# Patent

## Title
Methods of diagnosing or treating prostate cancer using the erg gene, alone or in combination with other over or under expressed genes in prostate cancer

## Abstract

The present invention relates to oncogenes or tumor suppressor genes, as well as other genes, involved in prostate cancer and their expression products, as well as derivatives and analogs thereof. Provided are therapeutic compositions and methods of detecting and treating cancer, including prostate and other related cancers. Also provided are methods of diagnosing and/or prognosing prostate cancer by determining the expression level of at least one prostate cancer-cell-specific gene, including, for example, the ERG gene or the LTF gene alone, or in combination with at least one of the AMACR gene and the DD3 gene.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a divisional of U.S. patent application Ser. No. 11/579,695, which is a national phase application of PCT/US2005/015926, filed May 6, 2005, and claims the benefit of U.S. provisional applications No. 60/568,822, filed May 7, 2004, and No. 60/622,021, filed Oct. 27, 2004, the entire disclosures of which are relied upon and incorporated by reference.


GOVERNMENT INTEREST
This invention was made with government support under grant numbers DK065977 and CA162383 awarded by the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates to oncogenes, tumor suppressor genes, as well as other genes, and their expression products, involved in prostate cancer, as well as derivatives and analogs thereof. The invention further relates to therapeutic compositions and methods of detecting, diagnosing, and treating cancer, including prostate and other related cancers.
BACKGROUND OF THE INVENTION
Prostate cancer (CaP) is the most common malignancy in American men and second leading cause of cancer mortality (Landis et al. (1999) Cancer J. Clin., 49:8-31; Jemal et al. (2004) Cancer J Clin 54:8-29). The molecular determinants in the development and progression of this disease are poorly understood. In recent years, there have been intensive investigations of molecular genetics of the CaP. To date, however, oncogene, tumor suppressor gene, or other gene alterations common to most CaPs have not been found. Alterations of tumor suppressors such as p53, PTEN and p27, or oncogenes such as BCL2, HER2 and C-MYC associate with only small subsets of primary CaP, with more frequent association observed in advanced CaP.
Current clinical parameters, including serum Prostate Specific Antigen (PSA), tumor stage, and Gleason score are routinely used as risk factors at the time of diagnosis, but have limited application to identify patients at a greater risk for developing aggressive CaP. Approximately 30-40% of patients treated with radical prostatectomy for localized CaP have been found to have microscopic disease that is not organ-confined and a significant portion of these patients relapse. (Singh et al., Cancer Cell (2000) 1:203-209; Henshell et at., Can. Res. (2003) 63: 4196-4203). Therefore, discovery of novel biomarkers or gene expression patterns defining CaP onset and progression is crucial in predicting patients with greater risk to develop aggressive CaP.
CaP-specific genetic alterations have been the subject of intensive research by several investigations in the past five years (Srikatan et al., In Prostate Cancer, Diagnosis and Surgical Treatment (2002) Springer-Verlag, 25-40; Karan et al., Int. J. Can. (2003) 103:285-293; Augustus et al., In Molecular Pathology of Early Cancer (1999) IOS press: 321-340; Moul et al., Clin Prostate Cancer (2002) 1:42-50; Lalani et al., Cancer and Mets Rev (1997) 16: 29-66; Issacs et al., Epidemiol Rev (2001) 23:36-41; Ozen et al., Anticancer Res (2000) 20:1905-1912; Morton et al., J Natl Med Assoc (1998) 90:S728-731). Promising leads both in biology and translational research areas are beginning to emerge from recent genomics and proteomics technology, as well as traditional approaches. However, the inherent heterogeneity of CaP has hampered the molecular characterization of CaP.
One of the challenges in studying molecular alterations in human cancers, including prostate tumors, is to define the relative contributions of genetic alterations in epithelial and non-epithelial components of the target organ in the process of tumorigeneis. Despite advances in technology, changes in human CaP-specific epithelial and stromal cell-associated gene expression are still not well understood.
Despite recent advances in the identification of molecular alterations associated with certain prostate cancers, the heterogeneous nature of prostate tissue has hindered the identification of genetic targets common to all, or at least the vast majority of, prostate cancers. The complexity and heterogeneity of prostate cancer has also hindered the identification of targets that allow differentiation between clinically aggressive and non-aggressive cancers at the time of diagnosis. Therefore, there remains a need to identify molecular alterations specific for a pathologically defined cell population that can provide important clues for optimal diagnosis and prognosis, and help to establish individualized treatments tailored to the molecular profile of the tumor.
Citation of references herein shall not be construed as an admission that such references are prior art to the present invention.
SUMMARY OF THE INVENTION
It is one of the objects of the present invention to provide methods and kits for detecting cancer, in particular prostate cancer. These methods and kits can be used to detect (either qualitatively or quantitatively) nucleic acids or proteins that serve as cancer markers. For example, the expression of the prostate cancer-cell-specific gene ERG, when detected in a biological sample from a subject, either alone or in combination with other cancer markers, including the expression of other prostate cancer-cell-specific genes, can be used to indicate the presence of prostate cancer in the subject or a higher predisposition of the subject to develop prostate cancer. Detecting ERG expression, alone or in combination with the expression of any gene identified in Tables 1-6, can thus be used to diagnose or prognose cancer, particularly prostate cancer.
According to one aspect of the invention, the method for detecting the expression of one or more prostate cancer cell-specific genes, such as ERG, AMACR, and LTF or the DD3 gene, in a biological sample, comprises:

    
    
        (a) combining a biological sample with at least a first and a second oligonucleotide primer under hybridizing conditions, wherein the first oligonucleotide primer contains a sequence that hybridizes to a first sequence in a target sequence from a prostate cancer cell-specific gene, such as ERG (SEQ ID NO:1), AMACR (SEQ ID NO:3), and/or LTF (SEQ ID NO:5) and/or DD3 (SEQ ID NO:4), and the second oligonucleotide primer contains a sequence that hybridizes to a second sequence in a nucleic acid strand complementary to the target sequence, wherein the first sequence does not overlap with the second sequence,
        (b) adding at least one polymerase activity to produce a plurality of amplification products when the target sequence is present in the biological sample,
        (c) adding an oligonucleotide probe that hybridizes to at least one amplification product of the target sequence, and
        (d) detecting whether a signal results from hybridization between the oligonucleotide probe and the at least one amplification product, wherein detection of the signal indicates the expression of a prostate cancer cell-specific gene in the biological sample.
    
    


The method preferably comprises detecting the expression of the following combinations of genes: 1) ERG and AMACR; 2) ERG and DD3; and 3) ERG, AMACR and DD3. In another embodiment, the method comprises detecting LTF and one or more of ERG, AMACR and DD3. Expression of these genes can also be detected by measuring ERG, AMACR or LTF polypeptides in the biological sample.
The biological sample is preferably a prostate tissue, blood, or urine sample. Detecting a signal resulting from hybridization between the oligonucleotide probe and the at least one amplification product can be used to diagnose or prognose cancer, particularly prostate cancer.
The oligonucleotide probe may be optionally fixed to a solid support. When detecting ERG expression in a biological sample, the oligonucleotide probe, first oligonucleotide primer, and second oligonucleotide primer, each comprise a nucleic acid sequence that is capable of hybridizing under defined conditions (preferably under high stringency hybridization conditions, e.g., hybridization for 48 hours at 65° C. in 6×SSC followed by a wash in 0.1×SSX at 50° C. for 45 minutes) to SEQ ID NO:1. Thus, the oligonucleotide probe, first oligonucleotide primer, and second oligonucleotide primer can include, for example, SEQ ID NO:1 itself, or a fragment thereof or a sequence complementary thereto. Preferably the oligonucleotide probe, first oligonucleotide primer, or second oligonucleotide primer is a fragment of SEQ ID NO:1 having at least about 15, at least about 20, or at least about 50 contiguous nucleotides of SEQ ID NO:1 or a sequence complementary thereto. When detecting ERG expression, the target sequence is preferably a fragment of SEQ ID NO:1. Probes, primers, and target sequences can be similarly derived from other genes of interest, such as DD3 (SEQ ID NO:4), and other prostate cancer-cell-specific genes, including, for example, AMACR (SEQ ID NO:3) and LTF (SEQ ID NO:5).
In another aspect of the invention, the method of diagnosing or prognosing prostate cancer comprises:

    
    
        measuring the expression level (e.g. mRNA or polypeptide) of an over expressed prostate cancer cell-specific gene, such as ERG and/or AMACR, and/or the DD3 gene in a biological sample, and
        correlating the expression level of the ERG, AMACR, and/or DD3 gene with the presence of prostate cancer or a higher predispo-sition to develop prostate cancer in the subject.
    
    


In a related aspect of the invention, the method of diagnosing or prognosing prostate cancer comprises:

    
    
        measuring the expression level (e.g. mRNA or polypeptide) of an under expressed prostate cancer cell-specific gene, such as LTF in a biological sample, and
        correlating the expression level of the LTF gene with the presence of prostate cancer or a higher predisposition to develop prostate cancer in the subject.
    
    


The skilled artisan will understand how to correlate expression levels or patterns of the desired genes with the presence of prostate cancer or a higher predisposition to develop prostate cancer. For example, the expression levels can be quantified such that increased or decreased expression levels relative to a control sample or other standardized value or numerical range indicate the presence of prostate cancer or a higher predisposition to develop prostate cancer.
The increased or decreased expression levels in the methods of the invention may be measured relative to the expression level of the prostate cancer cell-specific gene or polypeptide in normal, matched tissue, such as benign prostate epithelial cells from the same subject. Alternatively, the expression level of a gene or polypeptide may be measured relative to the expression of the gene or polypeptide in other noncancerous samples from the subject or in samples obtained from a different subject without cancer. Expression of a gene may also be normalized by comparing it to the expression of other cancer-specific markers. For example, in prostate cancer, a prostate-cell specific marker, such as PSA, can be used as a control to compare and/or normalize expression levels of other genes, such as ERG, LTF, DD3, and/or AMACR. By way of example, the method of diagnosing or prognosing prostate cancer comprises measuring the expression levels of the ERG, DD3 and/or AMACR gene and diagnosing or prognosing prostate cancer, where an increased expression level of the ERG, DD3, and/or AMACR gene of at least two times as compared to the control sample indicates the presence of prostate cancer or a higher predisposition in the subject to develop prostate cancer. Conversely, by way of example, in such a method of diagnosing or prognosing prostate cancer, a decreased expression of the LTF gene of at least two times as compared to the control sample indicates the presence of prostate cancer or a higher predisposition in the subject to develop prostate cancer.
The expression levels of prostate cancer cell-specific genes (e.g., mRNA or polypeptide expression) can be detected according to the methods described herein or using any other known detection methods, including, without limitation, immunohistochemistry, Southern blotting, Northern blotting, Western blotting, ELISA, and nucleic acid amplification procedures, including but not limited to PCR, transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), ligase chain reaction (LCR), strand displacement amplification (SDA), and Loop-Mediated Isothermal Amplification (LAMP).
It is yet another object of the present invention to provide a method of determining a gene expression pattern in a biological sample, where the pattern can be correlated with the presence or absence of tumor cells, particularly prostate tumor cells. For example, ERG is detected in combination with other prostate cancer cell-specific genes (identified in Tables 1-6), including AMACR and/or LTF, to obtain expression profiles from biological samples. The expression profiles of these prostate cancer-cell-specific genes are useful for detecing cancer, particularly prostate cancer. ERG can also be detected in combination with DD3, with or without other prostate cancer cell-specific genes, such as AMACR and/or LTF, to obtain expression profiles from biological samples. These expression profiles are also useful for detecting cancer, particularly prostate cancer. Increased levels of ERG, AMACR, and/or DD3 in a biological sample indicate the presence of prostate cancer or a higher predisposition in the subject to develop prostate cancer. Decreased levels of LTF in a biological sample indicate the presence of prostate cancer or a higher predisposition in the subject to develop prostate cancer.
It is yet another object of the present invention to provide a method of determining a gene expression pattern in a biological sample, where the pattern can be used to indicate or predict the pathologic stage of cancer, particularly prostate cancer. For example, the gene expression pattern can be used to indicate or predict a moderate risk prostate cancer or a high risk prostate cancer or to predict whether the prostate cancer is progressing or regressing or in remission. The gene expression pattern can also be used as a prognostic indictor of disease-free survival following radical prostatectomy. In a particular embodiment, gene expression patterns are derived from the expression level of the ERG gene, alone or in combination with other prostate cancer-cell-specific genes (identified in Tables 1-6), including AMACR and LTF, or DD3.
Kits for detecting cancer, particularly prostate cancer, are also provided. These kits comprise a nucleic acid probe, such as the ones described herein, that hybridizes to a prostate cancer-cell-specific gene. In one embodiment the nucleic acid probe hybridizes to SEQ ID NO:1 (ERG) or the complement thereof under defined hybridization conditions (preferably under high stringency hybridization conditions, e.g., hybridization for 48 hours at 65° C. in 6×SSC followed by a wash in 0.1×SSX at 50° C. for 45 minutes) and includes SEQ ID NO:1, itself, or a fragment of SEQ ID NO:1 having at least about 15, at least about 20, or at least about 50 contiguous nucleotides of SEQ ID NO:1 or a sequence complementary thereto. In a particular embodiment, the probe selectively hybridizes to the ERG1 and ERG2 isoforms but not to ERG isoforms 3-9. In another embodiment, the probe selectively hybridizes to the ERG1 isoform but not to ERG isoforms 2-9. The nucleic acid probe may be optionally fixed to a solid support.
The kit may also contain at least one additional nucleic acid probe that hybridizes (preferably high stringency hybridization conditions, e.g., hybridization for 48 hours at 65° C. in 6×SSC followed by a wash in 0.1×SSX at 50° C. for 45 minutes) to DD3 (SEQ ID NO:4) or a gene identified in Tables 1-6, including for example, AMACR (SEQ ID NO:3) or LTF (SEQ ID NO:5). In one embodiment, the kit comprises a first oligonucloetide probe capable of hybridizing to SEQ ID NO:1 (ERG) or a sequence complimentary thereto under conditions of high stringency and at least one other oligonucleotide probe capable of hybridizing to SEQ ID NO:3 (AMACR) or a sequence complimentary thereto, or to SEQ ID NO:4 (DD3) or a sequence complementary thereto, or to a gene identified in Tables 1-6 under conditions of high stringency. In a related embodiment, the kit having an ERG and AMACR probe further comprises a third oligonucleotide probe capable of hybridzing to SEQ ID NO:4 (DD3) or a sequence complementary thereto. The kits described herein may optionally contain an oligonucleotide probe capable of hybridizing to SEQ ID NO:5 (LTF) or a sequence complementary thereto under conditions of high stringency.
The kits may further comprise a first oligonucleotide primer and a second oligonucleotide primer, where the first oligonucleotide primer contains a sequence that hybridizes to a first sequence in SEQ ID NO:1, and the second oligonucleotide primer contains a sequence that hybridizes to a second sequence in a nucleic acid strand complementary to SEQ ID NO:1, wherein the first sequence does not overlap with the second sequence. The first and second oligonucleotide primers are capable of amplifying a target sequence of interest in SEQ ID NO:1. Similarly, the kits can further comprise first and second oligonucleotide primers derived from DD3 (SEQ ID NO:4) or a prostate cancer-cell-specific gene, including, for example AMACR (SEQ ID NO:3) or LTF (SEQ ID NO:5).
It is another object of the invention to provide therapeutic methods of treating cancer, in particular prostate cancer.
It is yet another object of the present invention to provide screening methods for identifying compounds that modulate expression of a CaP-cell-specific gene, such as ERG, in prostate cancer cells.
The present invention is based in part on the identification of gene expression signatures that correlate with a high risk of CaP progression. Over expression or under expression of specific genes are predictive of tumor progression. The invention provides genes, such as the ERG gene, and analogs of specific genes that can be used alone or in combination with DD3 or other CaP-cell-specific genes, such as AMACR or LTF, to function as diagnostic and prognostic targets for cancer, particularly prostate tumors. The invention further provides genes, such as the ERG gene, and analogs of specific genes that can be used alone or in combination as therapeutic targets for cancer, in particular prostate tumors.
The invention further discloses diagnostic kits comprised of an anti-CaP-cell-specific gene antibody, for example, an anti-ERG gene antibody, which is optionally, detectably labeled. A kit is also provided that comprises nucleic acid primer sequences and/or a nucleic acid probe capable of hybridizing under defined conditions (preferably high stringency hybridization conditions, e.g., hybridization for 48 hours at 65° C. in 6×SSC followed by a wash in 0.1×SSX at 50° C. for 45 minutes) to an ERG nucleic acid. The kits may also contain an anti-DD3 gene antibody or a second anti-CaP-cell-specific gene antibody, such as an anti-AMACR or anti-LTF gene antibody, or a second set of nucleic acid primer sequences and/or a nucleic acid probe capable of hybridizing under defined conditions to the DD3 gene or another CaP-cell-specific gene, such as the AMACR or LTF gene.
The disclosed CaP-cell-specific genes, such as ERG, can be used alone or in combination as biomarkers of cancer, and in particular, prostate cancers and other related diseases, as targets for therapeutic intervention, or as gene therapy agents.
The invention provides for treatment of disorders of hyperproliferation (e.g., cancer, benign tumors) by administering compounds that modulate expression of the specific genes.
Methods of screening cancer cells, and in particular, prostate cancer cells, for specific gene expression signatures, including ERG gene expression signatures, alone or in combination with DD3 gene expression signatures or other CaP-cell-specific gene expression signatures, such as AMACR or LTF, are provided.
Additional objects of the invention will be set forth in part in the description following, and in part will be understood from the description, or may be learned by practice of the invention.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Relative expression level of ERG (A), AMACR (B), GSTP1 (C), and LTF (D) in matched tumor and benign prostate epithelial cells analyzed by QRT-PCR (TaqMan) X-axis: CaP patients analyzed (1-20); Y-axis: Expression ratio between tumor versus benign laser capture microdissection (LCM) sample pairs.
FIG. 2: Identification of genes by a distance based MDS and weighted analysis that discriminates between cancerous and benign tissue. (A) Two-dimensional MDS plot elucidating discrimination of 18 tumor samples and 18 benign samples. (B) Hierarchical clustering dendrogram with two major clusters of 18 tumor samples in the right cluster and 18 benign samples in the left cluster.
FIG. 3: A distance based MDS and weighted gene analysis using the tumor over benign ratio (or fold change) data for the identification of genes that can discriminate between high risk CaP and moderate risk CaP. (A) A supervised MDS analysis of 18 samples (9 samples from high risk group and 9 samples from moderate risk group) that ranks the genes according to their impact on minimizing cluster volume and maximizing center-to-center inter cluster distance. (B) Hierarchical clustering of the first 55 genes of the top 200 obtained by the MDS analysis. Genes and samples are arranged as ordered by cluster and treeview. Expression of each gene in each sample is obtained by the tumor over benign ratio or fold change (T/N). Dendrogram at the top of the cluster shows two major clusters, 9 samples of the MR groups in the right cluster and 9 samples of the HR groups in the left cluster. (C) Two-dimensional MDS plot of 18 CaP tumor epithelia that shows the differentiation between the high risk group (9 tumor epithelia) and moderate risk group (9 tumor epithelia) on the basis of the impact of the rank of the genes that discriminate between the HR and MR groups. (D) Hierarchical clustering dendrogram with two major clusters of 9 samples of the MR groups in the left cluster and 8+1 samples of the HR groups in the right cluster. (E) Two-dimensional MDS plot of 18 CaP benign epithelia that shows the discrimination between the high risk group (9 benign epithelia) and moderate risk group (9 benign epithelia) samples.
FIG. 4: In silico validation: the discriminatory potential of the genes that we obtained from our supervised MDS analysis on two independent data sets (Welsh et al. 2001, Singh et al. 2002). Two-dimensional MDS plot that shows the discrimination between 7 tumor epithelia of the high risk group and 7 tumor epithelia of the moderate risk group using data from Welsh et al. (A), as well as discrimination between 4 tumor epithelia of the high risk group and 5 tumor epithelia of the moderate risk group using data from Singh et al. (B).
FIG. 5: Combined gene expression analysis of ERG, AMACR and DD3 in tumor and benign prostate epithelial cells of 55 CaP patients. The graphs represent patient distribution by tumor versus benign gene expression ratios according to five gene expression categories: 1) “Up:” greater than 2 fold over expression in tumor compared to benign; 2) “Down:” less than 0.5 fold under expression in tumor compared to benign; 3) “Same:” no significant difference (0.5 to 2 fold); 4) “No expr.:” no detectable gene expression; and 5) “Other:” collectively defines patients with expression category 2, 3 and 4 for the indicated genes (i.e., other than category 1). (A) ERG Expression. (B) AMACR Expression. (C) DD3 Expression. (D) ERG or AMACR Expression. (E) ERG or DD3 Expression. (F) ERG, AMACR, or DD3 Expression.
FIG. 6: Map of ERG1 and ERG2 isoforms with probe and primer locations. The numbered boxes represent exons, the darker boxes after exon 16 are the 3′ non-coding exon regions. Translational start and stop codons are indicated by star and pound signs, respectively. The locations of the Affymetrix probe set (213541_s_at), the TaqMan probes, the traditional RT-PCR primers, and the in situ hybridization probe are indicated.
FIG. 7: Correlation of ERG1 expression and PSA recurrence-free survival. Kaplan-Meier analysis of correlation with post-prostatectomy PSA recurrence-free survival was performed on 95 CaP patients having detectable levels of ERG1 mRNA by real time QRT-PCR (TaqMan). Kaplan-Meier survival curves were stratified by the following ERG1 expression categories: 1) greater than 100 fold over expression; 2) 2-100 fold over expression; and 3) less than 2 fold over expression or under expression of ERG1 in the prostate tumor cells. The p value was 0.0006.
FIG. 8. In situ hybridization images in 7 CaP patients were analysed by the Open-Lab image analysis software (Improvisation, Lexington, Mass.) coupled to a microscope via a cooled digital camera (Leica Microsystems, Heidelburg, Germany). Density (OD) values for tumor (dark columns) and benign (light columns) epithelium are shown on the Y axis, and patients 1-7 are shown in the X axis. Patient No. 7 was added as a control with no significant ERG1 expression difference between tumor and benign cells by QRT-PCR (TaqMan). Statistical analysis was performed with the SPSS software package.
FIG. 9. ERG1 is represented as a modular structure. The two conserved regions namely SAM-PNT Domain (Protein/RNA interaction domain) and ETS Domain (Interaction with DNA) are shaded.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “CaP-cell-specific gene,” or “prostate cancer-cell-specific gene,” refers to a gene identified in Tables 1-6. The definition further encompasses CaP-cell-specific gene analogs, e.g., orthologues and homologues, and functionally equivalent fragments of CaP-cell-specific genes or their analogs, the expression of which is either upregulated or downregulated in prostate cancer cells.
The term “CaP-cell-specific gene expression signature” refers to the pattern of upregulation or downregulation of product expression as measured by the Affymetrix GeneChip assay described in Example 1, the QRT-PCR assay described in Example 2, or any other quantitative expression assay known in the art.
The term “ERG” refers to the ERG gene or ERG cDNA or mRNA described herein, and includes ERG isoforms, such as ERG1 and ERG2. The cDNA sequence of the ERG1 gene is published in GenBank under the accession number M21535. The cDNA sequence of the ERG2 gene is published in GenBank under the accession number M17254.
The term “AMACR” refers to the AMACR gene or AMACR cDNA or mRNA described herein, and includes AMACR isoforms. The cDNA sequence of the AMACR gene is published in GenBank under the accession number NM_014324.
The term “DD3” refers to the DD3 gene or DD3 cDNA or mRNA described herein, and includes DD3 isoforms. The cDNA sequence of the DD3 gene is published in GenBank under the accession number AF 103907 and is also disclosed in WO 98/45420 (1998). Although DD3 was originally used to describe a fragment of exon 4 of the prostate cancer antigen 3 (PCA3) gene, the term, as used in herein, is not so limited. DD3 is intended to refer to the entire DD3 gene or cDNA or mRNA, which in the art is also commonly referred to as PCA3.
The term “LTF” refers to the LTF gene or LTF cDNA or mRNA described herein and includes LTF isoforms. The cDNA sequence of the LTF gene is published in GenBank under the accession number NM_002343.
The term “polypeptide” is used interchangeably with the terms “peptide” and “protein” and refers to any chain of amino acids, regardless of length or posttranslational modification (e.g., glycosylation or phosphorylation), or source (e.g., species).
The phrase “substantially identical,” or “substantially as set out,” means that a relevant sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 97, 98, or 99% identical to a given sequence. By way of example, such sequences may be allelic variants, sequences derived from various species, or they may be derived from the given sequence by truncation, deletion, amino acid substitution or addition. For polypeptides, the length of comparison sequences will generally be at least 20, 30, 50, 100 or more amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50, 100, 150, 300, or more nucleotides. Percent identity between two sequences is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al. (1990) J. Mol. Biol., 215:403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48:444-453, or the algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4:11-17.
The terms “specific interaction,” “specific binding,” or the like, mean that two molecules form a complex that is relatively stable under physiologic conditions. The term is also applicable where, e.g., an antigen-binding domain is specific for a particular epitope, which is carried by a number of antigens, in which case the specific binding member carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope. Specific binding is characterized by a high affinity and a low to moderate capacity. Nonspecific binding usually has a low affinity with a moderate to high capacity. Typically, the binding is considered specific when the affinity constant Ka is higher than 106M−1, more preferably higher than 107M−1, and most preferably 108M−1. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. Such conditions are known in the art, and a skilled artisan using routine techniques can select appropriate conditions. The conditions are usually defined in terms of concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of non-related molecules (e.g., serum albumin, milk casein), etc. The term “detectably labeled” refers to any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as 32P, 35S, or 125I) and nonradioactive labeling (e.g., fluorescent and chemiluminescent labeling).
The term “modulatory compound” is used interchangeably with the term “therapeutic” as used herein means any compound capable of “modulating” either CaP-cell-specific gene expression at the transcriptional, translational, or post-translational levels or modulating the biological activity of a CaP-cell-specific polypeptide. The term “modulate” and its cognates refer to the capability of a compound acting as either an agonist or an antagonist of a certain reaction or activity. The term modulate, therefore, encompasses the terms “activate” and “inhibit.” The term “activate,” for example, refers to an increase in the expression of the CaP-cell-specific gene or activity of a CaP-cell-specific polypeptide in the presence of a modulatory compound, relative to the activity of the gene or the polypeptide in the absence of the same compound. The increase in the expression level or the activity is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher. Analogously, the term “inhibit” refers to a decrease in the expression of the CaP-cell-specific gene or activity of a CaP-cell-specific polypeptide in the presence of a modulatory compound, relative to the activity of the gene or the polypeptide in the absence of the same compound. The decrease in the expression level or the activity is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher. The expression level of the CaP-cell-specific gene or activity of a CaP-cell-specific polypeptide can be measured as described herein or by techniques generally known in the art.
The term “treatment” is used interchangeably herein with the term “therapeutic method” and refers to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder.
The term “isolated” refers to a molecule that is substantially free of its natural environment. Any amount of that molecule elevated over the naturally occurring levels due to any manipulation, e.g., over expression, partial purification, etc., is encompassed with the definition. With regard to partially purified compositions only, the term refers to an isolated compound that is at least 50-70%, 70-90%, 90-95% (w/w), or more pure.
The term “effective dose,” or “effective amount,” refers to that amount of the compound that results in amelioration of symptoms in a patient or a desired biological outcome, e.g., inhibition of cell proliferation. The effective amount can be determined as described in the subsequent sections.
The terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “DNA” are used interchangeably herein and refer to deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include nucleotide analogs, and single or double stranded polynucleotides. Examples of polynucleotides include, but are not limited to, plasmid DNA or fragments thereof, viral DNA or RNA, anti-sense RNA, etc. The term “plasmid DNA” refers to double stranded DNA that is circular.
As used herein the term “hybridization under defined conditions,” or “hybridizing under defined conditions,” is intended to describe conditions for hybridization and washes under which nucleotide sequences that are significantly identical or homologous to each other remain bound to each other. The conditions are such that sequences, which are at least about 6 and more preferably at least about 20, 50, 100, 150, 300, or more nucleotides long and at least about 70%, more preferably at least about 80%, even more preferably at least about 85-90% identical, remain bound to each other. The percent identity can be determined as described in Altschul et al. (1997) Nucleic Acids Res., 25: 3389-3402.
Appropriate hybridization conditions can be selected by those skilled in the art with minimal experimentation as exemplified in Ausubel et al. (2004), Current Protocols in Molecular Biology, John Wiley & Sons. Additionally, stringent conditions are described in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press. A nonlimiting example of defined conditions of low stringency is as follows. Filters containing DNA are pretreated for 6 hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×106 cpm 32P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 hours at 40° C., and then washed for 1.5 hours at 55° C. in a solution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60° C. Filters are blotted dry and exposed for autoradiography. Other conditions of low stringency well known in the art may be used (e.g., as employed for cross-species hybridizations).
A non-limiting example of defined conditions of high stringency is as follows. Prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65° C. in the prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×106 cpm of 32P-labeled probe. Washing of filters is done at 37° C. for 1 hour in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes. Other conditions of high stringency well known in the art may be used. An oligonucleotide hybridizes specifically to a target sequence under high stringency conditions.
The term “solid support” means a material that is essentially insoluble under the solvent and temperature conditions of the assay method, comprising free chemical groups available for joining an oligonucleotide or nucleic acid. Preferably, the solid support is covalently coupled to an oligonucleotide designed to directly or indirectly bind a target nucleic acid. When the target nucleic acid is an mRNA, the oligonucleotide attached to the solid support is preferably a poly-T sequence. A preferred solid support is a particle, such as a micron- or submicron-sized bead or sphere. A variety of solid support materials are contemplated, such as, for example, silica, polyacrylate, polyacrylamide, a metal, polystyrene, latex, nitrocellulose, polypropylene, nylon or combinations thereof. More preferably, the solid support is capable of being attracted to a location by means of a magnetic field, such as a solid support having a magnetite core. Particularly preferred supports are monodisperse magnetic spheres (i.e., uniform size.+−.about 5%).
The term “detecting” or “detection” means any of a variety of methods for determining the presence of a nucleic acid, such as, for example, hybridizing a labeled probe to a portion of the nucleic acid. A labeled probe is an oligonucleotide that specifically binds to another sequence and contains a detectable group which may be, for example, a fluorescent moiety, a chemiluminescent moiety (such as an acridinium ester (AE) moiety that can be detected chemiluminescently under appropriate conditions (as described in U.S. Pat. No. 5,283,174)), a radioisotope, biotin, avidin, enzyme, enzyme substrate, or other reactive group. Other well know detection techniques include, for example, gel filtration, gel electrophoresis and visualization of the amplicons, and High Performance Liquid Chromatography (HPLC). As used throughout the specification, the term “detecting” or “detection” includes either qualitative or quantitative detection.
The term “primer” or “oligonculeotide primer” means an oligonucleotide capable of binding to a region of a target nucleic acid or its complement and promoting nucleic acid amplification of the target nucleic acid. Generally, a primer will have a free 3′ end that can be extended by a nucleic acid polymerase. Primers also generally include a base sequence capable of hybridizing via complementary base interactions either directly with at least one strand of the target nucleic acid or with a strand that is complementary to the target sequence. A primer may comprise target-specific sequences and optionally other sequences that are non-complementary to the target sequence. These non-complementary sequences may comprise a promoter sequence or a restriction endonuclease recognition site.
CaP-Cell-Specific Gene Expression Signature Identification
The present invention is based in part on the identification and validation of consistent CaP epithelial cell specific gene expression signatures. These gene expression signatures define patients with CaP who are at risk to develop advanced disease by identifying genes and pathways in prostate epithelial cells that differentiate between aggressive and non-aggressive courses of cancer development. Two patient groups were selected, a high risk (HR) group having, for example, PSA recurrence, Gleason score 8-9, T3c stage, seminal vesicle invasion, poor tumor differentiation, and a moderate risk (MR) group having, for example, no PSA recurrence, Gleason score 6-7, T2a-T3b stage, no seminal vesicle invasion, well or moderate tumor differentiation. The two patient groups were matched for known risk factors: age, race, and family history of CaP. LCM derived epithelial cells from tumor and normal prostate of the two patient groups were compared by GeneChip analyses, as described in the following Example 1. Results were validated using quantitative reverse transcriptase PCR (QRT-PCR), as described in the following Example 2. The group of genes identified and validated as having the highest association with aggressive or non-aggressive CaP can be used to reliably determine the likely course of CaP progression.
Strikingly, one of the most consistently over expressed genes in CaP cells identified in this study was the ERG (ETS related gene) oncogene, a member of the ETS transcription factor family. (Oikawa et al., Gene (2003) 303:11-34; Sharrocks, A D, Nat Rev Mol Cell Biol (2001) 2(11):827-37; Hart et al., Oncogene (1995) 10(7):1423-30; Rao et al., Science (1987) 237(4815): 635-639). Two isoforms of ERG, ERG1 and ERG2, are over expressed with the highest frequency. The ERG1 coding sequence (with start and stop codons underlined) is publicly available through GenBank under the accession number M21535, as follows:








