CA2328377A1 - A novel method of diagnosing, monitoring, and staging prostate cancer - Google Patents

Abstract

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

Description

A NOVEL METHOD OF DIAGNOSING, MONITORING, AND STAGING PROSTATE CANCER
FIELD OF THE INVENTION
This invention relates, in part, to newly developed assays for detecting, diagnosing, monitoring, staging, and prognosticating 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 in the United States, 41,400 deaths would result from this disease, 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 chance 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 classification 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 further subdivided into 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 both 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 further subdivided into two stages. In sub-stage Cl, 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. The fourth stage is metastatic cancer and is also subdivided into two 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 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 93o and 29%, respectively.
Accordingly, there is a great need for increasingly sensitive 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.
In the present invention, methods are provided for detecting, diagnosing, monitoring, staging and prognosticating cancers, particularly prostate cancer via seven (7) Prostate Specific Genes (PSG). The seven PSGs refer, among other things, to native proteins expressed by the genes comprising the polynucleotide sequences of any of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7. In the alternative, what is meant by the seven PSGs as used herein, means the native mRNAs encoded by the genes comprising any of the polynucleotide sequences of SEQ
ID N0: 1, 2, 3, 4, 5, 6 or 7 or levels of the genes comprising any of the polynucleotide sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7.
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.
SZJI~IAIEtY 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 in a patient which comprises measuring levels of PSG in a sample of cells, tissue or bodily fluid from the patient and comparing the measured levels of PSG with levels of PSG in preferably the same cells, tissue, or bodily fluid type of a control, wherein an increase in the measured PSG levels in the patient versus levels of PSG in the control is associated with prostate cancer.
Another object of the present invention is to provide a method of diagnosing metastatic prostate cancer in a patient which comprises measuring PSG levels in a sample of cells, tissue, or bodily fluid from the patient and comparing the measured PSG levels with levels of PSG in preferably the same cells, tissue, or bodily fluid type of a control, wherein an increase in measured PSG levels in the patient versus levels of PSG in the control is associated with a cancer which has metastasized.
Another object of the present invention is to provide a method of staging prostate cancer in a patient which comprises identifying a patient having prostate cancer, measuring levels of PSG in a sample of cells, tissues, or bodily fluid obtained from the patient, and comparing the measured PSG levels with levels of PSG in preferably the same cells, tissue or bodily fluid type of a control. An increase in measured PSG levels in the patient versus PSG levels in the control can be associated with a cancer which is progressing while a decrease or equivalent level of PSG measured in the patient versus the control can be associated with a cancer which is regressing or in remission. ' Another object of the present invention is to provide a method of monitoring prostate cancer in a patient for the onset of metastasis. The method comprises identifying a patient having prostate cancer that is not known to have metastasized, periodically measuring levels of PSG in a sample of cells, tissues, or bodily fluid obtained from the patient, and comparing the measured PSG levels with levels of PSG in preferably the same cells, tissue, or bodily fluid type of a control, wherein an increase in measured PSG levels versus control PSG levels is associated with a cancer which has metastasized.
Yet another object of the present invention is to provide a method of monitoring the change in stage of prostate cancer in a patient which comprises identifying a patient having prostate cancer, periodically measuring levels of PSG
in a sample of cells, tissue, or bodily fluid obtained from the patient, and comparing the measured PSG levels with levels of PSG in preferably the same cells, tissues, or bodily fluid type of a control wherein an increase in measured PSG levels versus the control PSG levels is associated with a cancer which is progressing and a decrease in the measured PSG levels versus the control PSG levels is associated with a cancer which is regressing or in remission.
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 _ 5 _ 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.
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 PSG measured in a patient with levels of PSG in a control. What is meant by "levels of PSG" as used herein, means levels of the native protein expressed by the gene comprising the polynucleotide sequence of any of SEQ ID
NO: 1, 2, 3, 4, 5, 6 or 7. In the alternative, what is meant by "levels of PSG" as used herein, is levels of the native mRNA encoded by the gene comprising any of the polynucleotide sequence of SEQ ID N0: 1, 2, 3, 4, 5, 6 or 7 or levels of the gene comprising any of the polynucleotide sequence of SEQ ID
N0: 1, 2, 3, 4, S, 6 or 7. Such levels are preferably measured in at least one of cells, tissues and/or bodily fluids, and includes determination of both normal and abnormal levels of PSGs. Thus, for instance, a diagnostic assay in accordance with the invention for diagnosing overexpression of PSG protein compared to control bodily fluids, cells, or tissue samples may be used to diagnose the presence of cancers, including prostate cancer. Any of the seven PSGs may be measured alone in the methods of the invention, all together or in various combinations of the seven PSGs.
