# Patent

## Title
Method of diagnosing, monitoring, staging, imaging and treating prostate cancer

## Abstract

The present invention provides new methods for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating prostate cancer.

## Background
This patent application is a divisional of U.S. application Ser. No. 09/807,201, filed Apr. 25, 2001, now issued as U.S. Pat. No. 6,902,892, which is the U.S. National Stage of PCT Application No. PCT/US1999/024331, filed Oct. 19, 1999, which claims the benefit of U.S. Provisional Application No. 60/104,737, filed Oct. 19, 1998, teachings of each of which are herein incorporated by reference in their entirety.


FIELD OF THE INVENTION
This invention relates, in part, to newly developed assays for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating cancers, particularly prostate cancer.
BACKGROUND OF THE INVENTION
Cancer of the prostate is the most prevalent malignancy in adult males, excluding skin cancer, and is an increasingly prevalent health problem in the United States. In 1996, it was estimated that 41,400 deaths would result from this disease in the United States alone, indicating that prostate cancer is second only to lung cancer as the most common cause of death in the same population. If diagnosed and treated early, when the cancer is still confined to the prostate, the chances of cure is significantly higher.
Treatment decisions for an individual are linked to the stage of prostate cancer present in that individual. A common classification of the spread of prostate cancer was developed by the American Urological Association (AUA). The AUA system divides prostate tumors into four stages, A to D. Stage A, microscopic cancer within prostate, is further subdivided into stages A1 and A2. Sub-stage A1 is a well-differentiated cancer confined to one site within the prostate. Treatment is generally observation, radical prostatectomy, or radiation. Sub-stage A2 is a moderately to poorly differentiated cancer at multiple sites within the prostate. Treatment is radical prostatectomy or radiation. Stage B, palpable lump within the prostate, is also further subdivided into sub-stages B1 and B2. In sub-stage B1, the cancer forms a small nodule in one lobe of the prostate. In sub-stage B2, the cancer forms large or multiple nodules, or occurs in both lobes of the prostate. Treatment for sub-stages B1 and B2 is either radical prostatectomy or radiation. Stage C is a large cancer mass involving most or all of the prostate and is also further subdivided into two sub-stages. In sub-stage C1, the cancer forms a continuous mass that may have extended beyond the prostate. In sub-stage C2, the cancer forms a continuous mass that invades the surrounding tissue. Treatment for both these sub-stages is radiation with or without drugs to address the cancer. The fourth stage, Stage D is metastatic cancer and is also subdivided into two sub-stages. In sub-stage D1, the cancer appears in the lymph nodes of the pelvis. In sub-stage D2, the cancer involves tissues beyond lymph nodes. Treatment for both of these sub-stages is systemic drugs to address the cancer as well as pain.
However, current prostate cancer staging methods are limited. As many as 50% of prostate cancers initially staged as A2, B, or C are actually stage D, metastatic. Discovery of metastasis is significant because patients with metastatic cancers have a poorer prognosis and require significantly different therapy than those with localized cancers. The five year survival rates for patients with localized and metastatic prostate cancers are 93% and 29%, respectively.
Accordingly, there is a great need for more sensitive and accurate methods for the staging of a cancer in a human to determine whether or not such cancer has metastasized and for monitoring the progress of a cancer in a human which has not metastasized for the onset of metastasis.
It has now been found that a number of proteins in the public domain are useful as diagnostic markers for prostate cancer. These diagnostic markers are referred to herein as cancer specific genes or CSGs and include, but are not limited to: Pro109 which is a human zinc-α2-glycoprotein (Freje et al. Genomics 1993 18(3):575-587); Pro112 which is a human cysteine-rich protein with a zinc-finger motif (Liebhaber et al. Nucleic Acid Research 1990 18(13):3871-3879; WO9514772 and WO9845436); Pro111 which is a prostate-specific transglutaminase (Dubbink et al. Genomics 1998 51(3):434-444); Pro115 which is a novel serine protease with transmembrane, LDLR, and SRCR domains and maps to 21q22.3 (Paoloni-Giacobino et al. Genomics 1997 44(3):309-320; WO9837418 and WO987093); Pro110 which is a human breast carcinoma fatty acid synthase (U.S. Pat. No. 5,665,874 and WO9403599); Pro113 which is a homeobox gene, HOXB13 (Steinicki et al. J. Invest. Dermatol. 1998 111:57-63); Pro114 which is a human tetraspan NET-1 (WO9839446); and Pro118 which is a human JM27 protein (WO9845435). ESTs for these CSGs are set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15 while the full length contigs for these CSGs are set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14 and 16, respectively. Additional CSGs for use in the present invention are depicted herein in SEQ ID NO: 17, 18, 19 and 20.
In the present invention, methods are provided for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating prostate cancer via the cancer specific genes referred to herein as CSGs. For purposes of the present invention, CSG refers, among other things, to native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. By “CSG” it is also meant herein polynucleotides which, due to degeneracy in genetic coding, comprise variations in nucleotide sequence as compared to SEQ ID NO: 1-20, but which still encode the same protein. In the alternative, what is meant by CSG as used herein, means the native mRNA encoded by the gene comprising the polynucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, levels of the gene comprising the polynucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or levels of a polynucleotide which is capable of hybridizing under stringent conditions to the antisense sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.
SUMMARY OF THE INVENTION
Toward these ends, and others, it is an object of the present invention to provide a method for diagnosing the presence of prostate cancer by analyzing for changes in levels of CSG in cells, tissues or bodily fluids compared with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control, wherein a change in levels of CSG in the patient versus the normal human control is associated with prostate cancer.
Further provided is a method of diagnosing metastatic prostate cancer in a patient having prostate cancer which is not known to have metastasized by identifying a human patient suspected of having prostate cancer that has metastasized; analyzing a sample of cells, tissues, or bodily fluid from such patient for CSG; comparing the CSG levels in such cells, tissues, or bodily fluid with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control, wherein an increase in CSG levels in the patient versus the normal human control is associated with prostate cancer which has metastasized.
Also provided by the invention is a method of staging prostate cancer in a human which has such cancer by identifying a human patient having such cancer; analyzing a sample of cells, tissues, or bodily fluid from such patient for CSG; comparing CSG levels in such cells, tissues, or bodily fluid with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in CSG levels in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of CSG is associated with a cancer which is regressing or in remission.
Further provided is a method of monitoring prostate cancer in a human having such cancer for the onset of metastasis. The method comprises identifying a human patient having such cancer that is not known to have metastasized; periodically analyzing a sample of cells, tissues, or bodily fluid from such patient for CSG; comparing the CSG levels in such cells, tissue, or bodily fluid with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in CSG levels in the patient versus the normal human control is associated with a cancer which has metastasized.
Further provided is a method of monitoring the change in stage of prostate cancer in a human having such cancer by looking at levels of CSG in a human having such cancer. The method comprises identifying a human patient having such cancer; periodically analyzing a sample of cells, tissues, or bodily fluid from such patient for CSG; comparing the CSG levels in such cells, tissue, or bodily fluid with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in CSG levels in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of CSG is associated with a cancer which is regressing or in remission.
Further provided are methods of designing new therapeutic agents targeted to a CSG for use in imaging and treating prostate cancer. For example, in one embodiment, therapeutic agents such as antibodies targeted against CSG or fragments of such antibodies can be used to detect or image localization of CSG in a patient for the purpose of detecting or diagnosing a disease or condition. Such antibodies can be polyclonal, monoclonal, or omniclonal or prepared by molecular biology techniques. The term “antibody”, as used herein and throughout the instant specification is also meant to include aptamers and single-stranded oligonucleotides such as those derived from an in vitro evolution protocol referred to as SELEX and well known to those skilled in the art. Antibodies can be labeled with a variety of detectable labels including, but not limited to, radioisotopes and paramagnetic metals. Therapeutics agents such as antibodies or fragments thereof can also be used in the treatment of diseases characterized by expression of CSG. In these applications, the antibody can be used without or with derivatization to a cytotoxic agent such as a radioisotope, enzyme, toxin, drug or a prodrug.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.


DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to diagnostic assays and methods, both quantitative and qualitative for detecting, diagnosing, monitoring, staging and prognosticating cancers by comparing levels of CSG in a human patient with those of CSG in a normal human control. For purposes of the present invention, what is meant be CSG levels is, among other things, native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. By “CSG” it is also meant herein polynucleotides which, due to degeneracy in genetic coding, comprise variations in nucleotide sequence as compared to SEQ ID NO: 1-20, but which still encode the same protein. The native protein being detected, may be whole, a breakdown product, a complex of molecules or chemically modified. In the alternative, what is meant by CSG as used herein, means the native mRNA encoded by the gene comprising the polynucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, levels of the gene comprising the polynucleotide sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or levels of a polynucleotide which is capable of hybridizing under stringent conditions to the antisense sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Such levels are preferably determined in at least one of, cells, tissues and/or bodily fluids, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for diagnosing overexpression of CSG protein compared to normal control bodily fluids, cells, or tissue samples may be used to diagnose the presence of prostate cancer.
All the methods of the present invention may optionally include determining the levels of other cancer markers as well as CSG. Other cancer markers, in addition to CSG, useful in the present invention will depend on the cancer being tested and are known to those of skill in the art.
Diagnostic Assays
The present invention provides methods for diagnosing the presence of prostate cancer by analyzing for changes in levels of CSG in cells, tissues or bodily fluids compared with levels of CSG in cells, tissues or bodily fluids of preferably the same type from a normal human control, wherein an increase in levels of CSG in the patient versus the normal human control is associated with the presence of prostate cancer.
Without limiting the instant invention, typically, for a quantitative diagnostic assay a positive result indicating the patient being tested has cancer is one in which cells, tissues or bodily fluid levels of the cancer marker, such as CSG, are at least two times higher, and most preferably are at least five times higher, than in preferably the same cells, tissues or bodily fluid of a normal human control.
The present invention also provides a method of diagnosing metastatic prostate cancer in a patient having prostate cancer which has not yet metastasized for the onset of metastasis. In the method of the present invention, a human cancer patient suspected of having prostate cancer which may have metastasized (but which was not previously known to have metastasized) is identified. This is accomplished by a variety of means known to those of skill in the art.
In the present invention, determining the presence of CSG levels in cells, tissues or bodily fluid, is particularly useful for discriminating between prostate cancer which has not metastasized and prostate cancer which has metastasized. Existing techniques have difficulty discriminating between prostate cancer which has metastasized and prostate cancer which has not metastasized and proper treatment selection is often dependent upon such knowledge.
In the present invention, the cancer marker levels measured in such cells, tissues or bodily fluid is CSG, and are compared with levels of CSG in preferably the same cells, tissue or bodily fluid type of a normal human control. That is, if the cancer marker being observed is just CSG in serum, this level is preferably compared with the level of CSG in serum of a normal human control. An increase in the CSG in the patient versus the normal human control is associated with prostate cancer which has metastasized.
Without limiting the instant invention, typically, for a quantitative diagnostic assay a positive result indicating the cancer in the patient being tested or monitored has metastasized is one in which cells, tissues or bodily fluid levels of the cancer marker, such as CSG, are at least two times higher, and most preferably are at least five times higher, than in preferably the same cells, tissues or bodily fluid of a normal patient.
Normal human control as used herein includes a human patient without cancer and/or non cancerous samples from the patient; in the methods for diagnosing or monitoring for metastasis, normal human control may preferably also include samples from a human patient that is determined by reliable methods to have prostate cancer which has not metastasized.
Staging
The invention also provides a method of staging prostate cancer in a human patient. The method comprises identifying a human patient having such cancer and analyzing cells, tissues or bodily fluid from such human patient for CSG. The CSG levels determined in the patient are then compared with levels of CSG in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in CSG levels in the human patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of CSG (but still increased over true normal levels) is associated with a cancer which is regressing or in remission.
Monitoring
Further provided is a method of monitoring prostate cancer in a human patient having such cancer for the onset of metastasis. The method comprises identifying a human patient having such cancer that is not known to have metastasized; periodically analyzing cells, tissues or bodily fluid from such human patient for CSG; and comparing the CSG levels determined in the human patient with levels of CSG in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in CSG levels in the human patient versus the normal human control is associated with a cancer which has metastasized. In this method, normal human control samples may also include prior patient samples.
Further provided by this invention is a method of monitoring the change in stage of prostate cancer in a human patient having such cancer. The method comprises identifying a human patient having such cancer; periodically analyzing cells, tissues or bodily fluid from such human patient for CSG; and comparing the CSG levels determined in the human patient with levels of CSG in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in CSG levels in the human patient versus the normal human control is associated with a cancer which is progressing in stage and a decrease in the levels of CSG is associated with a cancer which is regressing-in stage or in remission. In this method, normal human control samples may also include prior patient samples.
Monitoring a patient for onset of metastasis is periodic and preferably done on a quarterly basis. However, this may be more or less frequent depending on the cancer, the particular patient, and the stage of the cancer.
Assay Techniques
Assay techniques that can be used to determine levels of gene expression (including protein levels), such as CSG of the present invention, in a sample derived from a patient are well known to those of skill in the art. Such assay methods include, without limitation, radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches: two-dimensional gel electrophoresis (2D electrophoresis) and non-gel based approaches such as mass spectrometry or protein interaction profiling. Among these, ELISAs are frequently preferred to diagnose a gene's expressed protein in biological fluids.
An ELISA assay initially comprises preparing an antibody, if not readily available from a commercial source, specific to CSG, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds specifically to CSG. The reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase.
To carry out the ELISA, antibody specific to CSG is incubated on a solid support, e.g. a polystyrene dish, that binds the antibody. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the sample to be analyzed is incubated in the dish, during which time CSG binds to the specific antibody attached to the polystyrene dish. Unbound sample is washed out with buffer. A reporter antibody specifically directed to CSG and linked to a detectable reagent such as horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to CSG. Unattached reporter antibody is then washed out. Reagents for peroxidase activity, including a colorimetric substrate are then added to the dish. Immobilized peroxidase, linked to CSG antibodies, produces a colored reaction product. The amount of color developed in a given time period is proportional to the amount of CSG protein present in the sample. Quantitative results typically are obtained by reference to a standard curve.
A competition assay can also be employed wherein antibodies specific to CSG are attached to a solid support and labeled CSG and a sample derived from the host are passed over the solid support. The amount of label detected which is attached to the solid support can be correlated to a quantity of CSG in the sample.
Nucleic acid methods can also be used to detect CSG mRNA as a marker for prostate cancer. Polymerase chain reaction (PCR) and other nucleic acid methods, such as ligase chain reaction (LCR) and nucleic acid sequence based amplification (NASABA), can be used to detect malignant cells for diagnosis and monitoring of various malignancies. For example, reverse-transcriptase PCR (RT-PCR) is a powerful technique which can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species. In RT-PCR, an mRNA species is first reverse transcribed to complementary DNA (cDNA) with use of the enzyme reverse transcriptase; the cDNA is then amplified as in a standard PCR reaction. RT-PCR can thus reveal by amplification the presence of a single species of mRNA. Accordingly, if the mRNA is highly specific for the cell that produces it, RT-PCR can be used to identify the presence of a specific type of cell.
Hybridization to clones or oligonucleotides arrayed on a solid support (i.e. gridding) can be used to both detect the expression of and quantitate the level of expression of that gene. In this approach, a cDNA encoding the CSG gene is fixed to a substrate. The substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon or plastic. At least a portion of the DNA encoding the CSG gene is attached to the substrate and then incubated with the analyte, which may be RNA or a complementary DNA (cDNA) copy of the RNA, isolated from the tissue of interest. Hybridization between the substrate bound DNA and the analyte can be detected and quantitated by several means including but not limited to radioactive labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid. Quantitation of the level of gene expression can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards. The standards can be obtained by in vitro transcription of the target gene, quantitating the yield, and then using that material to generate a standard curve.
Of the proteomic approaches, 2D electrophoresis is a technique well known to those in the art. Isolation of individual proteins from a sample such as serum is accomplished using sequential separation of proteins by different characteristics usually on polyacrylamide gels. First, proteins are separated by size using an electric current. The current acts uniformly on all proteins, so smaller proteins move farther on the gel than larger proteins. The second dimension applies a current perpendicular to the first and separates proteins not on the basis of size but on the specific electric charge carried by each protein. Since no two proteins with different sequences are identical on the basis of both size and charge, the result of a 2D separation is a square gel in which each protein occupies a unique spot. Analysis of the spots with chemical or antibody probes, or subsequent protein microsequencing can reveal the relative abundance of a given protein and the identity of the proteins in the sample.
The above tests can be carried out on samples derived from a variety of cells, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a patient. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. By blood it is meant to include whole blood, plasma, serum or any derivative of blood.
In Vivo Targeting of CSGs
Identification of these CSGs is also useful in the rational design of new therapeutics for imaging and treating cancers, and in particular prostate cancer. For example, in one embodiment, antibodies which specifically bind to CSG can be raised and used in vivo in patients suspected of suffering from prostate cancer. Antibodies which specifically bind a CSG can be injected into a patient suspected of having prostate cancer for diagnostic and/or therapeutic purposes. The preparation and use of antibodies for in vivo diagnosis is well known in the art. For example, antibody-chelators labeled with Indium-111 have been described for use in the radioimmunoscintographic imaging of carcinoembryonic antigen expressing tumors (Sumerdon et al. Nucl. Med. Biol. 1990 17:247-254). In particular, these antibody-chelators have been used in detecting tumors in patients suspected of having recurrent colorectal cancer (Griffin et al. J. Clin. Onc. 1991 9:631-640). Antibodies with paramagnetic ions as labels for use in magnetic resonance imaging have also been described (Lauffer, R. B. Magnetic Resonance in Medicine 1991 22:339-342). Antibodies directed against CSG can be used in a similar manner. Labeled antibodies which specifically bind CSG can be injected into patients suspected of having prostate cancer for the purpose of diagnosing or staging of the disease status of the patient. The label used will be selected in accordance with the imaging modality to be used. For example, radioactive labels such as Indium-111, Technetium-99m or Iodine-131 can be used for planar scans or single photon emission computed tomography (SPECT). Positron emitting labels such as Fluorine-19 can be used in positron emission tomography. Paramagnetic ions such as Gadlinium (III) or Manganese (II) can be used in magnetic resonance imaging (MRI). Localization of the label permits determination of the spread of the cancer. The amount of label within an organ or tissue also allows determination of the presence or absence of cancer in that organ or tissue.
For patients diagnosed with prostate cancer, injection of an antibody which specifically binds CSG can also have a therapeutic benefit. The antibody may exert its therapeutic effect alone. Alternatively, the antibody can be conjugated to a cytotoxic agent such as a drug, toxin or radionuclide to enhance its therapeutic effect. Drug monoclonal antibodies have been described in the art for example by Garnett and Baldwin, Cancer Research 1986 46:2407-2412. The use of toxins conjugated to monoclonal antibodies for the therapy of various cancers has also been described by Pastan et al. Cell 1986 47:641-648. Yttrium-90 labeled monoclonal antibodies have been described for maximization of dose delivered to the tumor while limiting toxicity to normal tissues (Goodwin and Meares Cancer Supplement 1997 80:2675-2680). Other cytotoxic radionuclides including, but not limited to Copper-67, Iodine-131 and Rhenium-186 can also be used for labeling of antibodies against CSG.
Antibodies which can be used in these in vivo methods include polyclonal, monoclonal and omniclonal antibodies and antibodies prepared via molecular biology techniques. Antibody fragments and aptamers and single-stranded oligonucleotides such as those derived from an in vitro evolution protocol referred to as SELEX and well known to those skilled in the art can also be used.
Small molecules predicted via computer imaging to specifically bind to regions of CSGs can also be designed and synthesized and tested for use in the imaging and treatment of prostate cancer. Further, libraries of molecules can be screened for potential anticancer agents by assessing the ability of the molecule to bind to CSGs identified herein. Molecules identified in the library as being capable of binding to CSG are key candidates for further evaluation for use in the treatment of prostate cancer.
EXAMPLES
The present invention is further described by the following examples. These examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplifications, while illustrating certain aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.
All examples outlined here were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques of the following example can be carried out as described in standard laboratory manuals, such as Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Example 1
Identification of CSGs
Identification of CSGs were carried out by a systematic analysis of data in the LIFESEQ database available from Incyte Pharmaceuticals, Palo Alto, Calif, using the data mining Cancer Leads Automatic Search Package (CLASP) developed by diaDexus LLC, Santa Clara, Calif.
The CLASP performs the following steps: selection of highly expressed organ specific genes based on the abundance level of the corresponding EST in the targeted organ versus all the other organs; analysis of the expression level of each highly expressed organ specific genes in normal, tumor tissue, disease tissue and tissue libraries associated with tumor or disease; selection of the candidates demonstrating component ESTs were exclusively or more frequently found in tumor libraries. The CLASP allows the identification of highly expressed organ and cancer specific genes. A final manual in depth evaluation is then performed to finalize the CSGs selection.
Clones depicted in the following Table 1 are CSGs useful in diagnosing, monitoring, staging, imaging and treating prostate cancer.