(SEQ ID NO: 1)










   1
gaattccctc caaagcaaga caaatgactc acagagaaaa aagatggcag aaccaagggc



 


  61
aactaaagcc gtcaggttct gaacagctgg tagatgggct ggcttactga aggacatgat


 


 121
tcagactgtc ccggacccag cagctcatat caaggaactc tcctgatgaa tgcagtgtgg


 


 181
ccaaaggcgg gaagatggtg ggcagcccag acaccgttgg gatgaactac ggcagctaca


 


 241
tggaggagaa gcacatgcca cccccaaaca tgaccacgaa cgagcgcaga gttatcgtgc


 


 301
cagcagatcc tacgctatgg agtacagacc atgtgcggca gtggctggag tgggcggtga


 


 361
aagaatatgg ccttccagac gtcaacatct tgttattcca gaacatcgat gggaaggaac


 


 421
tgtgcaagat gaccaaggac gacttccaga ggctcacccc cagctacaac gccgacatcc


 


 481
ttctctcaca tctccactac ctcagagaga ctcctcttcc acatttgact tcagatgatg


 


 541
ttgataaagc cttacaaaac tctccacggt taatgcatgc tagaaacaca gatttaccat


 


 601
atgagccccc caggagatca gcctggaccg gtcacggcca ccccacgccc cagtcgaaag


 


 661
ctgctcaacc atctccttcc acagtgccca aaactgaaga ccagcgtcct cagttagatc


 


 721
cttatcagat tcttggacca acaagtagcc gccttgcaaa tccaggcagt ggccagatcc


 


 781
agctttggca gttcctcctg gagctcctgt cggacagctc caactccagc tgcatcacct


 


 841
gggaaggcac caacggggag ttcaagatga cggatcccga cgaggtggcc cggcgctggg


 


 901
gagagcggaa gagcaaaccc aacatgaact acgataagct cagccgcgcc ctccgttact


 


 961
actatgacaa gaacatcatg accaaggtcc atgggaagcg ctacgcctac aagttcgact


 


1021
tccacgggat cgcccaggcc ctccagcccc accccccgga gtcatctctg tacaagtacc


 


1081
cctcagacct cccgtacatg ggctcctatc acgcccaccc acagaagatg aactttgtgg


 


1141
cgccccaccc tccagccctc cccgtgacat cttccagttt ttttgctgcc ccaaacccat


 


1201
actggaattc accaactggg ggtatatacc ccaacactag gctccccacc agccatatgc


 


1261
cttctcatct gggcacttac tactaaagac ctggcggagg cttttcccat cagcgtgcat


 


1321
tcaccagccc atcgccacaa actctatcgg agaacatgaa tcaaaagtgc ctcaagagga


 


1381
atgaaaaaag ctttactggg gctggggaag gaagccgggg aagagatcca aagactcttg


 


1441
ggagggagtt actgaagtct tactgaagtc ttactacaga aatgaggagg atgctaaaaa


 


1501
tgtcacgaat atggacatat catctgtgga ctgaccttgt aaaagacagt gtatgtagaa


 


1561
gcatgaagtc ttaaggacaa agtgccaaag aaagtggtct taagaaatgt ataaacttta


 


1621
gagtagagtt tgaatcccac taatgcaaac tgggatgaaa ctaaagcaat agaaacaaca


 


1681
cagttttgac ctaacatacc gtttataatg ccattttaag gaaaactacc tgtatttaaa


 


1741
aatagtttca tatcaaaaac aagagaaaag acacgagaga gactgtggcc catcaacaga


 


1801
cgttgatatg caactgcatg gcatgtgctg ttttggttga aatcaaatac attccgtttg


 


1861
atggacagct gtcagctttc tcaaactgtg aagatgaccc aaagtttcca actcctttac


 


1921
agtattaccg ggactatgaa ctaaaaggtg ggactgagga tgtgtataga gtgagcgtgt


 


1981
gattgtagac agaggggtga agaaggagga ggaagaggca gagaaggagg agaccaggct


 


2041
gggaaagaaa cttctcaagc aatgaagact ggactcagga catttgggga ctgtgtacaa


 


2101
tgagttatgg agactcgagg gttcatgcag tcagtgttat accaaaccca gtgttaggag


 


2161
aaaggacaca gcgtaatgga gaaagggaag tagtagaatt cagaaacaaa aatgcgcatc


 


2221
tctttctttg tttgtcaaat gaaaatttta actggaattg tctgatattt aagagaaaca


 


2281
ttcaggacct catcattatg tgggggcttt gttctccaca gggtcaggta agagatggcc


 


2341
ttcttggctg ccacaatcag aaatcacgca ggcattttgg gtaggcggcc tccagttttc


 


2401
ctttgagtcg cgaacgctgt gcgtttgtca gaatgaagta tacaagtcaa tgtttttccc


 


2461
cctttttata taataattat ataacttatg catttataca ctacgagttg atctcggcca


 


2521
gccaaagaca cacgacaaaa gagacaatcg atataatgtg gccttgaatt ttaactctgt


 


2581
atgcttaatg tttacaatat gaagttatta gttcttagaa tgcagaatgt atgtaataaa


 


2641
ataagcttgg cctagcatgg caaatcagat ttatacagga gtctgcattt gcactttttt


 


2701
tagtgactaa agttgcttaa tgaaaacatg tgctgaatgt tgtggatttt gtgttataat


 


2761
ttactttgtc caggaacttg tgcaagggag agccaaggaa ataggatgtt tggcacccaa


 


2821
atggcgtcag cctctccagg tccttcttgc ctcccctcct gtcttttatt tctagcccct


 


2881
tttggaacag gaaggacccc ggggtttcaa ttggagcctc catatttatg cctggaagga


 


2941
aagaggccta tgaagctggg gttgtcattg agaaattcta gttcagcacc tggtcacaaa


 


3001
tcacccttaa ttctgctatg attaaaatac atttgttgaa cagtgaacaa gctaccactc


 


3061
gtaaggcaaa ctgtattatt actggcaaat aaagcgtcat ggatagctgc aatttctcac


 


3121
tttaca






Nucleotides 195-1286 of SEQ ID NO:1 represent the coding sequence of SEQ ID NO:1.
The ERG2 coding sequence is publicly available through GenBank under the accession number M17254, as follows (with start and stop codons underlined):








(SEQ ID NO: 2)










   1
gtccgcgcgt gtccgcgccc gcgtgtgcca gcgcgcgtgc cttggccgtg cgcgccgagc



 


  61
cgggtcgcac taactccctc ggcgccgacg gcggcgctaa cctctcggtt attccaggat


 


 121
ctttggagac ccgaggaaag ccgtgttgac caaaagcaag acaaatgact cacagagaaa


 


 181
aaagatggca gaaccaaggg caactaaagc cgtcaggttc tgaacagctg gtagatgggc


 


 241
tggcttactg aaggacatga ttcagactgt cccggaccca gcagctcata tcaaggaagc


 


 301
cttatcagtt gtgagtgagg accagtcgtt gtttgagtgt gcctacggaa cgccacacct


 


 361
ggctaagaca gagatgaccg cgtcctcctc cagcgactat ggacagactt ccaagatgag


 


 421
cccacgcgtc cctcagcagg attggctgtc tcaaccccca gccagggtca ccatcaaaat


 


 481
ggaatgtaac cctagccagg tgaatggctc aaggaactct cctgatgaat gcagtgtggc


 


 541
caaaggcggg aagatggtgg gcagcccaga caccgttggg atgaactacg gcagctacat


 


 601
ggaggagaag cacatgccac ccccaaacat gaccacgaac gagcgcagag ttatcgtgcc


 


 661
agcagatcct acgctatgga gtacagacca tgtgcggcag tggctggagt gggcggtgaa


 


 721
agaatatggc cttccagacg tcaacatctt gttattccag aacatcgatg ggaaggaact


 


 781
gtgcaagatg accaaggacg acttccagag gctcaccccc agctacaacg ccgacatcct


 


 841
tctctcacat ctccactacc tcagagagac tcctcttcca catttgactt cagatgatgt


 


 901
tgataaagcc ttacaaaact ctccacggtt aatgcatgct agaaacacag atttaccata


 


 961
tgagcccccc aggagatcag cctggaccgg tcacggccac cccacgcccc agtcgaaagc


 


1021
tgctcaacca tctccttcca cagtgcccaa aactgaagac cagcgtcctc agttagatcc


 


1081
ttatcagatt cttggaccaa caagtagccg ccttgcaaat ccaggcagtg gccagatcca


 


1141
gctttggcag ttcctcctgg agctcctgtc ggacagctcc aactccagct gcatcacctg


 


1201
ggaaggcacc aacggggagt tcaagatgac ggatcccgac gaggtggccc ggcgctgggg


 


1261
agagcggaag agcaaaccca acatgaacta cgataagctc agccgcgccc tccgttacta


 


1321
ctatgacaag aacatcatga ccaaggtcca tgggaagcgc tacgcctaca agttcgactt


 


1381
ccacgggatc gcccaggccc tccagcccca ccccccggag tcatctctgt acaagtaccc


 


1441
ctcagacctc ccgtacatgg gctcctatca cgcccaccca cagaagatga actttgtggc


 


1501
gccccaccct ccagccctcc ccgtgacatc ttccagtttt tttgctgccc caaacccata


 


1561
ctggaattca ccaactgggg gtatataccc caacactagg ctccccacca gccatatgcc


 


1621
ttctcatctg ggcacttact actaaagacc tggcggaggc ttttcccatc agcgtgcatt


 


1681
caccagccca tcgccacaaa ctctatcgga gaacatgaat caaaagtgcc tcaagaggaa


 


1741
tgaaaaaagc tttactgggg ctggggaagg aagccgggga agagatccaa agactcttgg


 


1801
gagggagtta ctgaagtctt actacagaaa tgaggaggat gctaaaaatg tcacgaatat


 


1861
ggacatatca tctgtggact gaccttgtaa aagacagtgt atgtagaagc atgaagtctt


 


1921
aaggacaaag tgccaaagaa agtggtctta agaaatgtat aaactttaga gtagagtttg


 


1981
aatcccacta atgcaaactg ggatgaaact aaagcaatag aaacaacaca gttttgacct


 


2041
aacataccgt ttataatgcc attttaagga aaactacctg tatttaaaaa tagtttcata


 


2101
tcaaaaacaa gagaaaagac acgagagaga ctgtggccca tcaacagacg ttgatatgca


 


2161
actgcatggc atgtgctgtt ttggttgaaa tcaaatacat tccgtttgat ggacagctgt


 


2221
cagctttctc aaactgtgaa gatgacccaa agtttccaac tcctttacag tattaccggg


 


2281
actatgaact aaaaggtggg actgaggatg tgtatagagt gagcgtgtga ttgtagacag


 


2341
aggggtgaag aaggaggagg aagaggcaga gaaggaggag accaggctgg gaaagaaact


 


2401
tctcaagcaa tgaagactgg actcaggaca tttggggact gtgtacaatg agttatggag


 


2461
actcgagggt tcatgcagtc agtgttatac caaacccagt gttaggagaa aggacacagc


 


