. C. water bath for 90 min.

[0162] The labeled DNA was brought up to 100 .mu.l Rnase-free water and then purified by use of a Bio-Spin 6 chromatography column (732-6002, Bio-Rad, Hercules, Calif.) following the manufacturer's instruction. DNA with more than 5% of .alpha.-.sup.33P incorporation was denatured for 5 min in a boiling water bath and added directly to the pre-hybridization. The hybridization was allowed to continue for 15 h at 42.degree. C. The washes were done to the final stringency of 0.5.times.SSC, 1% SDS at 50.degree. C. for 15 min. The filters were placed on ddH.sub.2O-moistened piece of Whatmann paper (28458-005, VWR, Bridgeport, N.J.), exposed onto a phosphor screen (Molecular Dynamics) for 5 h, and scanned for signals with the Storm 840 Scanner (Molecular Dynamics). The tiff images were transferred to software IPLab/ArraySuite v2.0 (NHGRI/NIH) for identification of differentially expressed genes as described previously (Su et al., Mol. Carcinog., 28:119-127, 2000).

[0163] Based on selection criterions of at least 800 expression intensities and 2-fold differences between the two cell lines, 651 of 19,592 genes (3.32%) (FIG. 1, panels C and D) were selected for making microarrays on glass slides to further investigate their expression in multiple breast cancer samples.

Example 2

Customized cDNA Microarrays on Glass Slides

[0164] In order to reproducibly measure gene expression, the resultant 651 differentially expressed genes and 117 controls were printed as double sets on the individual glass slides. The same batch microarrays were used to measure gene expression in MDA-MB-231 and MDA/H6 cell lines. Briefly, human sequence verified unigene cDNA clones were purchased from Research Genetics. Plasmid DNAs were isolated from bacterial clones. cDNA inserts were amplified by PCR using the vector sequence-specific primers flanking the inserts. 0.21 .mu.g/ml of the purified products including 651 cDNAs, 80 housekeeping genes for ratio control (Chen et al. Biomed. Optics, 2:364-374, 1997), 4 non-specific controls of E. coli DNA, and 33 negative controls of non-DNA sample were printed as double sets on the individual glass slides using GMS417 arrayer (Affymetrix).

[0165] The first strand cDNA was labeled by using MicroMax Kit (NEN, Boston, Mass.) following the manufacturer's instruction. All cancer samples were labeled with the fluorescent Cy3-dUTP and the reference sample (MDA/H6) with Cy5-dUTP. Very briefly, 50-ug total RNA was mixed with Cy3-dUTP (or Cy5-dUTP) and other reagents from the kit to synthesize the label first strand cDNA at 42.degree. C. for h. The reaction was stopped by addition of 2.5 ul 0.5M EDTA and 2.5 ul 1N NaOH and then incubated at 65.degree. C. for 30 min. After adding 6.2 ul 1M Tris-HCl (pH 7.5), the samples were purified by use of Microcon 100 (Cat. No. 42412, Millipore Corp., Bedford, Mass.) to remove unincorporated nucleotides and salts. The Cy3- and Cy5- labeled DNA samples of each pair were dissolved into 25 .mu.l Hybridization Buffer from the kit by heating at 50.degree. C. for 10 min. After overlaying a cover slip onto a microarrayed glass slide, the DNA sample was heated at 90.degree. C. for 2 min. After a quick spin, 25-ul sample was placed onto the edge of the coverslip. The sample was drawn underneath the coverslip by capillary action. Each slide was placed in a 50-ml conical tube with moisture Kimwipe. Hybridization was allowed to proceed at 65.degree. C. for 16 h. The slides were washed to a final stringency of 0.06.times.SSC at room temperature for 15 min.

[0166] Image and Statistic Analysis

[0167] Hybridized array slides were scanned by use of GenePix 4000A Laser Scanner (Axon Instruments, Inc., Foster City, Calif.). For each slide, two fluorescent intensities (Cy3, Cy5) were scanned separately and then placed into the red and green channel as the tiff images in software IPLab/ArraySuite v2.0 (NHGRI, NIH) for analysis.

[0168] Image segmentation, target detection and ratio calibration methods were employed to report the expression ratios of each gene on the slides (Sorlie Proc. Natl. Acad. Sci. U.S.A, 98:10869-10874, 2001). The ratio calibration on gene filters were performed based on signal intensities of all the targets; whereas the ratio calibration on glass slides were conducted based on 80 pre-selected internal control genes of which ratios were normalized close to a value of 1.0. A 99% confidence interval was used to determine significantly up- and down-expressed genes. In addition, an empirically determined intensity filter (greater than 800 on gene filters or greater than 2,000 of average intensities in red or green channels on glass slides, for an intensity range from 0 to 65,535) was applied to further strengthen the stringency for analysis. Scatter plots were drawn in which the calibrated ratios of genes from one set were plotted against those of the other on a log-scale. The linear regression and Pearson coefficient of correlation computed from the scatter plots were used to interpret the strength of the relations of gene expression detected by two sets of genes on the same slides and by genes on two different slides. Multidimensional scaling analysis was performed by use of software developed under MatLab 5.2.1 (The MathWorks, Inc.) platform for the Mac computer. Hierarchical dendrogram clustering analysis was conducted by using the software Cluster/TreeView (Eisen et al. Proc. Natl. Acad. Sci. U.S.A, 95:14863-14868, 1998).

[0169] Panels A and B of FIG. 2 show the representative image of 2 sets of genes on the same slide. The calibrated expression ratios of informative genes (>2,000 average intensities in either red or green channel) from these two cell lines were subjected to log-transformation to obtain approximate normal distribution. The log-transformed ratios from one set of genes were drawn against those from the other as a scatter plot, from which a linear regression and Pearson coefficient of correlation were computed. Panels C and D of FIG. 2 show the strong positive linear relations between Set A and Set B on Slide 1 and Slide 2, respectively. In addition, Pearson coefficient of correlation between the Set A and the Set B on Slide 1 and Slide 2 were 0.986 and 0.974, respectively. The expression ratios of genes from Set A and Set B were averaged for the same slides. The average values from Slide 1 were plotted against those from Slide 2. The results indicated, again, a strong positive linear relation with the high value of Pearson coefficient of correlation (r=0.982) (Panel E, FIG. 2), demonstrating the strength of reproducibility of the slides and the experiments.

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