By "control" it is meant a human patient without cancer-and/or non cancerous samples from the patient, also referred to herein as a normal human control; in the methods for diagnosing or monitoring for metastasis, control may also include samples from a human patient that is determined by reliable methods to have prostate cancer which has not metastasized.
All the methods of the present invention may optionally include measuring the levels of other cancer markers as well as PSG. Other cancer markers, in addition to PSG, useful in the present invention will depend on the cancer being tested and are known to those of skill in the art. For example, simultaneous testing for increases in PSA as well as increases in PSG are also within the scope of the present invention and believed to provide a higher level of assurance that such cancer being tested is metastatic or the onset of metastasis has occurred.
Diagnostic Assays The present invention provides methods for diagnosing the presence of prostate cancer by analyzing for changes in levels of PSG in cells, tissues or bodily fluids compared with levels of PSG in cells, tissues or bodily fluids of preferably the same type from a normal human control, wherein an increase in levels of PSG 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 PSG, 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 _ -7 _ variety of means known to those of skill in the art. For example, in the case of prostate cancer, patients are typically diagnosed with prostate cancer following traditional detection methods, In the present invention, determining the presence of PSG 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, tissue, or bodily fluid are PSGs, and are compared with levels of PSG 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 PSG in serum, this level is preferably compared with the level of PSG in serum of a normal human patient. An increase in the PSG 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 PSG, are at least two times higher, and most preferable are at least five times higher, than in preferably the same cells, tissues, or bodily fluid of a normal patient.
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 a sample of cells, tissues, or bodily fluid from such patient for PSG. Then, the method _ g _ compares PSG levels in such cells, tissues, or bodily fluid with levels of PSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in PSG levels in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of PSG is associated with a cancer which is regressing or in remission.
Monitoring 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 PSG; and comparing the PSG levels in such cells, tissue, or bodily fluid with levels of PSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in PSG
levels in the patient versus the normal human control is associated with a cancer which has metastasized.
Further provided by this invention is a method of monitoring the change in stage of prostate cancer in a human having such cancer. The method comprises identifying a human patient having such cancer; periodically analyzing a sample of cells, tissue, or bodily fluid from such patient for PSG;
comparing the PSG levels in such cells, tissue, or bodily fluid with levels of PSG in preferably the same patient.
Monitoring such 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 TecImique,s Assay techniques that can be used to determine levels of gene expression, such as PSG of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, WO 99/b0162 g _ reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses and ELISA
assays. 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 PSG, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds specifically to PSG. 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 PSG 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 PSG binds to the specific antibody attached to the polystyrene dish.
Unbound sample is washed out with buffer. A reporter antibody specifically directed to PSG and linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to PSG.
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 PSG
antibodies, produces a colored reaction product. The amount of color developed in a given time period is proportional to the amount of PSG protein present in the sample. Quantitative results typically are obtained by reference to a standard curve.
A competition assay may be employed wherein antibodies specific to PSG attached to a solid support and labeled PSG and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of PSG
in the sample.
Nucleic acid methods may be used to detect PSG 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 PSG 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 PSG
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.
The above tests can be carried out on samples derived from a variety of patients' cells, bodily fluids and/or tissue extracts (homogenates or solubilized tissue) such as from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva, or any other bodily secretion or derivative thereof. Blood can include whole blood, plasma, serum, or any derivative of blood.
EXAME?~ES
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 specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.
EXAI~I~E 1: PSGs Searches were carried out and PSGs identified using the following Search Tools as part of the LIFESEQ~ database available from Incyte Pharmaceuticals, Palo Alto, CA:
1. Library Comparison (compares one library to one other library) allows the identification of clones expressed in tumor and absent or expressed at a lower level in normal tissue.
2. Subsetting is similar to library comparison but allows the identification of clones expressed in a pool of libraries and absent or expressed at a lower level in a second pool of libraries.