2521
gtaatggaga aagggaagta gtagaattca gaaacaaaaa tgcgcatctc tttctttgtt


 


2581
tgtcaaatga aaattttaac tggaattgtc tgatatttaa gagaaacatt caggacctca


 


2641
tcattatgtg ggggctttgt tctccacagg gtcaggtaag agatggcctt cttggctgcc


 


2701
acaatcagaa atcacgcagg cattttgggt aggcggcctc cagttttcct ttgagtcgcg


 


2761
aacgctgtgc gtttgtcaga atgaagtata caagtcaatg tttttccccc tttttatata


 


2821
ataattatat aacttatgca tttatacact acgagttgat ctcggccagc caaagacaca


 


2881
cgacaaaaga gacaatcgat ataatgtggc cttgaatttt aactctgtat gcttaatgtt


 


2941
tacaatatga agttattagt tcttagaatg cagaatgtat gtaataaaat aagcttggcc


 


3001
tagcatggca aatcagattt atacaggagt ctgcatttgc acttttttta gtgactaaag


 


3061
ttgcttaatg aaaacatgtg ctgaatgttg tggattttgt gttataattt actttgtcca


 


3121
ggaacttgtg caagggagag ccaaggaaat aggatgtttg gcaccc






Nucleotides 257-1645 of SEQ ID NO:2 represent the coding sequence of SEQ ID NO:2.
Validation by QRT-PCR (TaqMan) in microdissected tumor and benign prostate epithelial cells of 20 CaP patients confirmed a consistent, tumor associated over expression of ERG isoforms ERG1 and/or ERG2 in 95% of patients (19 of 20) (FIG. 1A). As a quality test and comparison, the expression of AMACR, a recently identified CaP tissue marker (Rubin et al, JAMA (2002) 287:1662-1670; Luo et al., Cancer Res (2002) 62: 2220-2226), and of GSTP1, a gene known to have decreased expression in CaP (Nelson et al., Ann N Y Acad Sci (2001) 952: 135-144), was also determined (FIGS. 1B and 1C). Robust over expression in CaP cells of 95% of the patients, similarly to ERG, was observed for AMACR, while the GSTP1 expression was significantly decreased in the tumor cells of each CaP patient, confirming the high quality of the tumor and benign LCM specimens and the reliability of the QRT-PCR.
Recently a detailed mapping of the chromosomal region (21q22.2-q22.3) containing the ERG gene, as well as a complete exon-intron structure with 9 alternative transcripts (or isoforms) has been described. (Owczarek et al., Gene (2004) 324: 65-77). The probes on the Affymetrix GeneChip used in our initial discovery of consistent ERG over expression in CaP, as well as the TaqMan probe designed for the validation experiment, recognize a region specific to the ERG 1 and 2 isoforms only.
Both ERG and ETS are proto-oncogenes with mitogenic and transforming activity. (Sharrocks, A D, Nat Rev Mol Cell Biol (2001) 2(11):827-37; Seth et al., Proc Natl Acad Sci USA (1989) 86:7833-7837). Deregulation or chromosomal reorganization of ERG is linked to Ewing sarcoma, myeloid leukemia and cervical carcinoma. (DeAlva et al., Int J Surg Pathol (2001) 9: 7-17; Simpson et al., Oncogene (1997) 14: 2149-2157; Shimizu et al., Proc Natl Acad Sci USA (1993) 90:10280-284; Papas, et al., Am J Med Genet Suppl. (1990) 7:251-261). ETS2 has been implicated in CaP, but it is over expressed only in a small proportion of CaP specimens. (Liu et al., Prostate (1997) 30:145-53; Semenchenko, et al., Oncogene (1998) 17:2883-88). ERG over expression without amplification of DNA copy number was recently reported in acute myeloid leukemia. (Balduc et al., Proc. Natl. Acad. Sci. USA (2004) 101:3915-20). Gavrilov et al., Eur J Cancer (2001) 37:1033-40 examined the expression of various transcription factors, including several proteins from the ETS family, in a very limited number of high-grade prostate cancer samples. Antibodies against the ETS family proteins, Elf-1 and Fli-1, caused intense staining of most of the high-grade prostate cancer samples. In contrast, ERG protein, while being detected in the noncancerous endothelial cells (microvessels in the stroma) of most samples tested, was detected in only a minority of the high-grade prostate cancers. ETS family proteins have a variety of expression patterns in human tissues. (Oikawa et al., Gene (2003) 303:11-34). ERG is expressed in endothelial tissues, hematopoietic cells, kidney, and in the urogenital tract. ERG proteins are nuclear transcription factors that form homodimers, as well as heterodimers with several other members of the ETS family of transcription factors. (Carrere et al., Oncogene (1998) 16(25): 3261-68). A negative crosstalk observed between ERG and estrogen receptor (ER-alpha) may be relevant in urogenital tissues, where both transcription factors are expressed. (Vlaeminck-Guillem et al., Oncogene (2003) 22(50):8072-84). The present invention is based in part upon the surprising discovery that ERG is over expressed in the majority of CaP specimens analyzed, indicating that this oncogene plays a role in prostate tumorigenesis, most likely by modulating transcription of target genes favoring tumorigenesis in prostate epithelium.
The present invention is further based in part upon the over expression of the AMACR gene in prostate cancer epithelium. The cDNA sequence of the AMACR is publicly available through GenBank under the accession numbers NM_014324 and AF047020. The sequence (with start and stop codons underlined) corresponding to accession number NM_014324 is as follows:








(SEQ ID NO: 3)










   1
gggattggga gggcttcttg caggctgctg ggctggggct aagggctgct cagtttcctt



 


  61
cagcggggca ctgggaagcg ccatggcact gcagggcatc tcggtcgtgg agctgtccgg


 


 121
cctggccccg ggcccgttct gtgctatggt cctggctgac ttcggggcgc gtgtggtacg


 


 181
cgtggaccgg cccggctccc gctacgacgt gagccgcttg ggccggggca agcgctcgct


 


 241
agtgctggac ctgaagcagc cgcggggagc cgccgtgctg cggcgtctgt gcaagcggtc


 


 301
ggatgtgctg ctggagccct tccgccgcgg tgtcatggag aaactccagc tgggcccaga


 


 361
gattctgcag cgggaaaatc caaggcttat ttatgccagg ctgagtggat ttggccagtc


 


 421
aggaagcttc tgccggttag ctggccacga tatcaactat ttggctttgt caggtgttct


 


 481
ctcaaaaatt ggcagaagtg gtgagaatcc gtatgccccg ctgaatctcc tggctgactt


 


 541
tgctggtggt ggccttatgt gtgcactggg cattataatg gctctttttg accgcacacg


 


 601
cactggcaag ggtcaggtca ttgatgcaaa tatggtggaa ggaacagcat atttaagttc


 


 661
ttttctgtgg aaaactcaga aattgagtct gtgggaagca cctcgaggac agaacatgtt


 


 721
ggatggtgga gcacctttct atacgactta caggacagca gatggggaat tcatggctgt


 


 781
tggagcaata gaaccccagt tctacgagct gctgatcaaa ggacttggac taaagtctga


 


 841
tgaacttccc aatcagatga gcatggatga ttggccagaa atgaagaaga agtttgcaga


 


 901
tgtatttgca gagaagacga aggcagagtg gtgtcaaatc tttgacggca cagatgcctg


 


 961
tgtgactccg gttctgactt ttgaggaggt tgttcatcat gatcacaaca aggaacgggg


 


1021
ctcgtttatc accagtgagg agcaggacgt gagcccccgc cctgcacctc tgctgttaaa


 


1081
caccccagcc atcccttctt tcaaaaggga tcctttcata ggagaacaca ctgaggagat


 


1141
acttgaagaa tttggattca gccgcgaaga gatttatcag cttaactcag ataaaatcat


 


1201
tgaaagtaat aaggtaaaag ctagtctcta acttccaggc ccacggctca agtgaatttg


 


1261
aatactgcat ttacagtgta gagtaacaca taacattgta tgcatggaaa catggaggaa


 


1321
cagtattaca gtgtcctacc actctaatca agaaaagaat tacagactct gattctacag


 


1381
tgatgattga attctaaaaa tggttatcat tagggctttt gatttataaa actttgggta


 


1441
cttatactaa attatggtag ttattctgcc ttccagtttg cttgatatat ttgttgatat


 


1501
taagattctt gacttatatt ttgaatgggt tctagtgaaa aaggaatgat atattcttga


 


1561
agacatcgat atacatttat ttacactctt gattctacaa tgtagaaaat gaggaaatgc


 


1621
cacaaattgt atggtgataa aagtcacgtg aaacagagtg attggttgca tccaggcctt


 


1681
ttgtcttggt gttcatgatc tccctctaag cacattccaa actttagcaa cagttatcac


 


1741
actttgtaat ttgcaaagaa aagtttcacc tgtattgaat cagaatgcct tcaactgaaa


 


1801
aaaacatatc caaaataatg aggaaatgtg ttggctcact acgtagagtc cagagggaca


 


1861
gtcagtttta gggttgcctg tatccagtaa ctcggggcct gtttccccgt gggtctctgg


 


1921
gctgtcagct ttcctttctc catgtgtttg atttctcctc aggctggtag caagttctgg


 


1981
atcttatacc caacacacag caacatccag aaataaagat ctcaggaccc cccagcaagt


 


2041
cgttttgtgt ctccttggac tgagttaagt tacaagcctt tcttatacct gtctttgaca


 


2101
aagaagacgg gattgtcttt acataaaacc agcctgctcc tggagcttcc ctggactcaa


 


2161
cttcctaaag gcatgtgagg aaggggtaga ttccacaatc taatccgggt gccatcagag


 


2221
tagagggagt agagaatgga tgttgggtag gccatcaata aggtccattc tgcgcagtat


 


2281
ctcaactgcc gttcaacaat cgcaagagga aggtggagca ggtttcttca tcttacagtt


 


2341
gagaaaacag agactcagaa gggcttctta gttcatgttt cccttagcgc ctcagtgatt


 


2401
ttttcatggt ggcttaggcc aaaagaaata tctaaccatt caatttataa ataattaggt


 


2461
ccccaacgaa ttaaatatta tgtcctacca acttattagc tgcttgaaaa atataataca


 


2521
cataaataaa aaaa






Nucleotides 83-1231 of SEQ ID NO:3 represent the coding sequence of AMACR.
The present invention is further based in part upon the over expression of the DD3 gene in prostate cancer epithelium. The cDNA sequence of the DD3 gene is publicly available through GenBank under the accession number AF103907. The sequence corresponding to accession number AF103907 is as follows:








(SEQ ID NO: 4)










   1
acagaagaaa tagcaagtgc cgagaagctg gcatcagaaa aacagagggg agatttgtgt



 


  61
ggctgcagcc gagggagacc aggaagatct gcatggtggg aaggacctga tgatacagag


 


 121
gaattacaac acatatactt agtgtttcaa tgaacaccaa gataaataag tgaagagcta


 


 181
gtccgctgtg agtctcctca gtgacacagg gctggatcac catcgacggc actttctgag


 


 241
tactcagtgc agcaaagaaa gactacagac atctcaatgg caggggtgag aaataagaaa


 


 301
ggctgctgac tttaccatct gaggccacac atctgctgaa atggagataa ttaacatcac


 


 361
tagaaacagc aagatgacaa tataatgtct aagtagtgac atgtttttgc acatttccag


 


 421
cccctttaaa tatccacaca cacaggaagc acaaaaggaa gcacagagat ccctgggaga


 


 481
aatgcccggc cgccatcttg ggtcatcgat gagcctcgcc ctgtgcctgg tcccgcttgt


 


 541
gagggaagga cattagaaaa tgaattgatg tgttccttaa aggatgggca ggaaaacaga


 


 601
tcctgttgtg gatatttatt tgaacgggat tacagatttg aaatgaagtc acaaagtgag


 


 661
cattaccaat gagaggaaaa cagacgagaa aatcttgatg gcttcacaag acatgcaaca


 


 721
aacaaaatgg aatactgtga tgacatgagg cagccaagct ggggaggaga taaccacggg


 


 781
gcagagggtc aggattctgg ccctgctgcc taaactgtgc gttcataacc aaatcatttc


 


 841
atatttctaa ccctcaaaac aaagctgttg taatatctga tctctacggt tccttctggg


 


 901
cccaacattc tccatatatc cagccacact catttttaat atttagttcc cagatctgta


 


 961
ctgtgacctt tctacactgt agaataacat tactcatttt gttcaaagac ccttcgtgtt


 


1021
gctgcctaat atgtagctga ctgtttttcc taaggagtgt tctggcccag gggatctgtg


 


1081
aacaggctgg gaagcatctc aagatctttc cagggttata cttactagca cacagcatga


 


1141
tcattacgga gtgaattatc taatcaacat catcctcagt gtctttgccc atactgaaat


 


1201
tcatttccca cttttgtgcc cattctcaag acctcaaaat gtcattccat taatatcaca


 


1261
ggattaactt ttttttttaa cctggaagaa ttcaatgtta catgcagcta tgggaattta


 


1321
attacatatt ttgttttcca gtgcaaagat gactaagtcc tttatccctc ccctttgttt


 


1381
gatttttttt ccagtataaa gttaaaatgc ttagccttgt actgaggctg tatacagcac


 


1441
agcctctccc catccctcca gccttatctg tcatcaccat caacccctcc cataccacct


 


1501
aaacaaaatc taacttgtaa ttccttgaac atgtcaggac atacattatt ccttctgcct


 


1561
gagaagctct tccttgtctc ttaaatctag aatgatgtaa agttttgaat aagttgacta


 


1621
tcttacttca tgcaaagaag ggacacatat gagattcatc atcacatgag acagcaaata


 


1681
ctaaaagtgt aatttgatta taagagttta gataaatata tgaaatgcaa gagccacaga


 


1741
gggaatgttt atggggcacg tttgtaagcc tgggatgtga agcaaaggca gggaacctca


 


1801
tagtatctta tataatatac ttcatttctc tatctctatc acaatatcca acaagctttt


 


1861
cacagaattc atgcagtgca aatccccaaa ggtaaccttt atccatttca tggtgagtgc


 


1921
gctttagaat tttggcaaat catactggtc acttatctca actttgagat gtgtttgtcc


 


1981
ttgtagttaa ttgaaagaaa tagggcactc ttgtgagcca ctttagggtt cactcctggc


 


2041
aataaagaat ttacaaagag ctactcagga ccagttgtta agagctctgt gtgtgtgtgt


 


2101
gtgtgtgtgt gagtgtacat gccaaagtgt gcctctctct cttgacccat tatttcagac


 


2161
ttaaaacaag catgttttca aatggcacta tgagctgcca atgatgtatc accaccatat


 


2221
ctcattattc tccagtaaat gtgataataa tgtcatctgt taacataaaa aaagtttgac


 


2281
ttcacaaaag cagctggaaa tggacaacca caatatgcat aaatctaact cctaccatca


 


2341
gctacacact gcttgacata tattgttaga agcacctcgc atttgtgggt tctcttaagc


 


2401
aaaatacttg cattaggtct cagctggggc tgtgcatcag gcggtttgag aaatattcaa


 


2461
ttctcagcag aagccagaat ttgaattccc tcatctttta ggaatcattt accaggtttg


 


2521
gagaggattc agacagctca ggtgctttca ctaatgtctc tgaacttctg tccctctttg


 


2581
tgttcatgga tagtccaata aataatgtta tctttgaact gatgctcata ggagagaata


 


2641
taagaactct gagtgatatc aacattaggg attcaaagaa atattagatt taagctcaca


 


2701
ctggtcaaaa ggaaccaaga tacaaagaac tctgagctgt catcgtcccc atctctgtga


 


2761
gccacaacca acagcaggac ccaacgcatg tctgagatcc ttaaatcaag gaaaccagtg


 


2821
tcatgagttg aattctccta ttatggatgc tagcttctgg ccatctctgg ctctcctctt


 


2881
gacacatatt agcttctagc ctttgcttcc acgactttta tcttttctcc aacacatcgc


 


2941
ttaccaatcc tctctctgct ctgttgcttt ggacttcccc acaagaattt caacgactct


 


3001
caagtctttt cttccatccc caccactaac ctgaatgcct agacccttat ttttattaat


 


3061
ttccaataga tgctgcctat gggctatatt gctttagatg aacattagat atttaaagct


 


3121
caagaggttc aaaatccaac tcattatctt ctctttcttt cacctccctg ctcctctccc


 


3181
tatattactg attgcactga acagcatggt ccccaatgta gccatgcaaa tgagaaaccc


 


3241
agtggctcct tgtggtacat gcatgcaaga ctgctgaagc cagaaggatg actgattacg


 


3301
cctcatgggt ggaggggacc actcctgggc cttcgtgatt gtcaggagca agacctgaga


 


3361
tgctccctgc cttcagtgtc ctctgcatct cccctttcta atgaagatcc atagaatttg


 


3421
ctacatttga gaattccaat taggaactca catgttttat ctgccctatc aattttttaa


 


3481
acttgctgaa aattaagttt tttcaaaatc tgtccttgta aattactttt tcttacagtg


 


3541
tcttggcata ctatatcaac tttgattctt tgttacaact tttcttactc ttttatcacc


 


3601
aaagtggctt ttattctctt tattattatt attttctttt actactatat tacgttgtta


 


3661
ttattttgtt ctctatagta tcaatttatt tgatttagtt tcaatttatt tttattgctg


 


3721
acttttaaaa taagtgattc ggggggtggg agaacagggg agggagagca ttaggacaaa


 


3781
tacctaatgc atgtgggact taaaacctag atgatgggtt gataggtgca gcaaaccact


 


3841
atggcacacg tatacctgtg taacaaacct acacattctg cacatgtatc ccagaacgta


 


3901
aagtaaaatt taaaaaaaag tga






The DD3 gene appears to represent a non-coding nucleic acid. Therefore, no start and stop codons have been indicated.
The present invention is further based in part upon the under expression of the LTF gene in prostate cancer epithelium. The cDNA sequence of the lactotransferrin (LTF) gene is publicly available through GenBank under the accession number NM_002343. The sequence (with start and stop codons underlined) corresponding to accession number NM_002343 is as follows:








(SEQ ID NO: 5)










   1
agagccttcg tttgccaagt cgcctccaga ccgcagacat gaaacttgtc ttcctcgtcc



 


  61
tgctgttcct cggggccctc ggactgtgtc tggctggccg taggaggagt gttcagtggt


 


 121
gcgccgtatc ccaacccgag gccacaaaat gcttccaatg gcaaaggaat atgagaaaag


 


 181
tgcgtggccc tcctgtcagc tgcataaaga gagactcccc catccagtgt atccaggcca


 


 241
ttgcggaaaa cagggccgat gctgtgaccc ttgatggtgg tttcatatac gaggcaggcc


 


 301
tggcccccta caaactgcga cctgtagcgg cggaagtcta cgggaccgaa agacagccac


 


 361
gaactcacta ttatgccgtg gctgtggtga agaagggcgg cagctttcag ctgaacgaac


 


 421
tgcaaggtct gaagtcctgc cacacaggcc ttcgcaggac cgctggatgg aatgtcccta


 


 481
tagggacact tcgtccattc ttgaattgga cgggtccacc tgagcccatt gaggcagctg


 


 541
tggccaggtt cttctcagcc agctgtgttc ccggtgcaga taaaggacag ttccccaacc


 


 601
tgtgtcgcct gtgtgcgggg acaggggaaa acaaatgtgc cttctcctcc caggaaccgt


 


 661
acttcagcta ctctggtgcc ttcaagtgtc tgagagacgg ggctggagac gtggctttta


 


 721
tcagagagag cacagtgttt gaggacctgt cagacgaggc tgaaagggac gagtatgagt


 


 781
tactctgccc agacaacact cggaagccag tggacaagtt caaagactgc catctggccc


 


 841
gggtcccttc tcatgccgtt gtggcacgaa gtgtgaatgg caaggaggat gccatctgga


 


 901
atcttctccg ccaggcacag gaaaagtttg gaaaggacaa gtcaccgaaa ttccagctct


 


 961
ttggctcccc tagtgggcag aaagatctgc tgttcaagga ctctgccatt gggttttcga


 


1021
gggtgccccc gaggatagat tctgggctgt accttggctc cggctacttc actgccatcc


 


1081
agaacttgag gaaaagtgag gaggaagtgg ctgcccggcg tgcgcgggtc gtgtggtgtg


 


1141
cggtgggcga gcaggagctg cgcaagtgta accagtggag tggcttgagc gaaggcagcg


 


1201
tgacctgctc ctcggcctcc accacagagg actgcatcgc cctggtgctg aaaggagaag


 


1261
ctgatgccat gagtttggat ggaggatatg tgtacactgc aggcaaatgt ggtttggtgc


 


1321
ctgtcctggc agagaactac aaatcccaac aaagcagtga ccctgatcct aactgtgtgg


 


1381
atagacctgt ggaaggatat cttgctgtgg cggtggttag gagatcagac actagcctta


 


1441
cctggaactc tgtgaaaggc aagaagtcct gccacaccgc cgtggacagg actgcaggct


 


1501
ggaatatccc catgggcctg ctcttcaacc agacgggctc ctgcaaattt gatgaatatt


 


1561
tcagtcaaag ctgtgcccct gggtctgacc cgagatctaa tctctgtgct ctgtgtattg


 


1621
gcgacgagca gggtgagaat aagtgcgtgc ccaacagcaa cgagagatac tacggctaca


 


1681
ctggggcttt ccggtgcctg gctgagaatg ctggagacgt tgcatttgtg aaagatgtca


 


1741
ctgtcttgca gaacactgat ggaaataaca atgaggcatg ggctaaggat ttgaagctgg


 


1801
cagactttgc gctgctgtgc ctcgatggca aacggaagcc tgtgactgag gctagaagct


 


1861
gccatcttgc catggccccg aatcatgccg tggtgtctcg gatggataag gtggaacgcc


 


1921
tgaaacaggt gttgctccac caacaggcta aatttgggag aaatggatct gactgcccgg


 


1981
acaagttttg cttattccag tctgaaacca aaaaccttct gttcaatgac aacactgagt


 