TABLE 1







CSGs











Clone ID
Pro #
SEQ ID NO:







3424528H1
Pro109
1, 2



578349H1
Pro112
3, 4



1794013H1
Pro111
5, 6



2189835H1
Pro115
7, 8



3277219H1
Pro110
 9, 10



1857415
Pro113
11, 12



1810463H1
Pro114
13, 14



zr65G11
Pro118
15, 16



2626135H1

17



zd46d08

18



1712252H1

19



784583H1

20










Example 2
Relative Quantitation of Gene Expression
Real-Time quantitative PCR with fluorescent Taqman probes is a quantitation detection system utilizing the 5′-3′ nuclease activity of Taq DNA polymerase. The method uses an internal fluorescent oligonucleotide probe (Taqman) labeled with a 5′ reporter dye and a downstream, 3′ quencher dye. During PCR, the 5′-3′ nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, Cailf., USA).
Amplification of an endogenous control is used to standardize the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency. Either cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ATPase, or 18S ribosomal RNA (rRNA) is used as this endogenous control. To calculate relative quantitation between all the samples studied, the target RNA levels for one sample were used as the basis for comparative results (calibrator). Quantitation relative to the “calibrator” can be obtained using the standard curve method or the comparative method (User Bulletin #2: ABI PRISM 7700 Sequence Detection System).
The tissue distribution and the level of the target gene were evaluated for every sample in normal and cancer tissues. Total RNA was extracted from normal tissues, cancer tissues, and from cancers and the corresponding matched adjacent tissues. Subsequently, first strand cDNA was prepared with reverse transcriptase and the polymerase chain reaction was done using primers and Taqman probes specific to each target gene. The results were analyzed using the ABI PRISM 7700 Sequence Detector. The absolute numbers are relative levels of expression of the target gene in a particular tissue compared to the calibrator tissue.
Expression of Clone ID 3424528H1 (Pro109):
For the CSG Pro109, real-time quantitative PCR was performed using the following primers:











Forward Primer:





5′-ATCAGAACAAAGAGGCTGTGTC-3′
(SEQ ID NO:21)



 



Reverse Primer:



5′-ATCTCTAAAGCCCCAACCTTC-3′
(SEQ ID NO:22)






The absolute numbers depicted in Table 2 are relative levels of expression of the CSG referred to as Pro109 in 12 normal different tissues. All the values are compared to normal stomach (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.