3. Transcript Imaging lists all of the clones in a single library or a pool of libraries based on abundance.
Individual clones can then be examined using Electronic Northerns to determine the tissue sources of their component ESTs.
4. Protein Function: Incyte has identified subsets of ESTs with a potential protein function based on homologies to known proteins. Some examples in this database include Transcription Factors and Proteases. Some leads were identified by searching in this database for clones whose component ESTs showed disease specificity.
Electronic subtractions, transcript imaging and protein function searches were used to identify clones, whose component ESTs were exclusively or more frequently found in libraries from specific tumors. Individual candidate clones were examined in detail by checking where each EST originated.
Table l:
SEQ ID Clone ID # Gene ID

NO: #

1 1550426 244673 Protein Function (Transcription Factors) 2 1255804 14878 Subletting 3 1808432 255819 Subsetting 4 3930803 none Subsetting 5 645804 235032 Subletting 6 1862352 221558 Subletting 7 1450626 236019 Subletting EXAMPLE 2: Measurement of SEQ ID NO: l; Clone ID # 1550426;
Gene ID #244673 (pro101) The example is 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 are 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) .
Relative Quantitation of Gene Expression Real-time quantitative PCR with fluorescent Taqman probes is a quantitative detection system utilizing the 5' 3' nuclease activity of Taq DNA polymerise. 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 polymerise 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, CA, 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) 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 are used as the basis for comparative results (calibrator).
Quantitation relative to the "calibrator" is obtained using the standard curve method or the comparative method (User Bulletin #2: ABI PRISM 7700 Sequence Detection System).
To evaluate the tissue distribution, and the level of pro101 (SEQ ID N0:1) in normal and tumor tissue, total RNA
was extracted from tumor and matched normal adjacent tissues and from unmatched tumor and normal tissues. Subsequently, first strand cDNA was prepared with reverse transcriptase and the polymerise chain reaction carried out using primers and Taqman probe specific to pro101 (SEQ ID N0:1). The results WO 99/60162 PtvTYUS99/t 0548 were obtained using the ABI PRISM 7700 Sequence Detector. The absolute numbers are relative levels of expression of pro101 (SEQ ID NO: l) compared to the calibrator.
The absolute numbers are depicted in the following Table 2 as relative levels of expression in 12 normal tissues of pro101 (SEQ ID N0:1) compared to kidney (calibrator).
These RNA samples were generated by pooling samples from a particular tissue from different individuals.
Table 2: Relative levels of pro101 Expression in Pooled Samples Tissue NORMAL

Brain 1.2 Heart 2 Kidney 1 Liver 7.2 Lung 4g,2 Mamma r y 2 . 5 Prostate 1418.4 Spleen 1.6 Small 1.9 Testis 57.3 Thymus 1.3 Uterus J 7 , 6 '-The relative levels of expression in Table 2 show that for the PSG pro101 (SEQ ID NO: l) mRNA expression is more than 20 fold higher in the pool of normal prostate compared with the other 11 normal tissue pools analyzed. These results demonstrate that mRNA expression of the PSG is highly specific for prostate.
The tissues shown in Table 2 correspond to pools of samples from different individuals. The tissues shown in the following Table 3 were obtained from individuals and are not pooled. Hence the values for mRNA expression levels shown in Table 2 cannot be directly compared to the values shown in Table 3.

The absolute numbers in Table 3 are relative levels of expression of pro101 (SEQ ID N0:1) compared to kidney (calibrator), in 60 pairs of matching samples. Each matching pair contains the cancer sample for a particular tissue and the normal adjacent sample for that same tissue from the same individual. The results from 3 unmatched ovary tumor, 3 unmatched normal ovary, 1 unmatched mammary tumor and 1 unmatched normal mammary gland are also shown.
Table 3: Relative Levels of pro101 Expression in Individual Samples T SUE ANCER MATCHING U TCHED