2041
gtctggccag actccatggc aaaacaacat atgaaaaata tttgggacca cagtatgtcg


 


2101
caggcattac taatctgaaa aagtgctcaa cctcccccct cctggaagcc tgtgaattcc


 


2161
tcaggaagta aaaccgaaga agatggccca gctccccaag aaagcctcag ccattcactg


 


2221
cccccagctc ttctccccag gtgtgttggg gccttggcct cccctgctga aggtggggat


 


2281
tgcccatcca tctgcttaca attccctgct gtcgtcttag caagaagtaa aatgagaaat


 


2341
tttgttgata ttctctcctt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa






Nucleotides 39-2171 of SEQ ID NO:5 represent the coding sequence of LTF.
LTF is a non-heme iron binding glycoprotein and a member of the transferring gene family. Bowman et al., Adv. Genet. 25:1-38 (1988); Park et al., Proc. Natl. Acad. Sci. U.S.A., 82:3149-53 (1985). The concentration of LTF in human prostate is hormone dependent and its expression is regulated by estrogen. van Sande et al., Urol. Res., 9(5):241-244 (1981); Teng et al., Biochem. Cell Biol., 80:7-16 (2002); Teng et al., Mol. Human Reproduction., 8, (1):58-67 (2002). LTF has also been implicated in certain cancers. For example, bovine LTF inhibits colon, esophagus, lung, and bladder carcinomas in rats. Tsuda et al., Biochem. Cell Biol., 80:131-136 (2002); Tsuda et al., Biofactors., 12(1-4):83-8 (2000); Tsuda et al., Biofactors., 12(1-4):83-8 (2000); Tsuda et al., Mutat Res., 462(2-3):227-33 (2000). In a study published over 20 years ago, van Sande et al., Urol. Res. 9:241-244 (1981), examined lactoferrin protein levels in human benign prostatic hypertrophy samples. They also detected low levels of lactoferrin protein in 3 carcinoma samples. However, we are the first to report the consistent and significant under expression of LTF mRNA in prostate cancer epithelial cells from a large number of patient samples. The observed under expression of LTF mRNA in such a statistically significant sample size indicates that under expression of LTF is a useful diagnostic marker for prostate cancer.
In one experiment, when screened using the Affymetrix GeneChip, CaP tumor cells exhibited upregulated AMACR expression in comparison to matched benign cells. In this studied patient cohort (n=73), AMACR was upregulated in tumor compared to matched benign prostate epithelium in 89.04% of the patients (65 of 73), while ERG was upregulated in 78.08% (57 of 73). When these two markers were combined, we observed a 100% CaP detection rate (under the criteria that at least one marker was upregulated) in the studied patient cohort (73 of 73). These data indicate that the combination of ERG and AMACR screening provides a highly accurate tool for CaP detection.
In another experiment, 96.4% of patients showed upregulation of either the ERG or AMACR gene in laser microdissected matched tumor and benign prostate epithelial cells from 55 CaP patients (FIG. 5). Similarly, 96.4% of patients showed upregulation of either the ERG or DD3 gene (FIG. 5). When the expression data for the ERG, AMACR, and DD3 genes was combined, 98.2% of the CaP patients showed upregulation of at least one of the three genes in tumor cells (FIG. 5). Thus, the combination of ERG, AMACR, and DD3 screening also provides a highly accurate tool for CaP detection.
In yet another experiment, validation by QRT-PCR (TaqMan) in microdissected tumor and benign prostate epithelial cells of 20 CaP patients confirmed a consistent, tumor associated under expression of LTF in 100% of patients (20 of 20) (FIG. 1D). Further validation studies by QRT-PCR in microdissected tumor and benign prostate epithelial cells of 103 CaP patients were consistent with the initial results, showing tumor associated under expression in 76% of patients (78 of 103).
Diagnostic Uses
In one embodiment, the present invention comprises a method of CaP diagnosis comprising screening biological samples for CaP-cell-specific gene expression signatures. In particular, the invention comprises screening for at least one of the CaP-cell-specific genes listed in Tables 1-6, particularly the ERG gene, the AMACR gene, the LTF gene or a combination of the ERG gene and the AMACR genes. The invention also comprises methods of diagnosing CaP comprising screening biological samples for expression of the ERG and DD3 genes, or a combination of the ERG, DD3, and AMACR genes.
In a further embodiment, the present invention comprises a method of CaP diagnosis comprising screening biological samples for CaP-cell-specific gene expression signatures using methods known in the art, including, for example, immunohistochemistry, ELISA, in situ RNA hybridization, and any oligonucletide amplification procedure known or later developed, including PCR (including QRT-PCR), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), ligase chain reaction (LCR), strand displacement amplification (SDA), and Loop-Mediated Isothermal Amplification (LAMP). See, e.g., Mullis, U.S. Pat. No. 4,683,202; Erlich et al., U.S. Pat. No. 6,197,563; Walker et al., Nucleic Acids Res., 20:1691-1696 (1992); Fahy et al., PCR Methods and Applications, 1:25-33 (1991); Kacian et al., U.S. Pat. No. 5,399,491; Kacian et al., U.S. Pat. No. 5,480,784; Davey et al., U.S. Pat. No. 5,554,517; Birkenmeyer et al., U.S. Pat. No. 5,427,930; Marshall et al., U.S. Pat. No. 5,686,272; Walker, U.S. Pat. No. 5,712,124; Notomi et al., European Patent Application No. 1 020 534 A1; Dattagupta et al., U.S. Pat. No. 6,214,587; and HELEN H. LEE ET AL., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES: APPLICATION TO DISEASE DIAGNOSIS (1997). Each of the foregoing amplification references is hereby incorporated by reference herein. In particular, the invention comprises generating antibodies to CaP-cell-specific genes, including ERG, AMACR, LTF, and DD3 for use in a immunohistochemistry assay. Other known diagnostic assays may be used to detect gene expression.
In a specific embodiment, the present invention comprises a method of diagnosing CaP comprising screening biological samples for expression of the ERG and AMACR genes, the ERG and DD3 genes, or the ERG, AMACR, and DD3 genes, or the LTF gene using methods known in the art, including, for example, immunohistochemistry, ELISA, in situ hybridization, PCR (including QRT-PCR), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3SR), ligase chain reaction (LCR), strand displacement amplification (SDA), and Loop-Mediated Isothermal Amplification (LAMP).
ERG, LTF, or AMACR polypeptides, their fragments or other derivatives, or analogs thereof, may be used as immunogens in order to generate antibodies that specifically bind such immunogens. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain and Fab fragments. In a specific embodiment, antibodies to a human ERG, LTF or AMACR protein are produced. Antibodies can then be used in standard diagnostic assays to detect the protein produced by the desired gene.
Various procedures known in the art may be used for the production of polyclonal antibodies to an ERG, LTF, or AMACR protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of a ERG, LTF, or AMACR protein can be obtained. For the production of antibody, various host animals can be immunized by injection with the native ERG, LTF, or AMACR protein, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including, but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
For preparation of monoclonal antibodies directed toward a ERG, LTF, or AMACR protein sequence or analog thereof, any technique, which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler et al (1975) Nature, 256:495-497, as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al. (1983) Immunology Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al. (1983) Proc. Natl. Acad. Sci. U.S.A., 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). According to the invention, techniques developed for the production of chimeric antibodies (Morrison et al. (1984) Proc. Natl. Acad. Sci. U.S.A., 81:6851-6855; Neuberger et al. (1984) Nature, 312:604-608; Takeda et al. (1985) Nature, 314:452-454) by splicing the genes from a mouse antibody molecule specific for ERG, LTF, or AMACR together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.
Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be used to produce ERG-, LTF-, or AMACR-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al. (1989) Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for ERG, LTF or AMACR proteins, derivatives, or analogs.
Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments, including single chain Fv (scFv) fragments.
In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., ELISA. For example, to select antibodies that recognize a specific domain of a ERG, LTF, or AMACR protein, one may assay generated hybridomas for a product which binds to a ERG, LTF, or AMACR fragment containing such domain.
A second aspect of the invention provides for use of the expression profiles resulting from these methods in diagnostic methods, including, but not limited to, characterizing the treatment response to any therapy, correlating expression profiles with clinico-pathologic features, distinguishing indolent prostate cancers from those with a more aggressive phenotype (e.g. moderate risk versus high risk), analyzing tumor specimens of patients treated by radical prostate surgery to help define prognosis, screening candidate genes for the development of a polynucleotide array for use as a blood test for improved prostate cancer detection, and identifying further genes that may serve as biomarkers for response to treatment to screen drugs for the treatment of advanced prostate cancer.
As will be readily appreciated by persons having skill in the art, the ERG, LTF, DD3, and/or the AMACR nucleic acid sequences described herein can easily be synthesized directly on a support, or pre-synthesized polynucleotide probes may be affixed to a support as described, for example, in U.S. Pat. Nos. 5,744,305, 5,837,832, and 5,861,242, each of which is incorporated herein by reference.
Such arrays may be used to detect specific nucleic acid sequences contained in a target cell or sample, as described in U.S. Pat. Nos. 5,744,305, 5,837,832, and 5,861,242, each of which is incorporated herein by reference. More specifically, in the present invention, these arrays may be used in methods for the diagnosis or prognosis of prostate cancer, such as by assessing the expression profiles of genes, in biological samples. In a preferred embodiment, computer models may be developed for the analysis of expression profiles. Moreover, such polynucleotide arrays are useful in methods to screen drugs for the treatment of advanced prostate cancer. In these screening methods, the polynucleotide arrays are used to analyze how drugs affect the expression of the ERG, LTF, AMACR, and/or DD3 genes.
Therapeutic Uses
The invention provides for treatment or prevention of various diseases and disorders by administration of a therapeutic compound (termed herein “therapeutic”). “Therapeutics” include but are not limited to: ERG or LTF proteins and analogs and derivatives (including fragments) thereof (e.g., as described herein above); nucleic acids encoding the ERG or LTF proteins, analogs, or derivatives; ERG or LTF antisense nucleic acids, ERG or LTF dominant negative mutants, siRNA against ERG or LTF, ERG or LTF antibodies and ERG or LTF agonists and antagonists. ERG or LTF agonists and antagonists, including small molecules, can be identified using the methods disclosed in this application or any standard screening assay to identify agents that modulate ERG or LTF expression or function, particularly in prostate cancer cells. For example, ERG or LTF expression or function can be readily detected, e.g., by obtaining a biological sample from a patient, e.g., a tissue sample (e.g., from biopsy tissue), a blood sample, or a urine sample, and assaying it in vitro for mRNA or protein levels, structure and/or activity of the expressed ERG or LTF mRNA or protein. Many methods standard in the art can be employed, including but not limited to, kinase assays, immunoassays to detect and/or visualize ERG or LTF protein (e.g., Western blot, immunoprecipitation followed by SDS-PAGE, immunocytochemistry, etc.) and/or hybridization assays to detect ERG or LTF expression by detecting and/or visualizing ERG or LTF mRNA (e.g., Northern assays, dot blots, in situ hybridization, PCR (including RT-PCR), TMA, NASAB, 3SR, LCR, SDA, LAMP, etc.).
Hyperproliferative Disorders
Disorders involving hyperproliferation of cells are treated or prevented by administration of a therapeutic that antagonizes (reduces or inhibits) ERG function or expression or enhances LTF function or expression. In certain embodiments, ERG function is inhibited by use of ERG antisense nucleic acids. The present invention provides the therapeutic or prophylactic use of nucleic acids of at least 10, 15, 100, 200, 500, 1000, 1500, 2000, or 2500 contiguous nucleotides in antisense to any of the ERG nucleotides described herein. In a particular embodiment, the ERG antisense nucleic acid comprises at least 10, 15, 100, 200, 500, 1000, 1500, 2000, or 2500 contiguous nucleotides in antisense orientation to the ERG nucleotide sequence. An ERG “antisense” nucleic acid as used herein refers to a nucleic acid capable of hybridizing under defined conditions to a portion of an ERG nucleic acid by virtue of some sequence complementarity. The antisense nucleic acid may be complementary to a coding and/or noncoding region of an ERG nucleic acid. Such antisense nucleic acids have utility as therapeutics that inhibit ERG function, and can be used in the treatment or prevention of disorders as described herein.
The antisense nucleic acids of the invention can be oligonucleotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenously, introduced coding sequences.
The dominant negative mutants of the invention can be produced by expression plasmids containing a nucleic acid encoding a non-functional domain of ERG, such as the DNA binding domain of ERG. These expression plasmids can be introduced into a target cell or tissue and can induce tumor growth inhibition and apoptosis by acting as a dominant negative form against the wild-type ERG transcription factors influencing cell hyperproliferation (Oikawa, Cancer Sci (2004), 95:626-33).
RNA interference can be achieved using siRNA against the ERG gene. The siRNA is a short double stranded RNA molecule of about 18-25 nucleotides that comprises a nucleotide sequence complementary to a region of the target gene. The siRNA can be introduced into a target cell or tissue, for example using an expression plasmid, where it interferes with the translation of the ERG gene. RNA interference techniques can be carried out using known methods as described, for example, in published U.S. Patent Applications 20040192626, 20040181821, and 20030148519, each of which is incorporated by reference.
Therapeutics which are useful according to this embodiment of the invention for treatment of a disorder may be selected by testing for biological activity in promoting the survival or differentiation of cells. For example, in a specific embodiment relating to cancer therapy, including therapy of prostate cancer, a therapeutic decreases proliferation of tumor cells. These effects can be measured as described in the Examples or using any other method standard in the art.
In specific embodiments, malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or prevented in the prostate.
The therapeutics of the invention that antagonize ERG activity can also be administered to treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described herein, such as prostate cancer.
Gene Therapy
In a specific embodiment, nucleic acids comprising a sequence encoding an ERG or LTF protein or functional derivative thereof, are administered to promote ERG or LTF function, by way of gene therapy. Alternatively, nucleic acids comprising an antisense ERG sequence are administered to antagonize ERG expression or function. Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject.
Any of the methods for gene therapy available in the art can be used according to the present invention. For specific protocols, see Morgan (2001) Gene Therapy Protocols, 2nd ed., Humana Press. For general reviews of the methods of gene therapy, see Goldspiel et al. (1993) Clinical Pharmacy, 12:488-505; Wu et al. (1991) Biotherapy, 3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol., 32:573-596; Mulligan (1993) Science, 260:926-932; and Morgan et al. (1993) Ann. Rev. Biochem., 62:191-217; May (1993) TIBTECH, 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Current Protocols in Molecular Biology (2004), Ausubel et al., eds., John Wiley & Sons, NY; and Kriegler (1990) Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.
In one embodiment, the therapeutic comprises an ERG or LTF nucleic acid or antisense ERG nucleic acid that is part of a vector. In particular, such a nucleic acid has a regulatory sequence, such as a promoter, operably linked to the ERG or LTF coding region or antisense molecule, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the ERG or LTF coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the ERG or LTF nucleic acid (Koller et al. (1989) Proc. Natl. Acad. Sci. U.S.A., 86:8932-8935; Zijlstra et al. (1989) Nature, 342:435-438).
In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the desired nucleic acids, such that expression of the nucleic acid is controllable by the appropriate inducer of transcription.
Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid is directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Pat. No. 4,980,286, which is incorporated herein by reference), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, DuPont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem., 262:4429-4432). In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell-specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Pubs. WO 92/06180; WO 92/22635; WO92/20316; WO93/14188; WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller et al. (1989) Proc. Natl. Acad. Sci. U.S.A., 86:8932-8935; Zijlstra et al. (1989) Nature, 342:435-438).
In a specific embodiment, a viral vector that contains an ERG or LTF nucleic acid is used. For example, a retroviral vector can be used (see, Miller et al. (1993) Meth. Enzymol., 217:581-599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The ERG or LTF nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al. (1994) Biotherapy, 6:291-302, which describes the use of a retroviral vector to deliver the MDRL gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al. (1994) J. Clin. Invest., 93:644-651; Kiem et al. (1994) Blood, 83:1467-1473; Salmons et al. (1993) Hum. Gene Ther., 4:129-141; and Grossman et al. (1993) Curr. Opin. Gen. Devel., 3:110-114.
Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky et al. (1993, Curr. Opin. Gen. Devel., 3:499-503) present a review of adenovirus-based gene therapy. Bout et al. (1994, Hum. Gene Ther., 5:3-10) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al. (1991) Science, 252:431-434; Rosenfeld et al. (1992) Cell, 68:143-155; and Mastrangeli et al. (1993) J. Clin. Invest., 91:225-234.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al. (1993) Proc. Soc. Exp. Biol. Med., 204:289-300).
Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler et al. (1993) Meth. Enzymol., 217:599-618; Cohen et al. (1993) Meth. Enzymol., 217:618-644; Cline (1985) Pharmac. Ther., 29:69-92) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
The resulting recombinant cells can be delivered to a patient by various methods known in the art. In one preferred embodiment, epithelial cells are injected, e.g., subcutaneously. In another embodiment, recombinant skin cells may be applied as a skin graft onto the patient. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) may be administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. In certain embodiments, the cells used for gene therapy are autologous to the patient.
In one embodiment, an ERG or LTF nucleic acid or antisense molecule is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention. Such stem cells include, but are not limited to, hematopoietic stem cells (HSC), stem cells of epithelial tissues such as the skin and the lining of the gut, embryonic heart muscle cells, liver stem cells (PCT Pub. WO 94/08598), and neural stem cells (Stemple et al. (1992) Cell, 71:973-985).
Epithelial stem cells (ESCs) or keratinocytes can be obtained from tissues such as the skin and the lining of the gut by known procedures (Rheinwald (1980) Meth. Cell Bio., 21A:229). In stratified epithelial tissue such as the skin, renewal occurs by mitosis of stem cells within the germinal layer, the layer closest to the basal lamina. Stem cells within the lining of the gut provide for a rapid renewal rate of this tissue. ESCs or keratinocytes obtained from the skin or lining of the gut of a patient or donor can be grown in tissue culture (Rheinwald (1980) Meth. Cell Bio., 21A:229; Pittelkow et al. (1986) Mayo Clinic. Proc., 61:771). If the ESCs are provided by a donor, a method for suppression of host versus graft reactivity (e.g., irradiation, drug or antibody administration to promote moderate immunosuppression) can also be used.
With respect to hematopoietic stem cells (HSC), any technique which provides for the isolation, propagation, and maintenance in vitro of HSC can be used in this embodiment of the invention. Techniques by which this may be accomplished include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host, or a donor, or (b) the use of previously established long-term HSC cultures, which may be allogeneic or xenogeneic. Non-autologous HSC may be used in conjunction with a method of suppressing transplantation immune reactions of the future host/patient. In a particular embodiment, human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration (see, e.g., Kodo et al. (1984) J. Clin. Invest., 73:1377-1384). In one embodiment, the HSCs can be made highly enriched or in substantially pure form. This enrichment can be accomplished before, during, or after long-term culturing, and can be done by any techniques known in the art. Long-term cultures of bone marrow cells can be established and maintained by using, for example, modified Dexter cell culture techniques (Dexter et al. (1977) J. Cell Physiol., 91:335) or Witlock-Witte culture techniques (Witlock et al. (1982) Proc. Natl. Acad. Sci. U.S.A., 79:3608-3612).
Pharmaceutical Compositions and Administration
The invention further provides pharmaceutical compositions comprising an effective amount of an ERG or LTF therapeutic, including ERG or LTF nucleic acids (sense or antisense) or ERG or LTF polypeptides of the invention, in a pharmaceutically acceptable carrier, as described below.
Compositions comprising an effective amount of a polypeptide of the present invention, in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Company, Easton, Pa., 1980.
In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application.
The compositions of the invention can be administered in any suitable manner, e.g., topically, parenterally, or by inhalation. The term “parenteral” includes injection, e.g., by subcutaneous, intravenous, or intramuscular routes, also including localized administration, e.g., at a site of disease or injury. Sustained release from implants is also contemplated. One skilled in the art will recognize that suitable dosages will vary, depending upon such factors as the nature of the disorder to be treated, the patient's body weight, age, and general condition, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.
Compositions comprising nucleic acids of the invention in physiologically acceptable formulations, e.g., to be used for gene therapy are also contemplated. In one embodiment, the nucleic acid can be administered in vivo to promote expression of the encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular as described in other sections herein.
Various delivery systems are known in the art and can be used to administer a therapeutic of the invention. Examples include, but are not limited to encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the therapeutic, receptor-mediated endocytosis (see, e.g., Wu et al. (1987) J. Biol. Chem., 262:4429-4432), construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, a suppository, an implant, wherein the said implant is of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
In another embodiment, the therapeutic can be delivered in a vesicle, in particular a liposome (see Langer (1990) Science, 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein et al., eds., Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327. In yet another embodiment, the therapeutic can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng., 14:201; Buchwald et al. (1980) Surgery, 88:507; Saudek et al. (1989) New Engl. J. Med., 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer et al., eds., CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., eds., Wiley, N.Y., 1984; Ranger et al. (1983) J. Macromol. Sci. Rev. Macromol. Chem., 23:61; see also Levy et al. (1985) Science, 228:190; During et al. (1989) Ann. Neurol., 25:351; Howard et al. (1989) J. Neurosurg., 71:105. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson (1984) in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer (1990, Science, 249:1527-1533).
Diagnosis and Screening
ERG, LTF, and/or AMACR proteins, analogues, derivatives, and fragments thereof, and antibodies thereto; ERG, LTF, DD3, and/or AMACR nucleic acids (and their complementary and homologous sequences) and antibodies thereto, including anti-ERG, anti-DD3, anti-LTF and/or anti-AMACR antibodies, have uses in diagnostics. Such molecules can be used in assays, such as immunoassays, to detect, prognose, diagnose, or monitor various conditions, diseases, and disorders affecting ERG, LTF, DD3, and/or AMACR expression, or monitor the treatment thereof, particularly cancer, and more particularly prostate cancer. In particular, such an immunoassay is carried out by a method comprising contacting a sample derived from an individual with an anti-ERG, anti-LTF, anti-DD3, and/or anti-AMACR antibody (directed against either a protein product or a nucliec acid) under conditions such that specific binding can occur, and detecting or measuring the amount of any specific binding by the antibody. In one embodiment, such binding of antibody, in tissue sections, can be used to detect aberrant ERG, LTF, DD3, and/or AMACR localization or aberrant (e.g., high, low or absent) levels of ERG, LTF, DD3, and/or AMACR. In a specific embodiment, antibody to ERG, LTF, DD3, and/or AMACR can be used to assay in a biological sample (e.g., tissue, blood, or urine sample) for the presence of ERG, LTF, DD3, and/or AMACR where an aberrant level of ERG, LTF, DD3, and/or AMACR is an indication of a diseased condition, such as cancer, including, for example, prostate cancer.
Any biological sample in which it is desired to detect an oligonucloetide or polypeptide of interest can be used, including tissue, cells, blood, lymph, semen, and urine. The biological sample is preferably derived from prostate tissue, blood, or urine. The tissue sample comprises cells obtained from a patient. The cells may be found in a prostate tissue sample collected, for example, by a prostate tissue biopsy or histology section, or a bone marrow biopsy. The blood sample can include whole blood, plasma, serum, or any derivative thereof, including, for example, circulating cells, such as prostate cells, isolated from the blood sample, or nucleic acid or protein obtained from the isolated cells. Blood may contain prostate cells, particularly when the prostate cells are cancerous, and, more particularly, when the prostate cancer metastasizes and is shed into the blood. Similarly, the urine sample can be whole urine or any derivative thereof, including, for example, cells, such as prostate cells, obtained from the urine.
The immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
ERG, LTF, DD3, and/or AMACR genes and related nucleic acid sequences and subsequences, including complementary sequences, can also be used in hybridization assays. ERG, LTF, DD3, and/or AMACR nucleic acid sequences, or subsequences thereof comprising about at least 8, 15, 20, 50, 100, 250, or 500 nucleotides can be used as hybridization probes. Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with aberrant changes in ERG, LTF, DD3, and/or AMACR expression and/or activity as described above. In particular, such a hybridization assay is carried out by a method comprising contacting a sample containing nucleic acid with a nucleic acid probe capable of hybridizing under defined conditions (preferably under high stringency hybridization conditions, e.g., hybridization for 48 hours at 65° C. in 6×SSC followed by a wash in 0.1×SSX at 50° C. for 45 minutes) to an ERG, LTF, DD3, and/or AMACR nucleic acid, and detecting (i.e, measuring either qualitatively or quantitatively) the degree of the resulting hybridization. As described herein, any nucleic acid amplification procedure, including, PCR/RT-PCR, TMA, NASBA, 3SR, LCR, SDA, and LAMP can be used to detect the presence of the ERG, LTF, DD3 and/or AMACR gene and/or the level of its mRNA expression.
In some applications, probes exhibiting at least some degree of self-complementarity are desirable to facilitate detection of probe:target duplexes in a test sample without first requiring the removal of unhybridized probe prior to detection. Molecular torch probes are a type of self-complementary probes that are disclosed by Becker et al., U.S. Pat. No. 6,361,945. The molecular torch probes disclosed Becker et al. have distinct regions of self-complementarity, referred to as “the target binding domain” and “the target closing domain,” which are connected by a joining region and which hybridize to one another under predetermined hybridization assay conditions. When exposed to denaturing conditions, the complementary regions (which may be fully or partially complementary) of the molecular torch probe melt, leaving the target binding domain available for hybridization to a target sequence when the predetermined hybridization assay conditions are restored. And when exposed to strand displacement conditions, a portion of the target sequence binds to the target binding domain and displaces the target closing domain from the target binding domain. Molecular torch probes are designed so that the target binding domain favors hybridization to the target sequence over the target closing domain. The target binding domain and the target closing domain of a molecular torch probe include interacting labels (e.g., luminescent/quencher) positioned so that a different signal is produced when the molecular torch probe is self-hybridized as opposed to when the molecular torch probe is hybridized to a target nucleic acid, thereby permitting detection of probe:target duplexes in a test sample in the presence of unhybridized probe having a viable label or labels associated therewith.
Another example of detection probes having self-complementarity are the molecular beacon probes disclosed by Tyagi et al. in U.S. Pat. No. 5,925,517. Molecular beacon probes include nucleic acid molecules having a target complement sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target nucleic acid sequence, and a label pair that interacts when the probe is in a closed conformation. Hybridization of the target nucleic acid and the target complement sequence separates the members of the affinity pair, thereby shifting the probe to an open confirmation. The shift to the open confirmation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and quencher, such as DABCYL and EDANS.
By way of example, ERG, LTF, AMACR, or DD3 hybridization probes can comprise a nucleic acid having a contiguous stretch of at least about 8, 15, 20, 50, 100, 250, 500, 750, 1000, 1250, or 1500 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 or a sequence complementary thereto. Such contiguous fragments of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 may also contain at least one mutation so long as the mutant sequence retains the capacity to hybridize to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, or SEQ ID NO:5 under low or high stringency conditions (preferably under high stringency hybridization conditions, e.g., hybridization for 48 hours at 65° C. in 6×SSC followed by a wash in 0.1×SSX at 50° C. for 45 minutes).
In specific embodiments, diseases and disorders involving hyperproliferation of cells, such as cancers, including, for example, prostate cancer, can be diagnosed, or their suspected presence can be screened for, or a predisposition to develop such disorders can be predicted, by detecting levels of the ERG, LTF, and/or AMACR protein, ERG, DD3, and/or AMACR RNA, or ERG, DD3, and/or AMACR functional activity, or by detecting mutations in ERG, DD3, LTF and/or AMACR RNA, DNA, or protein (e.g., translocations in ERG, LFT, DD3, or AMACR nucleic acids, truncations in the ERG, LFT, DD3, or AMACR gene or protein, changes in nucleotide or amino acid sequence relative to wild-type ERG, LTF, DD3, or AMACR) that cause increased or decreased expression or activity of ERG, LTF, DD3, and/or AMACR. By way of example, levels of ERG, LTF, and/or AMACR protein can be detected by immunoassay, levels of ERG, LTF, DD3, and/or AMACR mRNA can be detected by hybridization assays (e.g., Northern blots, dot blots, or any nucleic acid amplification procedure, including, PCR/RT-PCR, TMA, NASBA, 3SR, LCR, SDA, and LAMP), translocations and point mutations in ERG, LTF, DD3, and/or AMACR nucleic acids can be detected by Southern blotting, RFLP analysis, any nucleic acid amplification procedure, including, PCR/RT-PCR, TMA, NASBA, 3SR, LCR, SDA, LAMP, sequencing of the ERG, LTF, DD3, and/or AMACR genomic DNA or cDNA obtained from the patient, etc.
In one embodiment, levels of the ERG, DD3, LTF and/or AMACR mRNA or protein in a subject sample are detected or measured and compared to the mRNA or protein expression levels of the corresponding gene in a control sample or to a standard numerical value or range. For example, increased expression levels of ERG, DD3, and/or AMACR or decreased levels of LTF, relative to a matched, normal tissue sample, indicate that the subject has a malignancy or hyperproliferative disorder, including, for example, prostate cancer, or a predisposition to develop the same. Other appropriate controls include other noncancerous samples from the subject, samples obtained from a different subject without cancer, or other cancer-specific markers. For example, in prostate cancer, a prostate-cell specific marker, such as PSA, can be used as a control to compare and/or normalize expression levels of other genes, such as ERG, LTF, DD3, and/or AMACR. In one embodiment, a method of diagnosing cancer, such as prostate cancer, comprises obtaining a biological sample from a subject (e.g., a tissue sample (e.g., from biopsy tissue), a blood sample, or a urine sample), determining the expression level of a ERG, LTF, DD3, and/or AMACR gene and/or ERG, LTF, DD3, and/or AMACR activity in the samples, and diagnosing or prognosing cancer in said subject. In further embodiments, the expression level of the ERG, LTF, DD3, and/or AMACR gene and/or ERG, LTF, DD3, and/or AMACR activity is determined by Southern blotting, Northern blotting, Western blotting, ELISA, any nucleic acid amplification procedure, including, PCR/RT-PCR, TMA, NASBA, 3SR, LCR, SDA, and LAMP, or other techniques as described herein or known in the art. Without limiting the instant invention, increased or decreased expression of at least two times, as compared to the control sample indicates the presence of prostate cancer or a higher predisposition to developing prostate cancer.
Another aspect of the invention provides a means for monitoring a response to “hormonal therapy” by evaluating the expression profiles of the ERG gene, alone or in combination with the AMACR and/or DD3 genes and/or LTF genes, and correlating these profiles with the clinical signs of the disease.
Kits for diagnostic use are also provided. A kit comprises an anti-ERG gene antibody or an antibody directed against the ERG protein and/or an anti-AMACR gene antibody or an antibody directed against the AMACR protein and/or an anti-DD3 gene antibody and/or and an anti-LTF gene antibody or an antibody directed against the LTF protein, which can be optionally detectably labeled. A kit is also provided that comprises a nucleic acid probe capable of hybridizing under defined conditions (preferably under high stringency hybridization conditions, e.g., hybridization for 48 hours at 65° C. in 6×SSC followed by a wash in 0.1×SSX at 50° C. for 45 minutes) to ERG, LTF, DD3, and/or AMACR nucleic acid. In a specific embodiment, a kit comprises at least a pair of primers (e.g., each in the size range of at least about 6, 17, 30, or 60 nucleotides) that are capable of priming amplification, by any nucleic acid amplification procedure (including e.g., PCR/RT-PCR, TMA, NASBA, 3SR, LCR, SDA, LAMP), of the ERG, LTF, DD3, and/or AMACR gene or a fragment thereof. A kit can comprise a predetermined amount of a purified ERG, LTF, DD3, and/or AMACR protein or nucleic acid for use, e.g., as a standard or control. The kit can also comprise one or more components for detecting the nucleic acid probe, including components described herein or known in the art.
In one embodiment, the kit comprises a nucleic acid that hybridizes under defined conditions (and preferably under conditions of high stringency, e.g., hybridization for 48 hours at 65° C. in 6×SSC followed by a wash in 0.1×SSX at 50° C. for 45 minutes) with at least one gene chosen from those genes identified in Tables 1-6 or the DD3 gene, and is affixed to a support, alone, or in combination with other nucleic acids. For example, an ERG and/or LTF nucleic acid can be affixed to the support, with or without other nucleic acids. In a specific embodiment, the support comprises at least an ERG nucleic acid and an AMACR nucleic acid or at least an ERG nucleic acid and a DD3 nucleic acid. In another embodiment, the support comprises at least an ERG nucleic acid, an AMACR nucleic acid, and a DD3 nucleic acid. This support can be used as part of a kit for detecting cancer, such as prostate cancer. These kits can further comprise at least a pair of primers (e.g., each in the size range of at least about 6, 17, 30, or 60 nucleotides) that are capable of priming amplification, by any nucleic acid amplification procedure (including e.g., PCR/RT-PCR, TMA, NASBA, 3SR, LCR, SDA, LAMP), of the ERG, LTF, DD3, and/or AMACR gene or a fragment thereof.
EXAMPLES
Example 1
Screening of CaP Cell-Specific Gene Expression Signatures Using Affymetrix GeneChip
Patient Selection
Specimens were obtained under an IRB-approved protocol from patients treated by radical prostatectomy (RP) at Walter Reed Army Medical Center (WRAMC). From over 300 patients two groups were selected which had prostate tumors with either moderate (MR) or high risk (HR) of disease progression after RP. The HR group had PSA recurrence, Gleason score 8-9, T3c stage, seminal vesicle invasion, and poorly differentiated tumor cells; the MR group had no PSA recurrence, Gleason score 6-7, T2a-T3b stage, no seminal vesicle invasion, and well to moderately differentiated tumor cells. LCM compatible specimens were selected from age and race matched HR or MR patients with no family history of CaP.
Tissue Samples and Laser-Capture Microdissection
Normal and cancer cells were laser capture microdissected (LCM) from OCT embedded and Hematoxylin-eosin (H&E) stained frozen prostate sections of radical prostatectomy specimens (2000 laser shots for one sample). Laser capture microdissection (LCM) facilitates the isolation of morphologically defined, homogenous cell populations from complex tissues by selectively adhering the cells of interest to a transparent film with focused pulses of low energy infrared laser under a microscope. Emmert-Buck et al., Science (1996); 274(5289): 921-922; Schutz et al., Nat Biotechnol (1998) 16(8): 737-742.
RNA Extraction and T7-Based Linear RNA Amplification
Total RNA was isolated from the LCM samples with the MicroRNA kit (Stratagene, La Jolla, Calif.), quantified using RiboGreen dye (Molecular Probes, Eugene, Oreg.) and VersaFluor fluorimeter (BioRad, Hercules, Calif.), and quality tested by RT-PCR using NKX3.1 and GAPDH primers. Linear RNA amplification was performed using RiboAmp RNA amplification kit (Arcturus, Mountain View, Calif.). Precisely, 2 nanograms of total RNA from LCM derived epithelial cells of normal as well as tumor tissue from each patient was used for the first round of amplification. During the second round of amplification after cDNA synthesis and purification the samples were biotinylated during in vitro transcription which was used for the GeneChip analysis.
Gene Chip Analysis
Linearly amplified aRNA was hybridized to high-density oligonucleotide human genome array (HG U133A array) (Affymetrix, Santa Clara, Calif., USA). The array contains 22,283 probe sets, about 18,000 of which represent well annotated genes, while the remainder represent various expressed sequence tags (EST) and hypothetical genes. Biotinylation was carried out using aRNA by in vitro transcription using MEGA script T7 in vitro Transcription Kit (Ambion, Austin, Tex., USA) cDNA and biotinylated UTP and biotinylated CTP (ENZO, Farmingdale, N.Y., USA) (34). The biotin labeled cRNA was purified using the QIAGEN RNeasy spin columns (QIAGEN, Valencia, Calif.) following the manufacturer's protocol. The biotin labeled cRNA was fragmented in a 40 μl reaction mixture containing 40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate incubated at 94° C. for 35 minutes and then put on ice.
Hybridization, Staining and Scanning of the GeneChip
The biotin labeled and fragmented aRNA was hybridized to the HG U133A array. Briefly, a 220 μl hybridization solution consisting of: 1M NaCl, 10 mM Tris pH 7.6, 0.005% Triton X-100, 50 pM control Oligo B2 (5′ bioGTCAAGATGCTACCGTTCAG 3′) (SEQ ID NO:6) (Affymetrix); the control cRNA cocktail of: Bio B (150 pM), Bio C (500 pM), Bio D (2.5 nM) and Cre X (10 nM) (American Type Tissue Collection, Manassas, Va. and Lofstrand Labs, Gaithersburg, Md.), 0.1 mg/ml herring sperm DNA and 0.05 μg/μl of the fragmented labeled sample cRNA was heated to 95° C. for 35 min., cooled to 40° C. and clarified by centrifugation. Hybridization was at 42° C. in a rotisserie hybridization oven (Model 320, Affymetrix) at 60 rpm for 16 hours. Following hybridization, the GeneChip arrays were washed 10 times at 25° C. with 6×SSPE-T buffer (1 M NaCl, 0.006 M EDTA, and 0.06 M Na3PO4, 0.005% Triton X-100, pH 7.6) using the automated fluidics station protocol. GeneChip arrays were incubated at 50° C. in 0.5×SSPE-T, 0.005% Triton X-100 for 20 minutes at 60 rpm in the rotisserie oven. GeneChip arrays were stained for 15 minutes at room temperature and at 60 rpm, with streptavidin phycoerythrin (Molecular Probes, Inc., Eugene, Oreg.) stain solution at a final concentration of 10 μg/ml in 6×SSPE-T buffer and 1.0 mg/ml acetylated bovine serum albumin (Sigma). GeneChip arrays were washed twice at room temperature with 6×SSPE-T buffer, and then were scanned with the HP GeneArray Scanner (Hewlett-Packard, Santa Clara, Calif.) controlled by GeneChip 3.1 Software (Affymetrix).
Example 2
Analysis of GeneChip Results by Supervised Multi-Dimensional Scaling (MDS)
Image Analysis and Data Collection
Affymetrix GeneChip Microarray Analysis Software, version 3.1 and Affymetrix Micro DB and Data Mining Tool version 2.0 (Affymetrix), Microsoft Excel 2000 (Microsoft, Seattle, Wash.) and Statistica version 4.1 (Stat Soft, Inc., Tulsa, Okla.) were used. In the Affymetrix system, the average difference fluorescence is the average of the difference between every perfect match probe cell and its control mismatch probe cell and is directly related to the level of expression of a transcript. A comparative file indicates the relative change in abundance (fold change) for each transcript between a baseline and an experimental sample. For further detail and advanced bioinformatic analysis we used the Microarray Data Analysis software from NHGRI and the GeneSpring software (Silicon Genetics, Calif.).
Data Analysis
For clustering analysis, National Human Genome Research Institute (NHGRI) Microarray Data Analysis software was used, which partitioned the samples of the high risk and moderate risk groups into well-separated and homogeneous groups based on the statistical behavior of their genes expression. To achieve the objective of clustering each of the groups, all pair-wise similarities between samples were evaluated, and then grouped via the average linkage algorithm. Pearson correlation coefficient or Euclidean distance were typically used to quantify the similarity. Unsupervised hierarchical and or non hierarchical clustering was also performed using the same distance matrix.
Using a matrix of Euclidean distance measurements from complete pair wise comparison of all the prostate specimens, a multidimensional scaling (MDS) method was performed using an implementation of MDS in the MATLAB package to determine the overall similarities and dissimilarities in gene expression profiles. A weighted gene analysis was performed to generate a subset of genes statistically significant in separating the high risk group from the moderate risk group.
Briefly, for two different groups e.g., epithelium of high risk tumor and epithelium of moderate risk tumor with a given number of samples 25 and 25, the discriminative weight for each gene is determined by the formula: w=dB/(k1dw1+k2dw2+α); where dB is the Euclidean distance between two groups (center-to-center or between cluster Euclidean distance), dw1 is the average Euclidean distance among all the epithelial samples of high risk group, dw2 is average Euclidean distance among all the epithelial samples of moderate risk group, k1=25/(25+25), k2=25/(25+25), and α is a small constant to ensure the denominator is never equal to zero. Genes were ranked according to their w values. Genes with high w values created greater separation between groups and denser compaction within the group. In other words, the subset of genes with high w values have the most discriminative power to differentiate a high risk group from a moderate risk group and vice versa. Sample labels were randomly permuted and the w value was computed again for each gene to test the statistical significance of the discriminative weights. Genes with the most significant expression differences were selected by p-values. A hierarchical clustering algorithm was used to verify the predictor model obtained from the supervised MDS analysis.
From this analysis, specific genes were identified whose expression signature in tumor tissue varied from their expression signature in benign matched tissue. Genes with a p-value of not more than 0.05 were selected and ranked by p-value, as shown in Tables 1-6.
In Silico Validation:
We have tested the discriminatory potential of the genes that we obtained from our analysis on some independent data sets. Affymetrix oligonucleotide GeneChip Hum95Av2 data were obtained from Welsh et al. 2001, Singh et al. Genes from these data bases that correspond with the genes of our discriminatory list were selected and their tumor specific expression intensities and/or tumor over normal ratio were used for an MDS analysis as described above in the data analysis section. MDS plots were obtained depicting the discriminatory capability of the genes on the independent data sets.