TABLE 2







Relative Levels of CSG Pro109 Expression in Pooled


Samples










Tissue
NORMAL














Colon
0.02



Endometrium
0.01



Kidney
0.48



Liver
14.83



Ovary
0.08



Pancreas
4.38



Prostate
11.24



Small Intestine
0.42



Spleen
0



Stomach
1



Testis
0.62



Uterus
0.02










The relative levels of expression in Table 2 show that with the exception of liver (14.83), Pro109 mRNA expression is higher (11.24) in prostate compared with all other normal tissues analyzed. Pancreas, with a relative expression level of 4.38, is the only other tissue expressing considerable mRNA for Pro109.

The absolute numbers in Table 2 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 3.
The absolute numbers depicted in Table 3 are relative levels of expression of Pro109 in 28 pairs of matching samples and 4 unmatched samples. All the values are compared to normal stomach (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual.







TABLE 3







Relative Levels of CSG Pro109 Expression in


Individual Samples















Matching






Normal



Sample ID
Tissue
Cancer
Adjacent
















Pro34B
Prostate 1
5.98
6.06



Pro65XB
Prostate 2
16.68
3.85



Pro69XB
Prostate 3
20.46
6.82



Pro78XB
Prostate 4
1.39
1.4



Pro101XB
Prostate 5
24.8
9.8



Pro12B
Prostate 6
9.1
0.2



Pro13XB
Prostate 7
0.5
9.7



Pro20XB
Prostate 8
13
12.5



Pro23B
Prostate 9
16.8
3



Ovr10005O
Ovary 1
0.4



Ovr1028
Ovary 2
1.9



Ovr18GA
Ovary 3

0.1



Ovr206I
Ovary 4

0.1



Mam12X
Mammary Gland 1
13.5
1.4



Mam47XP
Mammary Gland 2
0.7
0.2



Lng47XQ
Lung 1
2.36
0.03



Lng60XL
Lung 2
7.39
0.2



Lng75XC
Lung 3
0.77
0.27



StoAC44
Stomach 1
0.05
1.19



StoAC93
Stomach 2
0.55
0.8



StoAC99
Stomach 3
0.12
3.04



ColAS43
Colon 1
16.11
0.07



ColAS45
Colon 2
0.11
0.08



ColAS46
Colon 3
4.99
0.4



Liv15XA
Liver 1
8.43
10.97



Liv42X
Liver 2
1.57
20.82



Liv94XA
Liver 3
2.98
9.19



Pan77X
Pancreas 1
36
32



Pan82XP
Pancreas 2
0.09
7.09



Pan92X
Pancreas 3
0.7
0



Pan71XL
Pancreas 4
2.48
0.73



Pan10343
Pancreas 5
46
5.5







0 = Negative






In the analysis of matching samples, the higher levels of expression were in prostate, showing a high degree of tissue specificity for prostate tissue. Of all the samples different than prostate analyzed, only 4 cancer samples (the cancer sample mammary 1 with 13.5, colon 1 with 16.11, liver 1 with 8.43, and lung 2 with 7.39) showed an expression comparable to the mRNA expression in prostate. These results confirmed some degree of tissue specificity as obtained with the panel of normal pooled samples (Table 2).
Furthermore, the level of mRNA expression was compared in cancer samples and the isogenic normal adjacent tissue from the same individual. This comparison provides an indication of specificity for the cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 3 shows overexpression of Pro109 in 6 out of 9 primary prostate cancer tissues compared with their respective normal adjacents. Thus, overexpression in the cancer tissue was observed in 66.66% of the prostate matching samples tested (total of 9 prostate matching samples)
Altogether, the degree of tissue specificity, plus the mRNA overexpression in 66.66% of the primary prostate matching samples tested is indicative of Pro109 being a diagnostic marker for prostate cancer.
Expression of Clone ID 578349H1 (Pro112):
For the CSG Pro112, real-time quantitative PCR was performed using the following primers:
Forward Primer

    
    
        5′-TGCCGAAGAGGTTCAGTGC-3′ (SEQ ID NO:23)
    
    


Reverse Primer

    
    
        5′-GCCACAGTGGTACTGTCCAGAT-3′ (SEQ ID NO:24)
    
    


The absolute numbers depicted in Table 4 are relative levels of expression of the CSG Pro112 in 12 normal different tissues. All the values are compared to normal thymus (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.







TABLE 4







Relative Levels of CSG Pro112 Expression in Pooled


Samples










Tissue
NORMAL














Brain
2.9



Heart
0.1



Kidney
0.2



Liver
0.2



Lung
7.7



Mammary
4.2



Muscle
0.1



Prostate
5.5



Small Intestine
1.8



Testis
1



Thymus
1



Uterus
21










The relative levels of expression in Table 4 show that Pro112 mRNA expression is the 3rd most highly expressed gene (after uterus and mammary) in the pool of normal prostate tissue compared to a total of 12 tissues analyzed. The absolute numbers in Table 4 were obtained analyzing pools of samples of a particular tissue from different individuals. These results demonstrate that Pro112 mRNA expression is specific for prostate thus indicating Pro112 to be a diagnostic marker for prostate disease especially cancer.
Expression of Clone ID 1794013H1 (Pro111):
For the CSG Pro111, real-time quantitative PCR was performed using the following primers:











Forward Primer





5′-GCTGCAAGTTCTCCACATTGA-3′
(SEQ ID NO:25)



 



Reverse Primer



5′-CAGCCGCAGGTGAAACAC-3′
(SEQ ID NO:26)






The absolute numbers depicted in Table 5 are relative levels of expression of the CSG Pro111 in 12 normal different tissues. All the values are compared to normal testis (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.








TABLE 5







Relative Levels of CSG Pro111 Expression in Pooled


Samples










Tissue
NORMAL














Brain
0.04



Heart
0



Kidney
0



Liver
0



Lung
0.05



Mammary
0.14



Muscle
5166.6



Prostate
1483.72



Small Intestine
0.33



Testis
1



Thymus
0.49



Uterus
0.07










The relative levels of expression in Table 5 show that Pro111 mRNA expression is extraordinarily high in the pool of normal prostate (1483.72) compared to all the other tissues analyzed with the exception of muscle (5166.6). These results demonstrate that Pro111 mRNA expression shows specificity for prostate and muscle.

The absolute numbers in Table 5 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 6.
The absolute numbers depicted in Table 6 are relative levels of expression of Pro111 in 48 pairs of matching and 18 unmatched samples. All the values are compared to normal testis (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual.