Prostate 1 103.9 0 Prostate 2 2219 - 84.2 Prostate 3 5048.2 3623.6 Prostate 4 11052.3 2029.4 Prostate 5 229.1 41.1 Prostate 6 57.9 25.3 Prostate 7 58.5 57.069 Prostate 8 1074.6 610.8 Prostate 9 32.7 79.3 Prostate 10 15.8 2.09 Prostate 11 436.4 438 Prostate 12 49.5 59.3 _ Prostate 13 128 56 Bladder 1 0 0 Bladder 2 0 0 Bladder 3 0.7 0 Colon 1 0 0 Colon 2 0 0 Colon 3 0 0 Colon 4 3.3 1.9 Colon 5 0.1 0.8 Colon 6 0 0 Lung 1 0 0 Lung 2 0.5 1.6 Lung 3 1.4 2.1 Lung 4 0 0 Lung 5 0 0 Kidney 1 0 0 Kidne 2 0 0 Kidne 3 0 0 Kidne 4 0 0 Liver 1 1.5 5.7 Liver 2 26.9 7.9 Liver 3 0 0 Pancreas 1 0.9 0.9 Pancreas 2 3 0 Pancreas 3 0 0 Pancreas 4 0 0 Pancreas 5 0 0 Stomach 1 0 0 Stomach 2 0 0 Stomach 3 0 0 Stomach 4 0 0 Stomach 5 0 0 Sm Int 1 0 0 Sm Int 2 0 0 Testis 1 0 0 Mammar 1 4 0 Mammar 2 5.6 0 Mamma r 3 0 . 5 0 Mamma r 4 0 . 4 0 Mamma r 5 0 . 5 Mammar 6 0 Endo 1 1.6 7.6 Endo 2 0 0 Endo 3 0 0 Endo 4 0.3 0.2 Endo 5 5.8 5 Uterus 1 0 0 Uterus 2 0 0 Uterus 3 0 0 Uterus 4 2.2 2.6 Ovar 1 1.4 Ovar 2 11.6 Ovar 3 1.5 Ovar 4 22.9 Ovar 5 0 Ovar 6 1.8 Among 128 samples in Table 3 representing 14 different tissues, the higher levels of expression are consistently in prostate tissues. These results confirm the tissue specificity results obtained with normal samples shown in Table 2. Table 2 and Table 3 represent a combined total of 90 140 samples in 18 human tissue types. Sixty-eight samples representing 13 different tissue types excluding prostate had no detected pro101 mRNA (Table 3). In 4 tissues (stomach small intestine kidney and testis) no pro101 (SEQ ID N0:1) mRNA was detected for any sample tested from individuals (Table 3). Expression of this PSG was detected in testis in the pooled normal sample (Table 3). The median expression in prostate cancer samples in Table 3 is 166.5 units. Excluding Ovary 4 (Normal), only 1 sample in Table 3, Liver 2 (Cancer), is greater than 10% of this value.
Comparisons of the level of mRNA expression in prostate tumor samples and the normal adjacent tissue from the same individuals are also shown in Table 3. The PSG pro101 (SEQ ID NO:1) is expressed at higher levels in 9 of 13 (69$) prostate cancer tissues (Prostate 1, 2, 3, 4, 5, 6, 8, 10 and 13) compared with the corresponding normal adjacent tissue.
The level of expression of this PSG is lower in prostate tumor compared to normal adjacent tissue in two samples (Prostate 9 and 12). Equivalent levels of expression were detected in two matched samples (Prostate 7 and 11). Previous mRNA
expression analysis for genes coding for the diagnostic markers PSA and PLA2 showed higher expression of the mRNA in 40o to 800 of the tumor samples compared to matching normal adjacent tissue. Higher expression in the tumor sample compared to the corresponding normal adjacent tissue is observed for Bladder 3, Colon 4, Liver 2, Pancreas 2, Endometrium 5 and. Mammary 1, 2 and 3. Higher expression in the normal adjacent samples is observed for Colon 5, Lung 2, Lung 3, Liver 1, Endometrium 1 and Uterus 4. However, the levels detected are in most cases comparable amongst the different tissues and low compared to levels found in most prostate tissues.
The high level of tissue specificity, plus the mRNA
overexpression in 9 of 13 of the prostate tumor samples tested compared to the normal adjacent tissues are believed to make the PSG, pro101 (SEQ ID N0:1) a good diagnostic marker for detection of prostate cancer using mRNA.