TABLE 1







The first 50 genes obtained from the supervised MDS analysis of tumor versus benign tissues of all the


high risk and moderate risk CaP patients, ranked by p-value. (T vs B in All 18 Samples)











Expression



GenBank
Regulation














No.
Accession
Common Name of Genes
Description of Genes
Map
p-Value
Tumor
Benign

















1.
AF047020
AMACR
Alpha-methylacyl-CoA racemase
5p13.2-q11.1
0
Up
Down


2.
NM_002343
LTF
Lactotransferrin
3q21-q23
0
Down
Up


3.
NM_002275
KRT15
Keratin 15
17q21
0.000001
Down
Up


4.
BC000915
CLIM1, CLP36, CLP-36
PDZ and LIM domain 1 (elfin)
10q22-q26.3
0.000001
Down
Up


5.
X90579
CYP3A5
Cytochrome P450, subfamily 3A,
7
0.000001
Down
Up





polypeptide 5


6.
NM_003671
CDC14B1, CDC14B2
H. sapiens CDC14 cell division cycle 14
9q22.2-q22.31
0.000005
Down
Up





homolog B


7.
AI424243
CEGP1
H. sapiens cDNA clone
11
0.000005
Down
Up





IMAGE: 2094442


8.
NM_022370
Rbig1
Hypothetical protein FLJ21044 similar
11q24.2
0.000009
Down
Up





to Rbig1


9.
AI356398
ZFP36L2
TISD_HUMAN P47974 TIS11D
2
0.000018
Down
Up





PROTEIN


10.
NM_005213
STF1, STFA
Cystatin A (stefin A)
3q21
0.000018
Down
Up


11.
NM_006394
RIG
Regulated in glioma
11p15.1
0.000018
Down
Up


12.
AF275945
EVA1
Epithelial V-like antigen 1
11q23.3
0.000018
Down
Up


13.
NM_020186
DC11
DC11 protein
7q21.3
0.000018
Up
Down


14.
AI922538
TMEM1
Transmembrane protein 1
21
0.000018
Down
Up


15.
NM_014863
BRAG, KIAA0598
B cell RAG associated protein
10q26
0.000018
Down
Up


16.
AI669229
RARRES1
Homo sapiens cDNA clone
3q25.33
0.000036
Down
Up





IMAGE: 2315074


17.
NM_006017
AC133, CD133
Prominin (mouse)-like 1
4p15.33
0.000036
Down
Up


18.
NM_004503
HOXC6
Homeo box C6
12q12-q13
0.000036
Up
Down


19.
NM_005084
PAFAH, LDL-PLA2
Phospholipase A2, group VII
6p21.2-p12
0.000036
Up
Down


20.
NM_001511
MGSA, CXCL1, SCYB1
GRO1 oncogene
4q21
0.000071
Down
Up


21.
BG054844
ARHE
H. sapiens cDNA clone
2q23.3
0.000071
Down
Up





IMAGE: 3441573


22.
NM_007191
WIF-1
Wnt inhibitory factor-1
12q14.2
0.000071
Down
Up


23.
X99268
TWIST
Twist (Drosophila) homolog
7p21.2
0.000071
Up
Down


24.
AI826799
EFEMP1
EXTRACELLULAR PROTEIN S1-5
2p16
0.000071
Down
Up





PRECURSOR


25.
NM_001018
RPS15
Ribosomal protein S15
19p13.3
0.000071
Up
Down


26.
AV711904
LYZ
Lysozyme (renal amyloidosis)

0.000071
Down
Up


27.
AI433463
MME
NEPRILYSIN (HUMAN)
3q25.1-q25.2
0.000071
Down
Up


28.
BE908217
ANXA2
H. sapiens cDNA clone
15q21-q22
0.000071
Down
Up





IMAGE: 3902323


29.
NM_000441
PDS, DFNB4
Solute carrier family 26, member 4
7q31
0.000071
Down
Up


30.
BC003068
SLC19A1
Solute carrier family 19, member 1
21q22.3
0.000071
Up
Down


31.
NM_005950
MT1
Metallothionein 1G
16q13
0.000071
Down
Up


32.
NM_013281
FLRT3
Fibronectin leucine rich transmembrane
20p11
0.000071
Down
Up





protein 3


33.
AI351043
ESTs
H. sapiens cDNA clone
21
0.000145
Up
Down





IMAGE: 1948310


34.
NM_001099
PAP
Acid phosphatase, prostate
3q21-q23
0.000145
Down
Up


35.
NM_006113
VAV3
Vav 3 oncogene
1p13.1
0.000145
Down
Up


36.
NM_005980
S100P
S100 calcium-binding protein P
4p16
0.000145
Down
Up


37.
NM_000165
GJA1
Gap junction protein, alpha 1, 43 kD
6q21-q23.2
0.000145
Down
Up





(connexin 43)