TABLE 6







Relative Levels of CSG Pro111 Expression in


Individual Samples















Matching






Normal



Sample ID
Tissue
Cancer
Adjacent
















Pro101XB
Prostate 1
8.3
21.8



Pro12B
Prostate 2
2336
133



Pro13XB
Prostate 3
3.4
23



Pro20XB
Prostate 4
21.6
121.5



Pro23B
Prostate 5
19.4
3.7



Pro34B
Prostate 6
15
39



Pro65XB
Prostate 7
8
867



Pro69XB
Prostate 8
56
94



Pro78XB
Prostate 9
24
1515



Pro84XB
Prostate 10
119
15.35



Pro90XB
Prostate 11
8.08
112.2



Pro91XB
Prostate 12
0.88
51.8



ProC215
Prostate 13
0.3



ProC234
Prostate 14
0.35



ProC280
Prostate 15
436.5



Pro109XB
Prostate 16
3.43
265



Pro110
Prostate 17
18.2
8.73



Pro125XB
Prostate 18
0.34
186



Pro326
Prostate 19
1392
110



Pro10R
Prostate 20
0.5




(prostatitis)



Pro20R
Prostate 21
24.1




(prostatitis)



Pro258
Prostate 22 (BPH)
4610



Pro263C
Prostate 23 (BPH)
0



Pro267A
Prostate 24 (BPH)
1.46



Pro271A
Prostate 25 (BPH)
0



Pro460Z
Prostate 26 (BPH)
1.47



ProC032
Prostate 27 (BPH)
14.4



Tst39X
Testis 1
0
0



Bld32XK
Bladder 1
0.44
0.41



Bld46XK
Bladder 2
0
0



Bld66X
Bladder 3
0
0



BldTR14
Bladder 4
0
0



Kid106XD
Kidney 1
0
0



Kid107XD
Kidney 2
0
0



Kid109XD
Kidney 3
0
0



Pan10343
Pancreas 1
0
0



Pan71XL
Pancreas 2
0
0



Pan77X
Pancreas 3
0
0



Liv15XA
Liver 1
0
0



Liv42X
Liver 2
0
0



ClnAS43
Colon 1
0
0



ClnAS45
Colon 2
0
0



ClnAS46
Colon 3
0
0



ClnAS67
Colon 4
0
0



ClnAC19
Colon 5
0
0



ClnAS12
Colon 6
0
0



SmI21XA
Small Intestine 1
0
0



SmIH89
Small Intestine 2
0
0



Lng47XQ
Lung 1
0.7
0



Lng60XL
Lung 2
0
0



Lng75XC
Lung 3
0
0



Lng90X
Lung 4
0
0



Mam12X
Mammary Gland 1
0
1.4



Mam59X
Mammary Gland 2
0.2
0



MamA06X
Mammary Gland 3
0
0



MamS127
Mammary Gland 4
0
0



Mam162X
Mammary Gland 5
0
0



Mam42DN
Mammary Gland 6
0
0



Ovr103X
Ovary 1
0.14
0



Ovr1005O
Ovary 2
0.2



Ovr1028
Ovary 3
0



Ovr1040O
Ovary 4
0.2



Ovr18GA
Ovary 5

0



Ovr206I
Ovary 6

0



Ovr20GA
Ovary 7

0.2



Ovr25GA
Ovary 8

0







0 = Negative






In the analysis of matching samples, the higher levels of expression were in prostate showing a high degree of tissue specificity for prostate. These results confirm the tissue specificity results obtained with normal pooled samples (Table 5).
Furthermore, the level of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 6 shows overexpression of Pro111 in 5 out of 16 primary prostate cancer samples compared with their respective normal adjacent (prostate samples 2, 5, 10, 17, and 19). Similar expression levels were observed in 3 unmatched prostate cancers (prostate samples 13, 14, 15), 2 prostatitis (prostate samples 20, 21), and 6 benign prostatic hyperplasia samples (prostate samples 22 through 27). Thus, there is overexpression in the cancer tissue of 31.25% of the prostate matching samples tested (total of 16 prostate matching samples).
Altogether, the high level of tissue specificity, plus the mRNA overexpression in 31.25% of the prostate matching samples tested are indicative of Pro111 being a diagnostic marker for prostate cancer.
Expression of Clone ID 2189835H1 (Pro115):
For the CSG Pro115, real-time quantitative PCR was performed using the following primers:











Forward Primer





5′-TGGCTTTGAACTCAGGGTCA-3′
(SEQ ID NO:27)



 



Reverse Primer



5′-CGGATGCACCTCGTAGACAG-3′
(SEQ ID NO:28)






The absolute numbers depicted in Table 7 are relative levels of expression of the CSG Pro115 in 12 normal different tissues. All the values are compared to normal thymus (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.








TABLE 7







Relative Levels of CSG Pro115 Expression in Pooled


Samples










Tissue
NORMAL














Brain
0.016



Heart
0.002



Kidney
8.08



Liver
2.20



Lung
112.99



Mammary
29.45



Muscle
0.05



Prostate
337.79



Small Intestine
7.54



Testis
1.48



Thymus
1



Uterus
1.4










The relative levels of expression in Table 7 show that Pro115 mRNA expression is higher (337.79) in prostate compared with all the other normal tissues analyzed. Lung, with a relative expression level of 112.99, and mammary (29.446) are the other tissues expressing moderate levels of mRNA for Pro115. These results establish Pro115 mRNA expression to be highly specific for prostate.
The absolute numbers in Table 7 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 8.
The absolute numbers depicted in Table 8 are relative levels of expression of Pro115 in 17 pairs of matching and 21 unmatched samples. All the values are compared to normal thymus (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual.







TABLE 8







Relative Levels of CSG Pro115 Expression in


Individual Samples















Matching






Normal



Sample ID
Tissue
Cancer
Adjacent
















Pro12B
Prostate 1
1475.9
190.3



ProC234
Prostate 2
169.61



Pro109XB
Prostate 3

639.53



Pro101XB
Prostate 4
1985.2
2882.9



Pro13XB
Prostate 5
34.9
13.9



Pro215
Prostate 6
525.59



Pro125XB
Prostate 7

556.05



Pro23B
Prostate 8
1891.4
1118.6



ProC280
Prostate 9
454.3



Pro20XB
Prostate 10
1332.6



Pro34B
Prostate 11

362.91



Pro65XB
Prostate 12

135.06



Pro69XB
Prostate 13

179.67



Pro10R
Prostate 14
143.82




(prostatitis)



Pro20R
Prostate 15
397.79




(prostatitis)



Pro258
Prostate 16 (BPH)
216.6



Pro263C
Prostate 17 (BPH)
601.25



Pro267A
Prostate 18 (BPH)
200.28



Pro271A
Prostate 19 (BPH)
111.43



Pro460Z
Prostate 20 (BPH)
53.84



ProC032
Prostate 21 (BPH)
56.94



SmI21XA
Small Intestine 1
28.8
29.9



SmIH89
Small Intestine 2
70.8
348.5



ClnAC19
Colon 1
22.73
446.47



ClnAS12
Colon 2
116.97
493.18



Kid106XD
Kidney 1
86.13
41.14



Kid107XD
Kidney 2
0.26
35.14



Lng47XQ
Lung 1
5.13
20.98



Lng60XL
Lung 2
13.93
114.78



Lng75XC
Lung 3
16.47
53.79



Mam12X
Mammary Gland 1
6.25
10.75



Mam162X
Mammary Gland 2
1.84
2.54



Mam42DN
Mammary Gland 3
23.08
35.51



Ovr10050
Ovary 1
0.9



Ovr1028
Ovary 2
261.4



Ovr103X
Ovary 3
7
0.1



Ovr20GA
Ovary 4

0



Ovr25GA
Ovary 5

0







0 = Negative






Higher levels of expression were seen in prostate, showing a high degree of tissue specificity for prostate tissue. Of all the analyzed samples different from prostate, only two cancer samples (colon 2 with 116.97 and ovary 2 with 261.4), and 5 normal adjacent tissue samples (small intestine 2, colon 1, colon 2, kidney 1, and lung 2), showed an expression comparable to the mRNA expression in prostate. These results confirmed the tissue specificity results obtained with the panel of normal pooled samples (Table 7).
Furthermore, the levels of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for the cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 8 shows higher expression of Pro115 in 3 out of 4 matched prostate cancer tissues (prostate samples 1, 5 & 8).
Altogether, the high level of tissue specificity, plus the higher expression in 75% of the prostate matching samples tested, are indicative of Pro115 being a diagnostic marker for prostate cancer.
Expression of Clone ID 3277219H1 (Pro110):
For the CSG Pro110, real-time quantitative PCR was performed using the following primers:











Forward Primer





5′-CGGCAACCTGGTAGTGAGTG-3′
(SEQ ID NO:29)



 



Reverse Primer



5′-CGCAGCTCCTTGTAAACTTCAG-3′
(SEQ ID NO:30)






The absolute numbers depicted in Table 9 are relative levels of expression of the CSG Pro110 in 12 normal different tissues. All the values are compared to normal small intestine (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.








TABLE 9







Relative Levels of CSG Pro110 Expression in Pooled


Samples










Tissue
NORMAL














Brain
6.61



Heart
0.7



Kidney
0.74



Liver
7.94



Lung
11.88



Mammary
22.78



Muscle
6.77



Prostate
3.01



Small Intestine
1



Testis
2.58



Thymus
13.74



Uterus
2.61










The relative levels of expression in Table 9 show that Pro110 mRNA expression is not as high in normal prostate (3.01) compared with all the other normal tissues analyzed.

The absolute numbers in Table 9 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 10.
The absolute numbers depicted in Table 10 are relative levels of expression of Pro110 in 33 pairs of matching samples. All the values are compared to normal small intestine (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual.







TABLE 10







Relative Levels of CSG Pro110 Expression in


Individual Samples















Matching






Normal



Sample ID
Tissue
Cancer
Adjacent
















Pro12B
Prostate 1
11.8
0.3



Pro78XB
Prostate 2
14.3
6.3



Pro101XB
Prostate 3
33.2
10.7



Pro13XB
Prostate 4
0.3
0.4



Pro23XB
Prostate 5
25.5
14.4



Pro20XB
Prostate 6
43.3
4



Pro34XB
Prostate 7
31.8
18.7



Pro65XB
Prostate 8
26.9
3.4



Pro69XB
Prostate 9
12.5
7



Lng75XC
Lung 1
1.9
3



Lng90X
Lung 2
5.5
0.5



LngAC11
Lung 3
9.3
9.7



LngAC32
Lung 4
11.2
2.2



Lng47XQ
Lung 5
11.3
0.3



Lng60XL
Lung 6
29.1
6.8



Mam12B
Mammary Gland 1
19.8
0



Mam603X
Mammary Gland 2
13.7
0



Mam82XI
Mammary Gland 3
73.5
0



MamA04
Mammary Gland 4
0
24.6



MamB011X
Mammary Gland 5
17.4
2



MamC012
Mammary Gland 6
0
12.8



MamC034
Mammary Gland 7
0
61



Mam12X
Mammary Gland 8
14
2.2



Mam59X
Mammary Gland 9
33
2.2



MamA06X
Mammary Gland 10
16.4
0.8



Liv15XA
Liver 1
4.7
0.6



Liv42X
Liver 2
7.5
2.6



Liv94XA
Liver 3
0.4
1.4



ClnAS43
Colon 1
52.9
1.4



ClnAS45
Colon 2
2.1
0.8



ClnAS46
Colon 3
39.8
3.7



SmI21X
Small Intestine 1
0.9
0.1



SmIH89
Small Intestine 2
5.8
0.9







0 = Negative






The levels of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for the cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 10 shows overexpression of Pro110 in 8 of the 9 primary prostate cancer tissues compared with their respective normal adjacent (except prostate 4). Thus, there was overexpression in 88.88% of the cancer prostate tissue as compared to the prostate matching samples tested (total of 9 prostate matching samples).

Although not tissue specific, Pro110 mRNA expression is upregulated in prostate cancer tissues. The mRNA overexpression in 88.88% of the primary prostate matching cancer samples tested is indicative of Pro110 being a diagnostic marker for prostate cancer. Pro110 also showed overexpression in several other cancers tested including small intestine, colon, liver, mammary and lung (see Table 10). Accordingly Pro110 may be a diagnostic marker for other types of cancer as well.
Expression of Clone ID 1857415; Gene ID 346880 (Pro113):
For the CSG Pro113, real-time quantitative PCR was performed using the following primers:











Forward Primer





5′-CGGGAACCTACCAGCCTATG-3′
(SEQ ID NO:31)



 



Reverse Primer



5′-CAGGCAACAGGGAGTCATGT-3′
(SEQ ID NO:32)






The absolute numbers depicted in Table 11 are relative levels of expression of the CSG Pro113 in 12 normal different tissues. All the values are compared to normal thymus (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.








TABLE 11







Relative Levels of CSG Pro113 Expression in Pooled Samples










Tissue
NORMAL














Brain
0.03



Heart
0



Kidney
0.01



Liver
0



Lung
0



Mammary Gland
0



Muscle
0.04



Prostate
489.44



Small Intestine
0.02



Testis
0.35



Thymus
1



Uterus
0.13










The relative levels of expression in Table 11 show that Pro113 mRNA expression is higher (489.44) in prostate compared with all the other normal tissues analyzed. Testis, with a relative expression level of 0.35, uterus (0.13), thymus (1.0), kidney (0.01) and brain (0.03) were among the other tissues expressing lower mRNA levels for Pro113. These results establish that Pro113 mRNA expression is highly specific for prostate.

The absolute numbers in Table 11 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 12.
The absolute numbers depicted in Table 12 are relative levels of expression of Pro113 in 78 pairs of matching and 25 unmatched tissue samples. All the values are compared to normal thymus (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. In cancers (for example, ovary) where it was not possible to obtain normal adjacent samples from the same individual, samples from a different normal individual were analyzed.







TABLE 12







Relative Levels of CSG Pro113 Expression in Individual Samples













Matched or





Unmatched





Normal


Sample ID
Tissue
Cancer
Adjacent













Pro780B/781B
Prostate 1
375.58
446.29


Pro1291B/1292B
Prostate 2
1060
31


Pro139B96/140B96
Prostate 3
41
32


Pro209B96/210B96
Prostate 4
505
255


Pro1256B/1257B
Prostate 5
165.79
141.63


Pro1293B/1294B
Prostate 6
1613.7
874.61


Pro694B/695B
Prostate 7
458.6
142.21


Pro1012B/1013B
Prostate 8
1520
864


Pro1222B/1223B
Prostate 9
939
530


Pro845B/846B
Prostate 10
1552.4
374.6


Pro1094B/1095B
Prostate 11
278.37
135.89


Pro650B/651B
Prostate 12
532.81
640.85


Pro902B/903B
Prostate 13
609.05
415.86


Pro916B/917B
Prostate 14
699.42
401.24


Pro9821110A/110B
Prostate 15
156
487.8


ProS9821326A/26B
Prostate 16
744.4
472.8


Pro9407c215
Prostate 17
1389.2


Pro9407c234
Prostate 18
305.5


Pro9407c280A
Prostate 19
894.5


Pro9409C010R
Prostate 20
269.7



(prostatitis)


Pro9404C120R
Prostate 21
299.2



(prostatitis)


Pro1000258
Prostate 22
149.6



(BPH)