SEQUENCE LISTING
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<221> unsure <222> (1480) <220>
<221> unsure <222> (1532) <220>
<221> unsure <222> (1562)..(1566) <220>
<221> unsure <222> (1569) <220>
<221> unsure <222> (1571) <220>
<221> unsure <222> (1631) <900> 3 gctcctacag ccgcatctgc gttaacatag catccctatg gccactgtct cccttgatcc 60 ccacagccat cctaggagaa aggcagaatg tcataatttg ctaaaaggga tgctgaggct 120 ctgggaggga aagggacttg cctaaagccc cagggtgaag cagcatctct ggactcccag 180 tccagtgatc ttgcccaata ctttgctgct tgcctatacc cctctaactt ggtcaacagc 240 acatcacagg gcaagcccaa tccctgcttc atttttatat atgggcgctg gtccacagcc 300, ccactctcca gccatttgga aacaaaaaca gatgctattg ttcttcctta gagaacgtgg 360 ccagtggaga cggcacactg gaaatcagag tgaatgttct tgaaagaggg tcacgggtca 420 acaaggccca gccaaaggat gcagtagaac cattttcctt agaaatcttt gggagtgaag 480 taggcttcag ccactaccca tccctgccct tgcggctacc actaccccat tagtttagac 540 agggtcgggc ggggaggggt gtggagaaga aatgagcttg cctgtngccc ccaggctccc 600 tctgtcctag ctcaggtctg ggtgccattc tttacactcg tgtgctcgct cacgcacaca 660 tcacacacct tgctggtcac acagtcacag actcgcctct gctcctgtgg tccagtggcc 720 ggacaccccc tgggatggct caaaggagtc aggacttgga agtggggaca tcagggtagc 780 tgaaggaaat ccacacaccc agagcatctc ggagttcaga ctctcagacc tgaagtaggc 840 gcccccggga ctgggctagg agttggacgg aatggaggat ggaggacagc gagaagaaag 900 gaagagaaat gcaaagtgtg ggcagccgcc aagagtgaaa atagagggaa gtgtcatgca 960 agtgctggac agaaggcggc aggtgggacg agccccacag ccccctcctc aaaaacgacc 1020 acctccagga ctcagtgatc cctggggggc aggctctgcc agccctcggc cacacgtggc 1080 tccggcaccc atggtcccag tgccttggat ggagacggcc agttctggcg gccagatgtg 1190 gtgctctgga atccagtccc atttccttcc tggccacgcc tgttccagcg gcctctttgg 1200 ctgcattcag cccctactta cctggggacc ccggctgggg cacaagagca ccaggggggt 1260 agggcccaaa gggatcaggg gaagcctctg gcctggaggg tatggggcac gcttccccaa 1320 gggcggaccc ggcaggagga agcccaggag ctgggtcctg ccgcccagga gctgggccct 1380 gccacccagg ccgggctagg gacatggcag ggcctgggca tcctgacgct ggacttgggc 1440 gacctgggag gcacagggag gggagagatg ggcggccccn acccagcgca gtgccggcca 1500 caccccaagg cggttgccag agcttaaggc cnggccccag caggagaaca tcccagctcc 1560 annnnnccnc nccgcagcca gtgctccttg tcaagctccc cccgtcactc caggtgggag 1620 ccaccccggt nagggggtgt gccacttgcc cccagggcac tcctctgggc atcccgggtg 1680 ggggattttg gggccgtggg gggcagtctc tggtacctgt gtgcgtcagg gatgctctgc 1740 acctgcaacc aggtgtcgtc cacgggcggg ggcatgggca tggtgacagt ggtcctgttg 1800 atgtcaccga tgatgctgag cgcctccttc agcgcgtggt gcatgtgcag catctcgtcg 1860 tgctgctgtg cctgctctgc caactcctcc atcagtgtgt tctggttccc acatgagtac 1920 atattggcca gcggctccga gatgatgaac tccggggtct gagagtgggc aaacagggaa 1980 gaaggttggg acctggtgcc tgtgccgccc tggctgcctt gctgggccct tctgggactg 2040 tgcgctggac ttggagcccc ttggagtatg gcttttcaca cgggcttcta taccgcttcg 2100 actggaagat ccacctcccc actgcctttt ctcactcaga tggggacacc gaggtccaga 2160 ggaaaagaca cctgtcaaat gtcacagatc tgggagggga cttaagacct atcatgccaa 2220 gaggacacct gtctactcag tttttttttg gtggggcggg gggcgnnnnn nnnnnnnnnn 2280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnggagttgg 2520 agttgatgcc tggatacagg agctctgtgg gtgggagtga gacaaaacac agggtcctga 2580 gctctgggga ccaagcaatg tcctctggtg aaaaaaatcc tggacttgct ggcagaagat 2640 ttgcctctta cttgccatgt gctctgaata catttacctg ccctctggga aaa 2693 <210>4 <211>292 <212>DNA