38.
NM_003897
DIF2, IEX1, PRG1
Immediate early response 3
6p21.3
0.000145
Down
Up


39.
BC001388
ANX2, LIP2, CAL1H
Annexin A2
15q21-q22
0.000145
Down
Up


40.
BC003070
HDR, MGC5445
GATA-binding protein 3
10p15
0.000145
Down
Up


41.
NM_020139
LOC56898
Oxidoreductase UCPA
4
0.000145
Down
Up


42.
AK002207
KIAA0610
KIAA0610 protein
13
0.000145
Down
Up


43.
NM_000574
CR, TC, CD55
Decay accelerating factor for
1q32
0.000145
Down
Up





complement


44.
NM_006926
SP-A2, COLEC5
Surfactant, pulmonary-associated
10q22-q23
0.000145
Up
Down





protein A2


45.
U37546
API2, MIHC, CIAP2
Baculoviral IAP repeat-containing 3
11q22
0.000145
Down
Up


46.
AU148057
DKK3
H. sapiens cDNA clone
11pter-p15.5
0.000145
Down
Up





MAMMA1002489


47.
NM_002600
DPDE4, PDEIVB
Phosphodiesterase 4B, cAMP-specific
1p31
0.000145
Down
Up


48.
S59049
BL34, IER1, IR20
Regulator of G-protein signalling 1
1q31
0.0003
Down
Up


49.
NM_001275
CGA, CgA
Chromogranin A (parathyroid secretory
14q32
0.0003
Down
Up





protein 1)


50.
AL575509
ETS2
H. sapiens cDNA clone CS0DI059YP21 =
21q22.2
0.0003
Down
Up
















TABLE 2







The first 50 genes from the supervised MDS analysis of tumor over benign (T/B) tissues ratio (Fold Change)


of the high risk versus moderate risk CaP patients, ranked by p-value.: (T/B Fold Change in HR vs MR)













Genbank






No
accession
Common Name of Genes
Description of Genes
Map
p-Value















1.
NM_004522
KINN, NKHC, NKHC2, NKHC-2
Kinesin family member 5C
2q23.3
0.00011


2.
J03198
GNAI3
Guanine nucleotide binding protein G (K), alpha subunit
1p13
0.000981


3.
NM_018010
HIPPI, FLJ10147
Hypothetical protein FLJ10147
3q13.13
0.003257


4.
NM_005479
FRAT1
Frequently rearranged in advanced T-cell lymphomas
10q23.33
0.004964


5.
NM_021795
SAP1
ELK4, ETS-domain protein (SRF accessory protein 1)
1q32
0.004964


6.
NM_003113
LEU5, RFP2
Nuclear antigen Sp100
2q37.1
0.004964


7.
NM_002053
GBP1
Guanylate binding protein 1, interferon-inducible, 67 kD
1p22.1
0.004964


8.
AF064092
GSA, GSP, GPSA, GNAS1,
Guanine nucleotide regulatory protein
20q13.2-q13.3
0.007579


9.
BC003070
HDR, MGC5445
GATA-binding protein 3
10p15
0.007579


10.
NM_012245
SKIP, NCOA-62
SKI-interacting protein
14q24.3
0.007579


11.
NM_015895
LOC51053
Geminin
6p22.2
0.007579


12.
AA083478
TRIM22
Stimulated trans-acting factor (50 kDa)
11
0.007579


13.
NM_000100
PME, CST6, EPM1, STFB
Cystatin B (stefin B)
21q22.3
0.007579


14.
NM_003031
SIAH1
Seven in absentia (Drosophila) homolog 1
16q12
0.007579


15.
NM_003407
TTP, GOS24, TIS11, NUP475
Zinc finger protein 36, C3H type, homolog (mouse)
19q13.1
0.007579


16.
BF979419
ESTs
ESTs, Highly similar to 60S ribosomal protein 13A
19q13.33
0.007579





[H. sapiens]


17.
NM_021038
MBNL
Muscleblind (Drosophila)-like
3q25
0.007579


18.
NM_014454
PA26
P53 regulated PA26 nuclear protein
6q21
0.007579


19.
BC004399
DEME-6
DEME-6 protein
1p32.3
0.007579


20.
NM_018490
LGR4
G protein-coupled receptor 48
11p14-p13
0.007579


21.
NM_004328
BCS, BCS1, h-BCS, Hs.6719
BCS1 (yeast homolog)-like
2q33
0.007579


22.
D87445
KIAA0256
KIAA0256 gene product
15
0.007579


23.
NM_006326
NIFIE14
Homo sapiens seven transmembrane domain protein,
19q13.12
0.007579





mRNA


24.
D83077
TTC3
Tetratricopeptide repeat domain 3
Xq13.1
0.007579


25.
NM_006732
GOS3
FBJ murine osteosarcoma viral oncogene homolog B
19q13.32
0.007579


26.
NM_003760
EIF4G3
Eukaryotic translation initiation factor 4 gamma, 3
1pter-p36.13
0.007579


27.
NM_004905
AOP2
Anti-oxidant protein 2
1q24.1
0.01159


28.
NM_018439
IMPACT
Hypothetical protein IMPACT
18
0.01159


29.
BC000629
DARS
Aspartyl-tRNA synthetase
2q21.2
0.01159


30.
AK002064
DKFZP564A2416
DKFZP564A2416 protein
2
0.01159


31.
NM_013387
HSPC051
Ubiquinol-cytochrome c reductase complex (7.2 kD)
22
0.01159


32.
AA135522
KIAA0089
Homo sapiens KIAA0089 mRNA sequence.
3
0.01159


33.
NM_015545
KIAA0632
KIAA0632 protein
7q22.1
0.01159


34.
NM_005767
P2Y5
Purinergic receptor (family A group 5)
13q14
0.01159


35.
BC003682
G25K, CDC42Hs
Cell division cycle 42 (GTP-binding protein, 25 kD)
1p36.1
0.01159


36.
NM_005053
RAD23A
RAD23 (S. cerevisiae) homolog A
19p13.2
0.017805


37.
AI672541
IPW
Human non-translated mRNA sequence.
15q11-q12
0.017805


38.
AK023938
H. sapiens cDNA FLJ13876 clone
SELECTED MODEL ORGANISM PROTEIN
2q37.3
0.017805





SIMILARITIES


39.
NM_000062
C1IN, C1NH, C1-INH
Serine (or cysteine) proteinase inhibitor, clade G (C1
11q12-q13.1
0.017805





inhibitor)


40.
AA576961
PHLDA1
Pleckstrin homology-like domain, family A, member 1
12q15
0.017805


41.
AI796269
NBS1, ATV, NIBRIN
H. sapiens cDNA similar to Cell Cycle Regulatory Protein
8q21-q24
0.017805





P95.


42.
NM_000016
ACADM
Acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight
1p31
0.017805





chain


43.
AI867102
KIAA0906, NUP210, gp210
Nuclear pore membrane glycoprotein 210
3p25.2-p25.1
0.017805


44.
AI263909
ARHB, RHOB, RHOH6
Oncogene RHO6; Aplysia RAS-related homolog 6
2pter-p12
0.017805


45.
NM_016021
NCUBE1
Non-canonical ubquitin conjugating enzyme 1
6
0.017805


46.
NM_012192
TIM9B, TIM10B
Fracture callus 1 (rat) homolog
11p15.5-p15.3
0.017805


47.
NM_025087
FLJ21511
Hypothetical protein FLJ21511
4
0.017805


48.
NM_014959
CARD8, CARDINAL,
Tumor up-regulated CARD-containing antagonist of
19q13.33
0.017805




KIAA0955
caspase 9


49.
AA923354
MAOA
Monoamine oxidase A.
Xp11.4-p11.3
0.017805


50.
NM_021964
ZNF148
Zinc finger protein 148 (pHZ-52)
3q21
0.017805


51.
NM_001674
ATF3
Activating transcription factor 3
1q32.3
0.017805
















TABLE 3







The first 50 genes obtained from the supervised MDS analysis of tumor versus benign tissues of all the


high risk CaP patients, ranked by p-value. (T vs N Intensities of 9 HR)












Common
Expression



GenBank
Name of
Regulation














No.
Accession
Genes
Description of Genes
Map
p-Value
Tumor
Benign

















1.
U65585
HLA-DR1B
Major histocompatibility complex, class II,
6p21.3
0.00002
Down
Up





DR beta 1


2.
NM_002053
GBP1
Guanylate binding protein 1, interferon-inducible,
1p22.1
0.000076
Down
Up


3.
NM_021983
HLA-DRB4
Major histocompatibility complex, class II,
6
0.000076
Down
Up





DR beta 4


4.
AI424243
CEGP1
Homo sapiens cDNA clone IMAGE: 2094442
11
0.000102
Down
Up


5.
NM_002343
LTF
Lactotransferrin
3q21-q23
0.000138
Down
Up


6.
NM_014575
SCHIP-1
Schwannomin-interacting protein 1
3q26.1
0.000257
Down
Up


7.
BC001169
ESD
Esterase D/formylglutathione hydrolase
13q14.1-q14.2
0.000357
Up
Down


8.
BF970427
UGCG
UDP-glucose ceramide glucosyltransferase
9
0.000357
Down
Up


9.
NM_002275
KRT15
Keratin 15
17q21
0.000495
Down
Up


10.
AU148057
DKK3
H. sapiens cDNA clone MAMMA1002489
11pter-p15.5
0.000495
Down
Up


11.
AI922538
TMEM1
Transmembrane protein 1
21
0.000689
Down
Up


12.
NM_004481
GALNAC-T2
UDP-GalNAc transferase 2
1q41-q42
0.000689
Down
Up


13.
BC003070
HDR,
GATA-binding protein 3
10p15
0.00097
Down
Up




MGC5445


14.
BF979419
ESTs, similar
H. sapiens 60S Ribosomal protein L13A

0.00097
Up
Down




to RPL13A


15.
BG054844
ARHE
H. sapiens cDNA clone IMAGE: 3441573
2q23.3
0.00097
Down
Up


16.
L42024
HLA-B
Major histocompatibility complex, class I, B
6p21.3
0.00138
Down
Up


17.
AL545982
CCT2
H. sapiens cDNA clone CS0DI023YD15
12q15
0.001992
Up
Down


18.
NM_001993
TF, TFA,
Coagulation factor III (thromboplastin, tissue factor)
1p22-p21
0.001992
Up
Down




CD142


19.
NM_004198
CHRNA6
Cholinergic receptor, nicotinic, alpha polypeptide 6
8p11.1
0.001992
Down
Up


20.
AV711904
LYZ
Lysozyme (renal amyloidosis)
12q15
0.001992
Down
Up


21.
NM_013387
HSPC051
Ubiquinol-cytochrome c reductase complex (7.2 kD)
22
0.001992
Up
Down


22.
AW514210
HLA-F
HLA CLASS I HISTOCOMPATIBILITY
6p21.3
0.001992
Down
Up





ANTIGEN, F A


23.
NM_005032
PLS3
Plastin 3 (T isoform)
Xq24
0.002894
Down
Up


24.
NM_003407
TTP, GOS24,
Zinc finger protein 36, C3H type, homolog (mouse)
19q13.1
0.002894
Down
Up




NUP475


25.
NM_000165
GJA1
Gap junction protein, alpha 1, 43 kD (connexin 43)
6q21-q23.2
0.002894
Down
Up


26.
AF275945
EVA1
Epithelial V-like antigen 1
11q23.3
0.002894
Down
Up


27.
NM_002450
MT1
Metallothionein 1L
16q13
0.002894
Down
Up


28.
NM_005950
MT1
Metallothionein 1G
16q13
0.002894
Down
Up


29.
NM_006994
BTN3A3
Butyrophilin, subfamily 3, member A3
6p21.33
0.002894
Down
Up


30.
AI049962
KIAA0191
H. sapiens cDNA clone IMAGE: 1700970
1
0.002894
Down
Up


31.
X99268
TWIST
Twist (Drosophila) homolog
7p21.2
0.002894
Up
Down


32.
NM_016021
NCUBE1
Non-canonical ubquitin conjugating enzyme 1
6
0.002894
Up
Down


33.
NM_016205
SCDGF
Platelet derived growth factor C
4q32
0.002894
Up
Down


34.
AI681120
RANBP2
H. sapiens cDNA clone IMAGE: 2272403
2q11-q13
0.004205
Up
Down


35.
NM_000574
CR, TC, CD55
Decay accelerating factor for complement
1q32
0.004205
Down
Up


36.
NM_014937
KIAA0966
Sac domain-containing inositol phosphatase 2
10q26.13
0.004205
Down
Up


37.
NM_005213
STF1, STFA
Cystatin A (stefin A)
3q21
0.004205
Down
Up


38.
NM_005952
MT1
Metallothionein 1X
16q13
0.004205
Down
Up


39.
AF130095
FN1
Fibronectin 1
2q34
0.004205
Down
Up


40.
BE568219
PDE8A
H. sapiens cDNA clone IMAGE: 3683966
15q25.1
0.004205
Up
Down


41.
D50925
STK37,
PAS-serine/threonine kinase
2q37.3
0.004205
Down
Up




PASKIN,


42.
NM_006113
VAV3
Vav 3 oncogene
1p13.1
0.004205
Down
Up


43.
NM_001018
RPS15
Ribosomal protein S15
19p13.3
0.006189
Up
Down


44.
NM_021038
MBNL
Muscleblind (Drosophila)-like
3q25
0.006189
Down
Up


45.
NM_012323
U-MAF
V-maf musculoaponeurotic fibrosarcoma, protein F
22q13.1
0.006189
Down
Up


46.
NM_005138
SCO1L
SCO (cytochrome oxidase deficient, yeast)
22q13.33
0.006189
Down
Up





homolog 2


47.
AF186779
KIAA0959
RalGDS-like gene
1q25.2
0.006189
Down
Up


48.
D26054
FBP
Fructose-1,6-bisphosphatase 1
9q22.3
0.006189
Up
Down


49.
U37546
API2, MIHC,
Baculoviral IAP repeat-containing 3
11q22
0.006189
Down
Up




HIAP1


50.
AB046845
SMURF1
E3 ubiquitin ligase SMURF1
7q21.1-q31.1
0.006189
Down
Up
















TABLE 4







The first 50 genes obtained from the supervised MDS analysis of tumor versus benign tissues of all the


moderate risk CaP patients, ranked by p-value: (T vs N Intensities of 9 MR)













Expression



Genbank
Common Name
Regulation














No.
Accession
of Genes
Description of Genes
Map
p-Value
Tumor
Benign

















1.
NM_014324
AMACR
Alpha-methylacyl-CoA racemase
5p13.2-q11.1
0
Up
Down


2.
NM_006457
ENH
LIM protein (similar to rat protein kinase C-binding
4q22
0.000009
Up
Down





enigma)


3.
AI351043
ESTs
H. sapiens cDNA clone IMAGE: 1948310
21
0.000011
Up
Down


4.
AI433463
MME
H. sapiens cDNA clone similar to NEPRILYSIN
3q25.1-q25.2
0.000028
Down
Up





(HUMAN)


5.
BE256479
HSPD1
H. sapiens cDNA clone IMAGE: 3352031
12p13.31
0.000037
Up
Down


6.
NM_015900
PS-PLA1
Phosphatidylserine-specific phospholipase A1alpha
3q13.13-q13.2
0.000083
Up
Down


7.
NM_002343
LTF
Lactotransferrin
3q21-q23
0.000083
Down
Up


8.
NM_001099
PAP
Acid phosphatase, prostate
3q21-q23
0.000083
Down
Up


9.
T15991
CHRM3
IB2413 Infant brain, Bento Soares Homo sapiens cDNA
1q41-q44
0.00011
Up
Down


10.
NM_005084
PAFAH
Phospholipase A2, group VII
6p21.2-p12
0.00011
Up
Down


11.
NM_004503
HOXC6
Homeo box C6
12q12-q13
0.00011
Up
Down


12.
N74607
AQP3
H. sapiens cDNA clone IMAGE: 296424
9p13
0.000149
Down
Up


13.
BC003068
SLC19A1
Solute carrier family 19 (folate transporter), member 1
21q22.3
0.000149
Up
Down


14.
M21535
ERG (ets-related
ERG v-ets erythroblastosis virus E26 oncogene like (avian)
21q22.3
0.000149
Up
Down




gene)



15.
NM_013451
MYOF,
Fer-1 (C. elegans)-like 3 (myoferlin)
10q24
0.0002
Down
Up


16.
NM_006017
AC133, CD133
Prominin (mouse)-like 1
4p15.33
0.0002
Down
Up


17.
BE550599
CACNA1D
H. sapiens cDNA clone IMAGE: 3220210
3p14.3
0.0002
Up
Down


18.
U22178
PSP57, PSP94
Microseminoprotein, beta-
10q11.2
0.0002
Down
Up


19.
NM_015865
JK, UT1, UTE
Solute carrier family 14 (urea transporter), member 1
18q11-q12
0.000275
Down
Up


20.
NM_000441
PDS, DFNB4
Solute carrier family 26, member 4
7q31
0.000275
Down
Up


21.
AA877789
MYO6
H. sapiens cDNA clone IMAGE: 1161091
6q13
0.000275
Up
Down


22.
AI356398
ZFP36L2
H. sapiens cDNA clone IMAGE: 2028039
2
0.000275
Down
Up


23.
BC000915
CLIM1, CLP36
PDZ and LIM domain 1 (elfin)
10q22-q26.3
0.000275
Down
Up


24.
NM_000286
PEX12
Peroxisomal biogenesis factor 12
17q11.2
0.000275
Up
Down


25.
NM_003671
CDC14B1,
Homo sapiens CDC14 cell division cycle 14 homolog B
9q22.2-q22.31
0.000386
Down
Up




CDC14B2,
(S. cerevisiae) (CDC14B), transcript variant 1, mRNA


26.
NM_016545
SBB148
Immediate early response 5
1q24.3
0.000386
Down
Up


27.
NM_002443
PSP57, PSP94
Microseminoprotein, beta-
10q11.2
0.000386
Down
Up


28.
NM_004999
DFNA22
Myosin VI
6q13
0.000386
Up
Down


29.
X99268
TWIST
Twist (Drosophila) homolog
7p21.2
0.000386
Up
Down


30.
NM_023009
MACMARCKS
Macrophage myristoylated alanine-rich C kinase
1p34.3
0.000386
Up
Down





substrate


31.
AI721219
TRAF3
as68b11.x1 Barstead colon HPLRB7 Homo sapiens
14q32.33
0.000547
Down
Up





cDNA clone IMAGE: 2333853 3′, mRNA sequence.


32.
NM_001584
D11S302E
Chromosome 11 open reading frame 8
11p13
0.000547
Down
Up


33.
NM_018846
SBBI26
SBBI26 protein
7p15.3
0.000547
Up
Down


34.
M87771
BEK, KGFR,
Fibroblast growth factor receptor 2
10q26
0.000547
Down
Up


35.
AF275945
EVA1
Epithelial V-like antigen 1
11q23.3
0.000547
Down
Up


36.
AI791860
ESTs
H. sapiens cDNA clone 1MAGE: 1011110

0.000547
Up
Down


37.
BC001282
NHC
High-mobility group (nonhistone chromosomal) protein
6p21.3
0.000547
Down
Up





17-like 3


38.
NM_002015
FKH1, FKHR
Furkhead box O1A (rhabdomyosarcoma)
13q14.1
0.000547
Down
Up


39.
X15306
NF-H
H. sapiens NF-H gene, exon 1 (and joined CDS).
22q12.2
0.000547
Down
Up


40.
BE965029
EST
H. sapiens cDNA clone IMAGE: 3886131
11
0.000775
Up
Down


41.
NM_002275
KRT15
Keratin 15
17q21
0.000775
Down
Up


42.
NM_001511
MGSA, CXCL1
GRO1 oncogene
4q21
0.000775
Down
Up


43.
NM_005213
STF1, STFA
Cystatin A (stefin A)
3q21
0.000775
Down
Up


44.
NM_007191
WIF-1
Wnt inhibitory factor-1
12q14.2
0.000775
Down
Up


45.
H15129
MEIS3
EPIDERMAL GROWTH FACTOR-LIKE CRIPTO
17
0.000775
Down
Up





PROTEIN


46.
AW452623
EST
H. sapiens cDNA clone IMAGE: 3068608
13
0.000775
Up
Down


47.
X90579
EST
H. sapiens DNA for cyp related pseudogene
7
0.000775
Down
Up


48.
BC001388
ANX2,
Annexin A2
15q21-q22
0.001116
Down
Up




ANX2L4


49.
NM_014863
BRAG,
B cell RAG associated protein
10q26
0.001116
Down
Up


50.
NM_021076
NEFH
Neurofilament, heavy polypeptide (200 kD)
22q12.2
0.001116
Down
Up
















TABLE 5







Top 50 Unregulated Genes in All the 18 Samples (HR and MR) obtained from Tumor over Benign (T/B) ratio.