Pro4001263C
Prostate 23
576



(BPH)


Pro4001267A
Prostate 24
132.1



(BPH)


Pro9411C032
Prostate 25
118.2



(BPH)


Pro4001460Z
Prostate 26
276.3



(BPH)


Pro4001271A
Prostate 27
58.7



(BPH)


Kid1064D/65D
Kidney 1
0
0.1


Kid1079D/1080D
Kidney 2
0.3
0.02


Kid1097D/1098D
Kidney 3
35.14
0.32


Kid1024D/1025D
Kidney 4
1.31
0


Kid1183D/1184D
Kidney 5
24.79
0


Kid1242D/1243D
Kidney 6
0
0


Bld469K
Bladder 1

2.88


Bld467K/468K
Bladder 2
2.65


Bld327K/328K
Bladder 3
0
4.05


Bld470K
Bladder 4

1.64


Bld665T/664T
Bladder 5
0.21
1.99


Bld1496K/1497K
Bladder 6
13.55
1.14


Bld1721K/1722K
Bladder 7
120.16
1.34


Tst239X/240X
Testis 1
31.5
0.73


TstS9820647A/47B
Testis 2
15.7
0


TstS9820663A/663B
Testis 3
72
1.4


SknS9821248A/248B
Skin 1
1.8
0.5


SknS99448A/448B
Skin 2
251.6
0


Skn99816A/816B
Skin 3
33
0.7


Sto4004864A4/B4
Stomach 1
14.12
0


Sto4004509A3/B1
Stomach 2
40.74
39


SmI9807A212A/213A
Small
0.1
0



Intestine 1


SmI9802H008/H009
Small
5.8
0.1



Intestine 2


Cln9608B012/B011
Colon 1
4.5
0


Cln9709c074ra/073ra
Colon 2
65.8
3.1


Cln4004709A1/709B1
Colon 3
1.1
0.9


Cln9405C199/C200
Colon 4
34.76
0.73


Cln9707c004gb/006ga
Colon 5
90.26
0.96


Cln96-09-B004/B003
Colon 6
17.9
20.64


Cln9612B006/B005
Colon 7
17.56
0.3


Cln9705F002D/F001c
Colon 8
21.39
0


ClnCXGA
Colon 9
429.14
142.69


Pan10343a
Pancreas 1
0
0


Pan776P/777P
Pancreas 2
0
0.15


Pan921O/922O
Pancreas 3
7.36
0


Pan714L/715L
Pancreas 4
13.57
0.11


Pan824P/825P
Pancreas 5
0
0


Lng476Q/477Q
Lung 1
0
0


Lng605L/606L
Lung 2
0
0.1


Lng1114SB/11145C
Lung 3
85.9
0


Lng0008632A/32B
Lung 4
23.85
0


Lng750C/751C
Lung 5
0.32
0.25


Lng8890A/8890B
Lung 6
10.63
0


Lng8926A/8926B
Lung 7
15.37
0


Lng0010239A/39B
Lung 8
26.17
0


Lng9502C109R/110R
Lung 9
0.68
0


LngS9821944a/44b
Lung 10
0
0


Mam00042D01/42N01
Mammary Gland 1
8.5
0


Mam59XC
Mammary Gland 2
61.07
0


Mam9706A066G/67C
Mammary Gland 3
4.84
0


Mam14153alC
Mammary Gland 4
9.72
6.99


Mam1620F/1621F
Mammary Gland 5
0.91
0


Mam00014D05
Mammary Gland 6
2.45
0


End10479B/D
Endometrium 1
133.43
1.12


End9705A125A/126A
Endometrium 2
0
0.39


End9704C281A/282A
Endometrium 3
23.5
1.56


End680o97/681o97
Endometrium 4
88.89
79.02


Utr1359O/1358O
Uterus 1
0.2
0


Utr850U/851U
Uterus 2
0
0


Utr1417O/1418O
Uterus 3
14
0.4


Utr233U96/234U96
Uterus 4
8.65
4.64


CvxVNM00052D01/52N01
Cervix 1
0.82
77.15


CvxVNM00083D01/83N01
Cervix 2
0.78
221.48


CvxND00023D01/23N01
Cervix 3
3.25
15.22


Ovr1037O/1038O
Ovary 1
0.1
0


Ovr1005O
Ovary 2
18.96


Qvr1028
Ovary 3
0


Ovr14638A1C
Ovary 4
3.2


Ovr14603A1D
Ovary 5
882.3


Ovr773O
Ovary 6
0


Ovr9702C018GA
Ovary 7

0.15


Ovr206I
Ovary 8

0


Ovr9702C020GA
Ovary 9

0


Ovr9702C025GA
Ovary 10

0


Ovr9701C035GA
Ovary 11

0.07


Ovr9701C050GB
Ovary 12

0.58





0 = Negative






In the analysis of matching samples, the higher levels of expression were in prostate, showing a high degree of tissue specificity for prostate tissue. In addition to the higher expression levels in prostate cancer samples, Pro113 expression was found to be either induced (where not expressed in normal adjacent tissues) or somewhat upregulated in several other cancers. However, the relative expression and the fold increase in prostate cancer samples far exceeds that in other cancer tissues and is highly significant.
Furthermore, the levels of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for the cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 12 shows overexpression of Pro113 in 13 out of 16 primary prostate cancer tissues compared with their respective normal adjacent (prostate samples 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 13, 14, 16). Thus, there was overexpression in the cancer tissue for 81.25% of the prostate matching samples tested. The median for the level of expression in prostate cancer tissue samples is 609, whereas the median for all other cancers is only 7.93, with the exception of one colon sample, colon 9, whose expression was similar to that found in prostate cancer tissues.
Altogether, the high level of tissue specificity, plus the mRNA overexpression in 81.25% of the primary prostate matching samples tested are indicative of Pro113 being a diagnostic marker for prostate cancer. Expression was also found to be higher in other cancer tissues compared with their respective normal adjacent tissues (kidney, bladder, testis, skin, stomach, small intestine, colon, pancreas, lung, mammary, endometrium, uterus, and ovary) thus indicating Pro113 to be a pan cancer marker.
Expression of Clone ID 1810463H1 (Pro114):
For the CSG Pro114, real-time quantitative PCR was performed using the following primers:











Forward Primer





5′-TGGGCATCTGGGTGTCAA-3′
(SEQ ID NO:33)



 



Reverse Primer



5′-CGGCTGCGATGAGGAAGTA-3′
(SEQ ID NO:34)






The absolute numbers depicted in Table 13 are relative levels of expression of the CSG Pro114 in 12 normal different tissues. All the values are compared to normal muscle (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.







TABLE 13







Relative Levels of CSG Pro114 Expression in Pooled Samples










Tissue
NORMAL














Brain
9.7



Heart
0.7



Kidney
414.4



Liver
4



Lung
882.2



Mammary
44



Muscle
1



Prostate
1951



Small Intestine
22



Testis
367.1



Thymus
25.8



Uterus
139.6










The relative levels of expression in Table 13 show that Pro114 mRNA expression is higher (1951) in prostate compared with all the other normal tissues analyzed. Lung, with a relative expression level of 882.2, kidney 414.4, testis 367.1 and uterus 139.6, are the other tissues expressing higher levels of mRNA for Pro114. These results establish Pro114 mRNA expression to be more specific for prostate than other tissues examined.