Genbank
T/N
Common Name




No
ID
ratio
of Genes
Description
Map















1.
AF047020
39.86910
AMACR
Alpha-methylacyl-CoA racemase
5p13.2-q11.1


2.
M54886
20.86411
LOC51334
Mesenchymal stem cell protein DSC54
5p13.1


3.
AF070581
19.07263
ESTs
Homo sapiens cDNA clone IMAGE: 1948310
21


4.
NM_014324
18.04841
TRG@
T cell receptor gamma locus
7p15-p14


5.
NM_001669
15.98177
NPY
Neuropeptide Y
7p15.1


6.
NM_018360
13.34037
HOXC6
Homeo box C6
12q12-q13


7.
AF092132
9.588665
IMPD2
IMP (inosine monophosphate) dehydrogenase 2
3p21.2


8.
NM_023067
7.712272
HSPC028
HSPC028 protein
7p21.2


9.
NM_014439
7.031155
LTBP1
Latent transforming growth factor beta binding protein 1
2p22-p21


10.
AI613045
6.739595
GMF
Glia maturation factor, beta
14q22.1


11.
AB051446
6.563991
DSC2
HUMAN Q02487 DESMOCOLLIN 2A/2B PRECURSOR
18q12.1


12.
NM_005342
6.442383
TRG, TCRG
T cell receptor gamma locus
7p15-p14


13.
D87012
6.327042
PAWR
H. sapiens cDNA clone IMAGE: 1950862
12q21


14.
NM_018221
6.098105
SNX2
Sorting nexin 2
5q23


15.
NM_005114
5.769173
HS3ST1
Heparan sulfate (glucosamine)-3-O-sulfotransferase 1
11


16.
NM_022831
5.624385
RA70, SAPS, SKAP55R
Src family associated phosphoprotein 2
7p21-p15


17.
NM_014324
5.621786
TRG, TCRG
T cell receptor gamma locus
7p15-p14


18.
NM_006820
5.550019
BICD1
Bicaudal D (Drosophila) homolog 1
12p11.2-p11.1


19.
NM_005574
5.454622
FOLH1
Folate hydrolase (prostate-specific membrane antigen) 1
11p11.2


20.
AL365343
5.451875
KIAA0615
Homo sapiens mRNA for KIAA0615 protein, complete cds.
16q11.2


21.
NM_022580
5.318270
TBCE
Tubulin-specific chaperone e
1q42.3


22.
AK022765
5.315669
CLDN8
Claudin 8
21


23.
AF067173
5.272626
P21, NSG1, D4S234
Neuron-specific protein
4p16.3


24.
NM_006220
5.180025
SHMT2
Homo sapiens cDNA clone IMAGE: 2676158
12q12-q14


25.
AL133600
5.146792
ANK2
Homo sapiens cDNA clone by03a08
4q25-q27


26.
AY009108
5.097967
PSM
PROSTATE-SPECIFIC MEMBRANE ANTIGEN
2






(HUMAN)


27.
AL035603
5.076761
FLJ10907
Ribonuclease 6 precursor
6q27


28.
NM_014017
5.058610
MAPBPIP
Mitogen-activated protein-binding protein-interacting
13






protein


29.
BF247098
5.030722
PHLP,
Phosducin-like
9q12-q13





DKFZp564M1863


30.
U62296
4.992345
GOLPH2
Golgi phosphoprotein 2
9


31.
AF130082
4.988912
EST
Homo sapiens clone FLC1492 PRO3121 mRNA, complete






cds


32.
NM_020373
4.969535
C8orf4
Chromosome 8 open reading frame 4
8


33.
U90030
4.873056
BICD1
Bicaudal D homolog 1 (Drosophila)
6


34.
NM_021071
4.821960
KIAA0426
KIAA0426 gene product
6p22.2-p21.3


35.
NM_030817
4.753895
KIAA1157
KIAA1157 protein
12q13.3-q14.1


36.
NM_019844
4.700642
HPRT, HGPRT
Hypoxanthine phosphoribosyltransferase 1
Xq26.1


37.
NM_004721
4.689246
RPL29
Ribosomal protein L29
3p21.3-p21.2


38.
NM_004866
4.669274
EF2, EEF-2
Eukaryotic translation elongation factor 2
19pter-q12


39.
NM_014501
4.610132
BGN
Biglycan
Xq28


40.
NM_020655
4.575193
SDC2
Syndecan 2 (heparan sulfate proteoglycan 1, fibroglycan)
8q22-q23


41.
NM_006716
4.557526
ASK
Activator of S phase kinase
19p13.11


42.
NM_002968
4.541752
FOLH1
Folate hydrolase (prostate-specific membrane antigen) 1
11q14.3


43.
X06268
4.539479
NCUBE1
Non-canonical ubquitin conjugating enzyme 1
6


44.
AK021609
4.520464
PTH2, PTEN2,
Phosphatase and tensin homolog (mutated in multiple
9p21





PSIPTEN
advanced cancers 1), pseudogene 1


45.
NM_001133
4.479513
TCTEX1L
T-complex-associated-testis-expressed 1-like
Xp21


46.
D38491
4.477160
KIAA0461, POGZ,
Pogo transposable element with ZNF domain, KIAA0461
1q21.2






protein


47.
NM_006426
4.385531
DDX26
Deleted in cancer 1; RNA helicase HDB/DICE1
13q14.12-







q14.2


48.
AW058148
4.347362
SPHAR
S-phase response (cyclin-related)
1q42.11-q42.3


49.
U55209
4.293919
MYO7A
myosin VIIA (Usher syndrome 1B)
4


50.
NM_004610
4.275521
KIAA0634, ASTN2
Astrotactin 2
9q33.1
















TABLE 6







Top 35 Downregulated Genes in All the 18 Samples (HR and MR) obtained from Tumor over Benign (T/B) ratio.













Genbank
T/N
Common Name




No.
ID
Ratio
of the Genes
Description
Map















1.
X90579
0.181138
CYP3A5
Cytochrome P450, family 3, subfamily A,
7






polypeptide 5


2.
NM_005213
0.198502
STF1, STFA
Cystatin A (stefin A)
3q21


3.
NM_005864
0.254524
EFS1, HEFS
Signal transduction protein (SH3 containing)
14q11.2-q12


4.
X15306
0.291665
NF-H
H. sapiens NF-H gene, exon 1 (and joined CDS).
22q12.2


5.
BE908217
0.319347
ANXA2
Annexin A2
15q21-q22


6.
BC001388
0.320110
ANX2, LIP2, ANX2L4
Annexin A2
15q21-q22


7.
U22178
0.326560
PSP57, PSP94, PSP-94
Microseminoprotein, beta-
10q11.2


8.
NM_002443
0.338948
PSP57, PSP94, PSP-94
Microseminoprotein, beta-
10q11.2


9.
NM_021076
0.359039
NEFH
Neurofilament, heavy polypeptide (200 kD)
22q12.2


10.
AI433463
0.360636
MME, CD10, NEP,
Neprilysin
3q25.1-q25.2





CALLA


11.
AF275945
0.366939
EVA1
Epithelial V-like antigen 1
11q23.3


12.
NM_002343
0.370305
LTF
Lactotransferrin
3q21-q23


13.
NM_013451
0.378555
MYOF, KIAA1207
Fer-1 (C. elegans)-like 3 (myoferlin)
10q24


14.
NM_001584
0.385272
239FB, D11S302E
Chromosome 11 open reading frame 8
11p13


15.
AL390736
0.391520
BA209J19.1, GW112
GW112(differentially expressed in hematopoietic






lineages)


16.
NM_000441
0.392117
PDS, DFNB4
Solute carrier family 26, member 4
7q31


17.
AL031602
0.399115
ESTs
ESTs
1p34.1-35.3


18.
NM_004039
0.399796
ANXA2
Annexin A2
15q21-q22


19.
NM_001546
0.402261
ID4
DNA binding inhibitor protein of ID-4
6p22-p21


20.
NM_001099
0.406234
PAP
Acid phosphatase, prostate
3q21-q23


21.
X57348
0.422692
9112
H. sapiens mRNA (clone 9112).
1p35.2


22.
NM_020139
0.440648
LOC56898
Oxidoreductase UCPA
4


23.
AU148057
0.444528
DKK3, REIC
Dickkopf related protein-3 precursor (Dkk-3)
11pter-p15.5






(Dickkopf-3) (hDkk-3)


24.
BF059159
0.446108
ROBO1, DUTT1, SAX3
Roundabout, axon guidance receptor, homolog 1
3p12






(Drosophila)


25.
BC001120
0.448109
MAC2, GALBP, MAC-2,
Lectin, galactoside-binding, soluble, 3 (galectin 3)
14q21-q22


26.
N74607
0.451123
AQP3
Aquqporin 3
9p13


27.
NM_013281
0.454835
FLRT3
Fibronectin leucine rich transmembrane protein 3
20p11


28.
NM_000700
0.456566
ANX1, LPC1
Annexin A1
9q12-q21.2


29.
X57348
0.458169
9112
H. sapiens mRNA (clone 9112).
1p35.2


30.
AI356398
0.467028
ZFP36L2, ERF-2, TIS11D
EGF-respons factor 2
2


31.
AF016266
0.467787
DR5, TRAILR2,
Tumor necrosis factor receptor superfamily, member
8p22-p21





TRICK2A,
10b


32.
S59049
0.467913
BL34, IER1, IR20
Regulator of G-protein signalling 1
1q31


33.
NM_000165
0.470393
GJA1
Gap junction protein, alpha 1, 43 kD (connexin 43)
6q21-q23.2


34.
AI826799
0.471081
EFEMP1, DRAD, FBNL
EGF-CONTAINING FIBULIN-LIKE EXTRACELLULAR
2p16






MATRIX PROTEIN 1


35.
AL575509
0.476538
ETS2
V-ets erythroblastosis virus E26 oncogene homolog 2
21q22.2






(avian)









Classification between Tumor and Benign Prostate Epithelium:

A class prediction analysis using distance based Multi Dimensional Scaling (MDS) was used to determine expression differences between tumor and benign epithelial cells in 18 patients with radical prostatectomy. All the genes that meet a minimum level of expression were included in the analysis. We used the normalized intensities of all the 18 tumor and 18 normal samples for a class prediction analysis by distance based MDS to determine differentiation between tumor and benign tissue specific gene expression profile among all the 18 patients. Using a matrix of Pearson correlation coefficients from the complete pair-wise comparison of all the experiments we observed a significant overall difference in gene expression pattern between the tumor and benign tissue as displayed as a two-dimensional MDS plot in FIG. 2A. The position of the each tumor and benign samples is displayed in the MDS plot in two-dimensional Euclidean space with the distance among the samples reflecting correlation among the samples in each individual group (distance within the cluster) and as well as reflecting distinct separation between the two groups (center-to center distance) (FIG. 2A). The MDS plot was obtained from the top 200 genes obtained by 10,000 permutations of the tumor and benign intensities of 4566 genes. Out of these 200 genes that define the tumor specific alteration of gene expression, 53 genes had higher expression in the tumor samples and the remaining 147 genes had higher expression in the benign samples. A partial list of genes that distinctly discriminate the tumor and benign samples from all the 18 patients is shown in Table 1. We also performed a hierarchical clustering analysis using the 200 discriminatory genes. The hierarchical clustering algorithm resulted in a hierarchical dendrogram that identified two major distinct clusters of 16 tumor samples and 17 benign samples (FIG. 2B).
Classification of CaP into HR and MR Groups Using the Ratio of Tumor over Benign Gene Expression Intensities
We used the tumor over benign gene expression intensity ratio (T/B ratio) (FIG. 3A) from the HR (9 patients) and MR (9 patients) groups for a class prediction analysis using distance based MDS method to determine if the 18 patients can be differentiated into the two patient groups. Pathological and clinical features of the 18 tumors used in our study were clearly distinguishable between the HR and MR groups. We observed a significant overall difference in expression pattern between the HR and MR groups. The distance between the samples reflects both the extent of correlation within each individual selected group (distance within the cluster) as well as distinct separation between the two selected groups (center-to-center distance) (FIG. 3A). The MDS plot obtained from top 200 genes by 10,000 permutations of the 4868 genes based on the T/B ratio is shown in FIG. 3A. Out of the top 200 genes of the MDS analysis 135 were over expressed in the HR group and 65 genes were over expressed in the MR group, The top 50 genes with best p-values identified by the T/B ratio based MDS analysis discriminating the HR and MR groups are listed in FIG. 3B. The approach we used for the interpretation of discrimination between the HR and MR groups was empirical. The ‘weighted list’ (FIG. 3B) of individual genes whose variance of change across all the tumor samples defines the boundary of a given cluster to predict a class that correlates with the pathological and clinical features of CaP. We also performed a hierarchical clustering to verify the results of the MDS analysis and also to test the potential of those 200 genes to predict class/group (HR and or MR) using another approach of analysis. The resulting hierarchical dendrogram of TB ratio demonstrates that 9 samples of the HR group formed a very distinct and tight cluster, as did the 9 samples of MR group (FIG. 3B).
Classification of CaP into HR and MR groups based on Gene Expression Intensities in Tumor Cells
MDS analysis was used to determine differentiation among 18 patients into HR and MR groups. An overall difference in tumor specific expression between the HR and MR groups is displayed as a two-dimensional MDS plot (FIG. 3C). The MDS plot obtained from 10,000 permutations of the gene expression intensities of 4115 genes from the tumor samples of 18 patients differentiated them into HR and MR groups based on the selected top 200 genes (FIG. 3C). Out of this 200 genes, 94 had higher expression in the HR groups and the remaining 106 genes had higher expression in the MR groups. We performed a hierarchical clustering analysis using the 200 discriminatory genes obtained from the supervised MDS analysis. The resulting hierarchical dendrogram of 18 tumor samples demonstrates that 9 tumor samples of the HR group and 9 tumor samples of the MR group were separated into two tight clusters. (FIG. 3D). The approach we utilized on the basis of the linear correlation of global gene expression in FIG. 3 to obtain ‘gene cluster’ interpretation to discriminate the HR and MR groups was empirical. Genes that discriminate the HR and MR groups are shown in Table 7.







TABLE 7







Top 17 genes analysis based on T/B fold change of HR vs MR groups

















Gene











Bank ID
Common Name
Description
Map
p-Value
HR
MR
Absent
Positive




















1
NM_004522
KINN, NKIIC
Kinesin family member 5C
2q23.3
0.0001
Up
Down
4
60%


3
NM_018010
HIPPI, FLJ10
Hypothetical protein FLJ10147
3q13.13
0.0033
Up
Down
0
56%


10
NM_012245
SKIP, NCOA-
SKI-interacting protein
14q24.3
0.0076
Up
Down
2
42.80%  


11
NM_015895
LOC51053
Geminin
6p22.2
0.0076
Up
Down
2
71.40%  


14
NM_003031
SIAH1
Seven in absentia (Drosophila) homolog 1
16q12
0.0076
Up
Down
3
66%


42
NM_000016
ACADM
Acyl-Coenzyme A dehydrogenase, C-4 to C-12
1p31
0.0178
Up
Down
1
75%





straight


47
NM_025087
FLJ21511
Hypothetical protein FLJ21511
4
0.0178
Up
Down
1
50%


17
NM_021038
MBNL
Muscleblind (Drosophila)-like
3q25
0.0076
Down
Up
2
71


25
NM_006732
GOS3
FBJ murine osteosarcoma viral oncogene
19q13.32
0.0076
Down
Up
3
83%





homolog B


51
NM_001674
ATF3
Activating transcription factor 3
1q32.3
0.0178
Down
Up
0
100% 


7
NM_002053
GBP1
Guanylate binding protein 1, interferon-inducible,
1p22.1
0.005
Down
Up
4
83%





67 KD


15
NM_003407
TTP, GOS24
Zinc finger protein 36, C3H type, homolog (mouse)
19q13.1
0.0076
Down
Up
1
62%


26
NM_003760
EIF4G3
Eukaryotic translation initiation factor 4 gamma, 3
1pter-p3
0.0076
Down
Up
4
40%


38
AK023938
Homo sapien
SELECTED MODEL ORGANISM PROTEIN
2q37.3
0.0178
Down
Up
4
80%





SIMILARITIES


45
NM_016021
NCUBE1
Non-canonical ubquitin conjugating enzyme 1
6
0.0178
Down
Up ?
3
66%


5
NM_021795
SAP1
ELK4, ETS-domain protein (SRF accessory protein
1q32
0.005
Up
Down
4
80%





1)


18
NM_014454
PA26
P53 regulated PA26 nuclear protein
6q21
0.0076
Up
Down
3
83%









Classification of CaP into High Risk and Medium Risk Groups Based on Gene Expression Intensities in Benign Prostate Epithelial

We used a similar MDS and Cluster analysis as in the tumor versus tumor sample gene expression intensities for the normalized intensities of 9 benign samples of HR group and 9 benign samples of MR group for a class prediction. Strikingly the MDS plot of the benign samples depicted distinct separation between the HR and MR groups (FIG. 3E). We observed a significant overall difference in expression pattern between the HR and MR groups. The MDS plot obtained from the top 200 genes by 10,000 permutations of the 3358 genes from the benign versus benign intensities (FIG. 3E). Out of this 200 genes 61 were over expressed in benign samples of the HR groups and the remaining 139 genes were over expressed in the MR groups. The ‘weighted list’ of individual genes whose variance of expression alteration across all the normal samples depicts the capability of a given cluster to predict classification. The hierarchical clustering algorithm identified a similar major cluster of the 9 benign samples of the HR group and a cluster of 9 benign samples of the MR group.
The weighted gene analysis by distance based supervised multidimensional scaling method we used, (depicted in FIGS. 3A, 3C, and 3E) utilizing the gene expression ratio of tumor and benign intensities, gene expression intensities of tumor samples and as well as normal for obtaining a ‘weighted list’ of individual genes, whose variance of change across all the tumor and benign samples distinctly delineate the boundary of a given cluster, to predict a class that correlates with the pathological and clinical features of CaP.
Independent In silico Cross Validation
In silico analysis for the predicted classifier was carried out using two independent data sets. The HR and MR groups were selected on the basis of Gleason score as that was the only criterion available for these data. At least 200 genes were extracted from all the MDS analysis (see methods for detail description). This subset of 200 classifier genes were found in the data of Welsh et al. 2001 and Sing et al. 2002. Exactly similar MDS analysis (p<0.001 as measured by 10,000 permutation testing) as described above was performed using the expression intensities of these 200 genes from Welsh and Singh data. MDS analysis using tumor over benign ratio of as low as 50 genes from the subset of 200 genes from Welsh data (FIG. 4A) as well as Singh data (FIG. 4B) clearly separated samples from HR group and samples from MR group. Thus, this observation elucidates that the differential expression profile of this small set of genes can be used to predict the identity or class or group of unknown prostate cancer samples on the basis of their clinico-pathological features. The outcome of this analysis depicts that the expression profile of this small number of genes is conserved across the independent data sets.
Validation of GeneChip Results by Real-Time PCR
To further validate the expression alterations of genes identified by GeneChip analysis with an indicated biological relevance to prostate cancer, primers and probes were obtained for real-time PCR analysis using AMACR and GSTP1. These genes were chosen for validation purposes because it has been reported previously by several investigators that AMACR is elevated and GSTP1 decreased in CaP. Each sample demonstrated a unique pattern of down-regulation of GSTP1 gene in 18 of 20 samples as well as up-regulation of AMACR (FIG. 1) the other two samples did show significant change (fold change less than 1.5).
One ng of total RNA samples from paired tumor and normal specimens was reverse-transcripted using Omnisensecript RT-kit (Qiagene, Valencia, Calif.) according to the manufacturer's protocol.
Quantitative gene expression analysis was performed using TaqMan Master Mix Reagent and an ABI prism 7700 Sequence Detection System (PE Applied Biosystems Foster, Calif.). All sets of primer and probe for tested genes were Assays-on-Demand Gene expression products obtained from PE Applied Biosystems. The expression of house keeping gene, GAPDH was simultaneously analyzed as the endogenous control of same batch of cDNA, and the target gene expression of each sample was normalized to GAPDH. For each PCR run, a master-mix was prepared on ice with 1× TaqMan Master Mix, 1× target gene primer/probe and 1×GAPDH primer/probe. Two microliters of each diluted cDNA sample was added to 28 μl of PCR master-mix. The thermal cycling conditions comprised an initial denaturation step at 95° C. for 10 minutes and 50 cycles at 95° C. for 15 seconds and 60° C. for 1 minute. RNA samples without reverse transcription were included as the negative control in each assay. All assays were performed in duplicate. Results were plotted as average CT (threshold cycle) of duplicated samples. The relative gene expression level was presented as “Fold Change” of tumor versus matched normal cells, which is calculated as: Fold change=2(ΔCTnormal-ΔCTtumor), where ΔCT means normalized CT value of target genes to GAPDH.
Example 3
Distinguishing Between ERG1 and ERG2 Isoforms
The Affymetrix GeneChip probe set (213541_s_at) and TaqMan probes used in the experiments described above recognize a region specific for both ERG1 and ERG2 isoforms (FIG. 6), but exclude isoforms 3 to 9. Although other primers and probes could be used, by way of example, TaqMan primers and probe recognizing both ERG1 and ERG2, but not other ERG isoforms were as follows:









Fwd primer:



(SEQ ID NO: 7)



5′-AGAGAAACATTCAGGACCTCATCATTATG-3′



 



Reverse primer:



(SEQ ID NO: 8)



5′-GCAGCCAAGAAGGCCATCT-3′



 



Probe:



(SEQ ID NO: 9)



5′-TTGTTCTCCACAGGGT-3′






The probe has the reporter dye, 6-FAM, attached to the 5′ end and TAMRA attached to the 3′ end. The 3′-TAMRA effectively blocks extension during PCR.

To further distinguish between these two ERG isoforms, the expression of the ERG1 and ERG2 isoforms were tested in PC3 cells and in normal prostate tissue (pooled prostate RNA from 20 men, Clontech), as well as in microdissected tumor and normal prostate epithelial cells from 5 CaP patients (data not shown). Only ERG1 was expressed in the prostate cells and in PC3 cells. ERG2 expression was not detectable. A TaqMan QRT-PCR probe and primers were designed that specifically recognize only the ERG1 isoform (FIG. 6). Although other primers and probes could be used, by way of example, we designed TaqMan primers and probes recognizing only the ERG1 isoform as follows:









Forward primer:



(SEQ ID NO: 10)



5′-CAGGTCCTTCTTGCCTCCC-3′



 



Reverse primer:



(SEQ ID NO: 11)



5′-TATGGAGGCTCCAATTGAAACC-3′



 



Probe:



(SEQ ID NO: 12)



5′-TGTCTTTTATTTCTAGCCCCTTTTGGAACAGGA-3′.






The probe has the reporter dye, 6-FAM, attached to the 5′ end and TAMRA attached to the 3′ end. The 3′-TAMRA effectively blocks extension during PCR.

ERG1 expression was determined in 228 RNA specimens from microdissected matched tumor and benign prostate epithelial cells of 114 CaP patients. Overall, 62.4% of the 114 CaP patients analyzed had significant over expression of ERG1 isoform in their tumor cells (i.e., greater than 2 fold ERG1 expression in tumor versus benign cells), while 16.6% of CaP patients had no detectable ERG1 expression, 15.0% had under expression of ERG1 (less than 0.5 fold difference in ERG1 expression in tumor versus benign cells), and 6.0% had no significant difference (0.5 to 2 fold difference in ERG1 expression between tumor versus benign cells).
In a further study, ERG expression was analyzed in 82 CaP patients. Using the TaqMan primers and probes discussed above, we observed tumor-associated over expression of ERG1 (isoform 1 only) and ERG (isoforms 1 and 2) in 63.4% and 72.0% of the patients, respectively. Therefore, ERG1 isoform specific expression may actually reflect an underestimate of the overall ERG expression in CaP.
Example 4
Correlation of ERG1 Expression With Various Clinico-Pathologic Features
Since the ERG1 tumor versus benign expression ratio data did not have normal distribution, the Wilcoxon Rank Sum Test was used to analyze its relationship with various clinico-pathologic features, as shown in Table 8.







TABLE 8







Relationship of ERG1 expression ratios in tumor versus benign


prostate epithelial cells with patient clinical factors













Median of
Mean scores of



Clinical

ERG1 fold
ERG1 fold


factors
N
changes
changes
P














PSA recurrence






No
75
142.2
52.19
0.0042


Yes
20
1.2
32.30


Tumor Differentiation



0.0020


Well & Moderate
40
362.3
57.62


Poor
54
13.9
40.00


Pathologic T stage



0.0136


pT2
38
502.0
53.45


pT3-4
52
33.5
39.69


Margin status



0.0209


Negative
64
197.0
52.55


Positive
31
20.4
38.61


Seminal vesicle



0.2555


Negative
82
106.7
49.28


Positive
13
6.9
39.92


Race



0.0086


Caucasian
73
172.1
52.08


African American
22
3.8
34.45


Family history



0.3887


No
70
106.7
49.46


Yes
25
4.8
43.92


Diagnostic PSA (ng/ml)


<=4
13
101.3
57.15
0.1801


>4-10
62
112.1
48.03


>10
19
20.5
39.16


Gleason sum



0.2923


<7
33
112.0
52.06


=7
45
118.1
47.16


>7
16
21.0
39.06









As shown in Table 8, 95 CaP patients with detectable ERG1 expression were analyzed by Wilcoxon rank sum test. N represents the number of CaP patients falling into the indicated clinical factor category. Significant p values (<0.05) are in bold face.
We also found a significant correlation of high ERG1 over expression with Caucasian over African American ethnicity (p=0.0086) (Table 8). To further explore the correlation with PSA recurrence, Kaplan-Meier survival analysis was performed based on three patient groups: 1) CaP patients with tumor versus benign ERG1 expression ratio of less than 2 fold; 2) CaP patients with tumor versus benign ERG1 expression ratio of 2-100 fold; and 3) CaP patients with tumor versus benign ERG1 expression ratio of greater than 100 fold (FIG. 7). The results show that patients with higher ERG1 over expression in their prostate tumor tissue had significantly longer PSA recurrence-free survival (log rank test, P=0.0006) (FIG. 7). The 36-months PSA recurrence-free survival for patients with less than 2 fold ERG1 expression ratio (n=24) was 54.4%, while for patients with greater than 100 fold ERG1 expression ratio (n=47) it was 87.7%. From a univariate COX proportional hazard ratio regression analysis for PSA recurrence-free time using ERG1 tumor/benign cells expression ratio, race, diagnostic PSA, Gleason sum, pathologic T stage, margin status, and seminal vesicle invasion status, we found that five of these variables (ERG1 tumor/benign cells expression ratio, Gleason sum, pathologic T stage, margin status, seminal vesicle invasion) had a significant p value (Table 9).







TABLE 9







Correlation of clinical parameters and ERG1 expression ratios


in tumor versus benign prostate epithelial cells with PSA


recurrence-free time after radical prostatectomy









Factors
Crude Hazard Ratio (95% CI)
P





ERG1 fold changes

0.0024


2-100 fold vs. <2 fold
0.291 (0.093-0.915)
0.0347


>100 fold vs. <2 fold
0.173 (0.060-0.498)
0.0011


Race


Caucasian vs. African American
1.092 (0.395-3.016)
0.8657


Diagnostic PSA

0.8723


>4-10 vs. <=4
0.976 (0.275-3.468)
0.9705


>10 vs. <=4
1.285 (0.307-5.378)
0.7313


Gleason Sum

0.0001


7 vs. 2-6
1.574 (0.393-6.296)
0.5215


8-10 vs. 2-6
9.899 (2.752-35.610)
0.0004


Pathologic T stage


pT3/4 vs. pT2
6.572 (1.517-28.461)
0.0118


Margin status


Positive vs. Negative
2.825 (1.169-6.826)
0.0210


Seminal Vesicle


Positive vs. Negative
3.792 (1.487-9.672)
0.0053









In Table 9, crude hazard ratios with 95% confidence interval are shown for ERG1 fold change (tumor versus benign) and six clinical parameter categories in a univariate COX proportional hazard ratio analysis. Significant p values are in bold face. The multivariate COX proportional hazard ratio regression analysis of the significant variables from the univariate analysis shows that ERG1 overexpression (greater than 100 fold vs. less than 2 fold: p=0.0239, RR=0.274, overall p value 0.0369), and Gleason sum (Gleason 8-10 vs. Gleason 2-6: p=0.0478, RR=4.078, overall p value 0.0148) are independent predictors of PSA recurrence after radical prostatectomy (Table 10). These results demonstrate that the status of ERG1 expression ratios (tumor vs. benign) in radical prostatectomy specimens carries a predictive value for patient prognosis.









TABLE 10





Factors
Crude Hazard Ratio (95% CI)
P







ERG1 fold changes

0.0369


2-100 fold versus <2 fold
0.320 (0.097-1.059)
0.0620


>100 fold versus <2 fold
0.274 (0.089-0.843)
0.0239


Gleason Sum

0.0148


7 versus 2-6
0.948 (0.223-4.033)
0.9424


8-10 versus 2-6
4.078 (1.014-16.401)
0.0478


Pathologic T stage


PT3/4 versus pT2
3.306 (0.636-17.177)
0.1550


Margin status


Positive versus Negative
1.116 (0.421-2.959)
0.8254


Seminal Vesicle


Positive versus Negative
1.308 (0.466-3.670)
0.6098









ERG1 expression in prostate tumor tissue showed highly significant association with longer PSA recurrence free survival (p=0.0042), well and moderately differentiated grade (p=0.0020), lower pathologic T stage (p=0.0136), and negative surgical margin status (p=0.0209), suggesting that ERG1 over expression in tumor cells is generally higher in less aggressive CaP than in more aggressive CaP (Table 8).
The ERG1 over expression in tumor cells identified by GeneChip analysis and verified by real time QRT-PCR assays was further validated by in situ hybridization. Based on the real time QRT-PCR data, 6 patients with high ERG1 over expression in their tumor cells (and as a control one patient with no ERG1 over expression) were selected for in situ hybridization and quantitative image analysis in a blinded fashion. As expected, in each case the in situ expression data confirmed the over expression of ERG1 in the tumor epithelial cells (FIG. 8).
Example 5
Generation and Characterization of ERG Antibody
Cloning of ERG1 into Tetracycline Regulated Mammalian Expression Vectors:
ERG1 cDNA was subcloned into tetracycline-regulated mammalian expression vectors (pTet-off, EC1214A). The constructs generated include, pTet-off-ERG1 (sense), pTet-off-ERG1 (antisense), pTet-off-FlagERG1 (sense) and pTet-off-FlagERG1 (antisense). Originally, ERG1 construct in a riboprobe vector pGEM was obtained from Dr. Dennis K. Watson, Medical University of South Carolina. The constructs were verified by dideoxy sequencing and agarose gel analysis.
Generation of Polyclonal ERG Antibody:
Antibodies against ERG were generated using peptide antigens derived from the full length ERG1 coding sequence. The epitope for the antigen were carefully selected such that the antibody recognizes specifically ERG1/2/3 and not other members of the ETS family (FIG. 9). The following peptides, having the highest hydrophilicity (−1.26 and −0.55) and antigenicity in the desired region, were used to generate antibodies:







Peptide M-50-mer:


(SEQ ID NO: 13)


CKALQNSPRLMHARNTDLPYEPPRRSAWTGHGHPTPQSKAAQPSPSTVP


 


K-[NH2]


 


Peptide C-49-mer:


(SEQ ID NO: 14)


CDFHGIAQALQPHPPESSLYKYPSDLPYMGSYHAHPQKMNFVAPHPPAL






Cysteine was added to each peptide for conjugation. Peptide M is amidated at the C-terminal residue because it is an internal pepetide.
The synthesis of the peptide epitopes and the immunization of rabbits were carried out in collaboration with Bio-Synthesis Inc. Two rabbits were immunized for each of the two epitopes. Bleeds obtained post immunization were collected and tested. Subsequently, bleeds from one of the rabbits from each epitope were affinity purified using SulfoLink kit (Pierce) and were verified by immunoblot analysis.
Characterization of Polyclonal ERG Antibody by Immunoblot Analysis:
To characterize the affinity purified antibody, we transiently transfected HEK-293 (Human embryonic kidney cell line, ATCC, Manassas, Va.) with ERG1 constructs pTet-off-ERG1 (sense) and pTet-off-FlagERG1 (sense) using Lipofectamine reagent (Invitrogen, Carlsbad, Calif.) as per manufacturers instructions. HEK-293 that were not transfected with the plasmid served as a transfection control. The cells were harvested 48 hours post-transfection and processed for immunoblot analysis. Expression of ERG1 following transfection was determined by immunoblotting using the affinity purified polyclonal antisera generated against the unique M- and C-ERG epitopes described above. Endogenous ERG1 expression was not detected in non-transfected HEK-293 cells. However, the ERG antibodies detected ERG1 expression in HEK-293 cells transfected with the various ERG1 constructs. Tetracycline (2 ug/ml) abolished ERG1 expression in both tetracycline-regulated constructs, pTet-off-ERG1 (sense) and pTet-off-FlagERG1 (sense). The M2-Flag antibody specifically recognized only the Flag-tagged ERG1 protein.
Example 6
Combined Expression of ERG, AMACR, and DD3 Genes in Prostate Tumors
The strikingly high frequency of ERG over expression in CaP cells led to a comparison of ERG expression with two other genes, AMACR and DD3, that are also over expressed in CaP cells. We have evaluated quantitative gene expression features of AMACR and DD3, along with the ERG gene, in laser microdissected matched tumor and benign prostate epithelial cells from 55 CaP patients.
Although other primers and probes can be used, by way of example, we designed the following TaqMan primers and probe recognizing the DD3 gene:









Forward primer:



(SEQ ID NO: 15)



5′-CACATTTCCAGCCCCTTTAAATA-3′



 



Reverse primer:



(SEQ ID NO: 16)



5′-GGGCGAGGCTCATCGAT-3′



 



Probe:



(SEQ ID NO: 17)



5′-GGAAGCACAGAGATCCCTGGGAGAAATG-3′.






The probe has the reporter dye, 6-FAM, attached to the 5′ end and TAMRA attached to the 3′ end. The 3′-TAMRA effectively blocks extension during PCR.

AMACR TaqMan primers and probe were purchased from Applied Biosystems.
AMACR and DD3 showed upregulation in tumor cells of 78.2% and 87.3% of CaP patients, respectively (FIG. 5). ERG over expression in tumor cells was detected in 78.2% of the same group of CaP patients (FIG. 5). Comparative expression analysis revealed that when the AMACR and ERG expression data are combined, 96.4% of the CaP patients showed upregulation of either of the two genes in tumor cells (FIG. 5). Similarly, the combination of the ERG and DD3 expression data improved the cancer detection power of either of the genes to 96.4% (FIG. 5). When combining the expression data from all the three genes, 98.2% of the CaP patients showed upregulation of at least one of the three genes in tumor cells (FIG. 5). Thus, screening for ERG gene expression, alone, or in combination with other genes that are over expressed in CaP, such as AMACR and DD3, provides a new, powerful diagnostic and prognostic tool for CaP.
Example 7
Under Expression of LTF in Malignant Prostate Epithelium
One of the most consistently under expressed genes in CaP cells was LTF (Table 1). Validation by QRT-PCR (TaqMan) in LCM-derived tumor and benign prostate epithelial cells confirmed a consistent, tumor associated LTF under expression in 100% of CaP cells tested (FIG. 1D). As a quality control, the expression of AMACR, a recently identified CaP tissue marker, and of GSTP1, a gene showing commonly reduced or absent expression in CaP (Nelson et al., Ann. N.Y. Acad. Sci., 952:135-44 (2001)), was also determined (FIGS. 1B and 1C, respectively). Robust under expression similar to LTF, was observed for GSTP1, while the increased expression of AMACR was noted in 95% of the tumor cells tested, confirming the high quality of the tumor and benign LCM specimens and the reliability of the QRT-PCR. In a further study, LTF expression was analyzed by QRT-PCR in in microdissected tumor and benign prostate epithelial cells of 103 CaP patients. The results were consistent with the initial results, showing tumor associated under expression in 76% of patients (78 of 103).
LTF under expression was also validated at the protein level with anti-LTF goat polyclonal antibody (Santa Cruz, Calif., sc-14434) using Western blot analysis on protein lysates and immunohistochemistry techniques. Hematotoxylin-eosin (H&E) and LTF staining was performed on tissue samples from 30 CaP patients by immunocytochemical analysis. In 30 of 30 (100%) cases, benign epithelial cells adjacent to tumor cells were highly positive for LTF, whereas, on average, less than 10% of prostate tumor cells revealed LTF positive cytoplasmic staining.
The specification is most thoroughly understood in light of the teachings of the references cited within the specification which are hereby incorporated by reference. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments are encompassed by the invention.

## Claims

1. A method of diagnosing prostate cancer in a subject, comprising:
a) measuring the expression level of a human ERG polypeptide in a biological sample from the subject, wherein the biological sample comprises prostate epithelial cells and wherein the expression level of the human ERG polypeptide is measured by an immunoassay using a monoclonal antibody that binds to the human ERG1 polypeptide and does not bind to the human ETS domain; and
b) correlating the expression level of the human ERG polypeptide with the presence of prostate cancer in the subject, wherein increased expression of the human ERG polypeptide in the biological sample as compared to the expression of the human ERG polypeptide in a control sample indicates the presence of prostate cancer in the subject.

2. The method of claim 1, wherein the monoclonal antibody is generated using a fragment of the human ERG1 polypeptide as an immunogen.

3. The method of claim 1, wherein the monoclonal antibody binds to the human ERG1, ERG2, and ERG3 polypeptides.

4. A method of diagnosing prostate cancer in a subject, comprising:
a) measuring the expression level of a human ERG polypeptide in a biological sample from the subject, wherein the biological sample comprises prostate epithelial cells and wherein the expression level of the human ERG polypeptide is measured by an immunoassay using a monoclonal antibody that binds to the human ERG1 polypeptide; and
b) correlating the expression level of the human ERG polypeptide with the presence of prostate cancer in the subject, wherein increased expression of the human ERG polypeptide in the biological sample as compared to the expression of the human ERG polypeptide in a control sample indicates the presence of prostate cancer in the subject, wherein the monoclonal antibody is generated using a fragment of the human ERG1 polypeptide as an immunogen, and wherein the monoclonal antibody binds to an epitope formed by amino acids 275-322 of the polypeptide encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1.

5. The method of claim 1, wherein the monoclonal antibody binds to an epitope formed by amino acids 118-166 of the polypeptide encoded by a nucleic acid comprising the sequence of SEQ ID NO:1.

6. The method of claim 1, wherein the biological sample is chosen from a tissue sample, a blood sample, or a urine sample.

7. The method of claim 6, wherein the biological sample is a prostate tissue sample.

8. The method of claim 1, further comprising measuring the expression levels of a human AMACR polypeptide and correlating the expression levels of the human ERG polypeptide and the human AMACR polypeptide with the presence of prostate cancer in the subject.

9. The method of claim 8, wherein the human AMACR polypeptide is encoded by a nucleic acid comprising the sequence of SEQ ID NO. 3.

10. The method of claim 1, wherein the control sample is a noncancerous biological sample from the subject.

11. The method of claim 8, wherein the expression levels of the human ERG polypeptide and the human AMACR polypeptide in the biological sample are compared to the expression levels of the human ERG polypeptide and the human AMACR polypeptide in a control sample and wherein increased expression of the human ERG polypeptide and the human AMACR polypeptide in the biological sample as compared to the expression of the ERG polypeptide and the human AMACR polypeptide in the control sample indicates the presence of prostate cancer in the subject.

12. The method of claim 1, wherein the immunoassay is an immunohistochemistry assay.

13. A method of identifying prostate cancer in a subject, comprising:
a) providing a prostate tissue sample from the subject;
b) contacting the prostate tissue sample with a monoclonal antibody that binds to a human ERG polypeptide and does not bind to the human ETS domain; and
c) measuring the level of expression of the human ERG polypeptide in the prostate tissue sample by detecting the amount of binding between the monoclonal antibody and the human ERG polypeptide in the prostate tissue sample;
d) comparing the level of expression of the ERG polypeptide in the prostate tissue sample to the level of expression of the ERG polypeptide in a control sample; and
e) identifying prostate cancer in the prostate tissue sample from the subject if the level of expression of the ERG polypeptide in the prostate tissue sample is increased as compared to the expression level of the ERG polypeptide in the control sample.

14. The method of claim 13, wherein the monoclonal antibody binds to the ERG1, ERG2, and ERG3 polypeptides.

15. A method of identifying prostate cancer in a subject, comprising:
a) providing a prostate tissue sample from the subject;
b) contacting the prostate tissue sample with a monoclonal antibody that binds to a human ERG polypeptide; and
c) measuring the level of expression of the human ERG polypeptide in the prostate tissue sample by detecting the amount of binding between the monoclonal antibody and the human ERG polypeptide in the prostate tissue sample;
d) comparing the level of expression of the ERG polypeptide in the prostate tissue sample to the level of expression of the ERG polypeptide in a control sample; and
e) identifying prostate cancer in the prostate tissue sample from the subject if the level of expression of the ERG polypeptide in the prostate tissue sample is increased as compared to the expression level of the ERG polypeptide in the control sample, wherein the monoclonal antibody binds to an epitope formed by amino acids 275-322 of the polypeptide encoded by a nucleic acid comprising the sequence of SEQ ID NO:1.

16. The antibody of claim 13, wherein the monoclonal antibody binds to an epitope formed by the amino acid sequence of SEQ ID NO:14.

17. The method of claim 13, wherein the control sample is a noncancerous biological sample from the subject.

18. The method of claim 17, wherein the noncancerous biological sample from the subject is normal prostate tissue comprising benign prostate epithelial cells.

19. A method of detecting the expression of a human ERG polypeptide in a biological sample, wherein the biological sample comprises human prostate epithelial cells, the method comprising:
a) contacting the human prostate epithelial cells with a monoclonal antibody that binds to a human ERG1, ERG2, and ERG3 polypeptide, wherein the monoclonal antibody binds to an epitope formed by amino acids 275-322 of the polypeptide encoded by a nucleic acid comprising the sequence of SEQ ID NO:1 or to an epitope formed by the amino acid sequence of SEQ ID NO:14; and
b) measuring the level of expression of the human ERG polypeptide in the human prostate epithelial cells sample by detecting the amount of binding between the monoclonal antibody and the human ERG polypeptide in the human prostate epithelial cells.

20. The method of claim 19, further comprising a step of comparing the level of expression of the human ERG polypeptide in the human prostate epithelial cells to the level of expression of the human ERG polypeptide in a control sample.

21. The method of claim 19, wherein the biological sample is obtained from a patient that has prostate cancer or is suspected of having prostate cancer.

22. The method of claim 21, wherein the patient has been treated by radical prostate surgery.

23. The method of claim 19, further comprising detecting the expression of a human AMACR polypeptide in the biological sample.

24. The method of claim 1, wherein the expression level of the ERG polypeptide is correlated with longer PSA recurrence free survival, well and moderate tumor differentiation, a pathologic T stage of pT2 or lower, or a negative surgical status.

25. The method of claim 1, wherein the monoclonal antibody does not bind to a SMAP-PNT domain.

26. The method of claim 13, wherein the monoclonal antibody does not bind to a SMAP-PNT domain.

27. The method of claim 1, wherein the monoclonal antibody specifically binds to ERG1, ERG2, and ERG3, but does not specifically bind to another protein of the ETS family.

28. The method of claim 13, wherein the monoclonal antibody specifically binds to ERG1, ERG2, and ERG3, but does not specifically bind to another protein of the ETS family.