The high level of tissue specificity is indicative of Pro114 being a diagnostic marker for diseases of the prostate, especially cancer.
Expression of Clone ID zr65g11 (Pro118):
For the CSG Pro118, real-time quantitative PCR was performed using the following primers:











Forward Primer





5′-GCCCATCTCCTGCTTCTTTAGT-3′
(SEQ ID NO:35)



 



Reverse Primer



5′-CGTGGAGATGGCTCTGATGTA-3′
(SEQ ID NO:36)






The absolute numbers depicted in Table 14 are relative levels of expression of the CSG Pro118 in 12 normal different tissues. All the values are compared to normal kidney (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.







TABLE 14







Relative Levels of CSG Pro118 Expression in Pooled Samples










Tissue
NORMAL














Colon
0.87



Endometrium
19282



Kidney
1



Liver
0



Ovary
86.22



Pancreas
0



Prostate
962.1



Small Intestine
0



Spleen
0.75



Stomach
0.54



Testis
343.7



Uterus
1064










The relative levels of expression in Table 14 show that Pro118 mRNA expression is the 3rd highest in prostate (962.1) next to endometrium (19282) and uterus (1064), which are female-specific tissues. Testis, with a relative expression level of 343.7 is the only other male tissue expressing moderate levels of mRNA for Pro118. These results establish Pro118 mRNA expression to be highly specific for reproductive tissues including the prostate.
The absolute numbers in Table 14 were obtained analyzing pools of samples of a particular tissue from different individuals. They cannot be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 15.
The absolute numbers depicted in Table 15 are relative levels of expression of Pro118 in 59 pairs of matching and 21 unmatched samples. All the values are compared to normal kidney (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual.







TABLE 15







Relative Levels of CSG Pro118 Expression in Individual Samples















Matching






Normal



Sample ID
Tissue
Cancer
Adjacent
















Pro12B
Prostate 1
41700.7
22242.83



ProC234
Prostate 2
40087



Pro78XB
Prostate 3
4075.6
7066.7



Pro109XB
Prostate 4
334.4
777.2



Pro84XB
Prostate 5
11684
58290



Pro101XB
Prostate 6
21474.13
100720.8



Pro91X
Prostate 7
14849
33717



Pro13XB
Prostate 8
202.57
146.91



ProC215
Prostate 9
73243



Pro125XB
Prostate 10
629.6
521.4



Pro23B
Prostate 11
157532.6
110654.4



Pro90XB
Prostate 12
2317
64134



ProC280
Prostate 13
42020



Pro20XB
Prostate 14
2909.31



Pro34B
Prostate 15
29610
23264



Pro110
Prostate 16
13354
30991



Pro65XB
Prostate 17
10126
11270



Pro69XB
Prostate 18

2671.42



Pro326
Prostate 19
9962.3
19231



Pro10R
Prostate 20
27355




(prostatitis)



Pro20R
Prostate 21
21081




(prostatitis)



Pro258
Prostate 22(BPH)
79916.32



Pro263C
Prostate 23(BPH)
108924.5



Pro267A
Prostate 24(BPH)
92910.22



Pro271A
Prostate 25(BPH)
57004.4



Pro460Z
Prostate 26(BPH)
57449.23



ProC032
Prostate 27(BPH)
45781.44



Kid106XD
Kidney 1
3.08
217.36



Kid107XD
Kidney 2
0
38.36



Kid109XD
Kidney 3
0
123.5



Kid10XD
Kidney 4
17.69
67.8



Kid11XD
Kidney 5
16.74
360.8



Kid124D
Kidney 6
0
167.4



Bld32XK
Bladder 1
0
0



Bld47K
Bladder 2

36.38



Bld66X
Bladder 3
0
4.52



BldTR14
Bladder 4
0
12.17



BldTR17
Bladder 5
0
0



Bld46XK
Bladder 6
16.5
0



Tst39X
Testis 1
116.6
24.35



Tst647T
Testis 2
856.16
43.5



StoAC44
Stomach 1
0
0



StoAC93
Stomach 2
0
0



SmI21XA
Small Intestine 1
68.45
0



SmIH89
Small Intestine 2
0
0



ClnAC19
Colon 1
149
21.33



ClnAS12
Colon 2
0
0



ClnB34
Colon 3
0
0



ClnB56
Colon 4
13.04
5.22



ClnAS43
Colon 5
0
0



Lng47XQ
Lung 1
0
0



Lng60XL
Lung 2
0
0



Lng75XC
Lung 3
0
3.38



Lng90X
Lung 4
0
0



LngBR26
Lung 5
0
26.82



Pan10343
Pancreas 1
50.47
0



Pan77X
Pancreas 2
281.1
0



Pan92X
Pancreas 3
18.41
0



Pan71XL
Pancreas 4
0
0



Pan82XP
Pancreas 5
0
0



PanC044
Pancreas 6
0
0



Mam12X
Mammary Gland 1
0
0



Mam162X
Mammary Gland 2
0
0



Mam42DN
Mammary Gland 3
0
0



MamS127
Mammary Gland 4
12.58
0



Mam14DN
Mammary Gland 5
0
0



End28XA
Endometrium 1
331.9
1824



End3AX
Endometrium 2
27825
65839



End4XA
Endometrium 3
10.3
15935



Utr141O
Uterus 1
18885
18116



Utr23XU
Uterus 2
3358
7674



CvxKS52
Cervix 1
0
0



CvxKS83
Cervix 2
0
0



Ovr1005O
Ovary 1
72.86



0vr1028
Ovary 2
0



0vr638A
Ovary 3
0



Ovr63A
Ovary 4
90.88



0vr773O
Ovary 5
1.21



Ovr1040O
Ovary 6
5.08



Ovr105O
Ovary 7
0



Ovr1118
Ovary 8
7.41



Ovr103X
Ovary 9

32.78



Ovr20GA
Ovary 10

0



Ovr25GA
Ovary 11

1173.83



Ovr35GA
Ovary 12

313.4



Ovr50GB
Ovary 13

823.1



Ovr18GA
Ovary 14

40.6



0vr206I
Ovary 15

1264



Ovr230A
Ovary 16

1285







0 = Negative






In the analysis of matching samples, the higher levels of expression were in prostate, endometrium, testis, and ovary showing a high degree. of tissue specificity for reproductive tissues. These results confirmed the tissue specificity results obtained with the panel of normal pooled samples (Table 14).

Furthermore, the levels of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual were compared. This comparison provides an indication of specificity for the cancer (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). Table 15 shows overexpression of Pro118 in 5 out of 14 primary prostate cancer tissues (prostate samples 1, 8, 10, 11, 15) compared with their respective normal adjacent. Thus, there was overexpression in the cancer tissue for 35.71% of the prostate matching samples tested (total of 14 prostate matching samples). Expression of Pro118 was similarly higher in 3 unmatched cancer tissues (prostate samples 9, 13, 14), 2 prostatitis (prostate samples 20, 21), and 6 benign hyperplasia tissues (prostate samples 22 through 27).
Altogether, the high level of tissue specificity, plus the mRNA overexpression in 35.71% of the primary prostate matching samples tested are indicative of Pro118 being a diagnostic marker for prostate cancer.

## Claims

1. A method for diagnosing the presence of prostate cancer in a patient comprising:
(a) determining levels of CSG in prostate cells or tissue or bodily fluids in a patient; and
(b) comparing the determined levels of CSG with levels of CSG in prostate cells or tissue or bodily fluids from a non-cancerous control, wherein an increase in determined levels of CSG in said patient versus said control is associated with the presence of prostate cancer,

wherein said CSG comprises the protein expressed by the nucleic acid comprising SEQ ID NO:7.

2. A method of imaging prostate cancer in a patient comprising administering to the patient an antibody which specifically binds a CSG comprising the protein expressed by the nucleic acid comprising SEQ ID NO:7.

3. The method of claim 2 wherein said antibody is labeled with paramagnetic ions or a radioisotope.