<213>Homo Sapiens <220>
<221> unsure <222> (289) <400> 9 aagaatatga gatttgctta gaaatgaagg actggaagga gcccacagag ttatttttta 60 aactatccag taaggcttag agggtttcaa tcagaaatat gtgttagggg aaaaaatgca 120 ctttttctat attaaaaaat attattttct tcttttaaat gtaaagcatt cctattgtga 180 agaattgaga aaatacagaa aagtacaaag aaaaacatta cctacaactc caccatccgt 240 gattatcact gttcacattt gtggctcatt tttcagtatk tctnttattt as 292 <210> 5 <211> 2699 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> (52) <220>
<221> unsure <222> (74) <220>
<221> unsure <222> (76) <220>
<221> unsure <222> (80) <220>
<221> unsure <222> (92) <220>
<221> unsure <222> (97) <220>
<221> unsure <222> (123) <220>
<221> unsure <222> (132) <220>
<221> unsure <222> (173) <220>
<221> unsure <222> (217) <220>
<221> unsure <222> (257) <220>
<221> unsure <222> (2539) <400> 5 tactatattg ctcagcattt ctaagtattc tctaagtgct ctttatttat gntttaaaat 60 agctctctta cccngntgcg ncgactagaa gancttgntt taggaaacaa tgaaatatat 120 WO 991601b2 PCT/US99/10548 aanttgccag antcaattgg agccctctta catctaaaag atctctggtt ggntggaaat 180 caactgtcag aattacctca ggaaatagga aatctgnaga acctgctgtg tttagatgtc 240 tctgaaaaca ggttggnaag acttcctgaa gaaatcagtg gcctgacttc attaacggat 300 ttagtcattt cccagaactt attagaaacg attccggatg gcattggaaa actaaagaaa 360 ctgtcaatct tgaaggtgga tcagaataga ctcacacagt tgcctgaagc agttggggaa 420 tgtgaaagtc tcactgagtt agttcttaca gaaaatcagc tcctgaccct gcctaaaagc 480 attggaaaac taaagaagtt gagcaacttg aatgcagaca gaaataaatt agtgtcctta 540 ccaaaagaga tcggcgggtg ctgcagcctc actgtgttct gtgtacgtga caacagacta 600 actcggatac ctgcagaggt gtcacaggca acagaacttc atgtcctgga tgtggcaggg 660 aacaggttgc tgcatctacc tttatccctg actgccttga agttgaaggc tctgtggcta 720 tctgacaacc agtcccagcc cctgcttaca ttccagacag acacagacta caccacagga 780 gagaagattt taacctgtgt cttacttcct cagctgcctt ctgaacctac ttgtcaagag 840 aatctgcctc gctgtggtgc actggagaac ttggtaaatg atgtctctga tgaagcctgg 900 aacgagcgtg ctgtcaacag agtcagtgcg atccgatttg tggaggatga gaaagatgaa 960 gaagacaatg agacgagaac acttctaagg cgagccactc cacacccagg ggagttaaag 1020 cacatgaaaa agacagtgga gaatttacgg aatgacatga atgctgctaa aggactggac 1080 tcaaacaaaa acgaggtcaa tcatgccatt gaccgagtga ccacttctgt gtagagtttc 1140 acctccaagt tttacctcct gtgtcttcct ctgctgtcga gacgttcctg tctgcttccc 1200 gggagcctca cgtgctcctt gtcctaacca gcccccgcgc gccatcttcc cgtggagtgt 1260 ggggaagctg ctgtctccca ggaagtgcct tactcatccc gcaaccagtc agcgcaccag 1320 tggtctcccg gtgtgatttt tttttttttt aatttcagtt gtttgtaata agtagaatac 1380 actactgtaa acatacgacc tttgtttttg tcttatgttg gggtaaagga aagcaggaag 1440 gggaattttt atcctcctcc cttccgtaaa gtgctgggat attttgaatc ccccaagttc 1500 ccttggacct actgatgaga gatagtttta tgtatgggga aaaatggata ctttttaaac 1560 cttttttggc agctcagatg gtgtaaattt taaaattttg tataggtatt tcataacaaa 1620 aatatgtatt tcttttttgt tattttatct tgaaaacggt acatatttta gtatttgtgc 1680 agaaaaacaa gtcctaaagt atttgttttt atttgtacca tccacttgtg ccttactgta 1740 tcctgtgtca tgtccaatca gttgtaaaca atggcatctt tgaacagtgt gatgagaata 1800 ggaatgtggt gttttaaagc agtgttgcat tttaatcagt aatctacctg gtggatttgt 1860 ttttaaccaa aaagatgaat tatcaatgat ttgtaattat atcggttgat tttttttgaa 1920 aagatgaacc aaaggatttg actgctaata ttttattcct tacacttttt ttctgaataa 1980 gtctctcata atgagtgcag tgtcagactg tgcctactct gatggtatgt gccatttgta 2040 aaataaaata gagcagaaaa acacaaaaag agaacactgg ttcagacatt cagtgggcaa 2100 gtaaattatg gactgcaaaa taatgatttt tattcaagaa agctttaaaa gttttatatc 2160 cagatataca accacaataa agcaaaataa cctactatca aaatagaaat gttgctatct 2220 ttataagtgc aatttaattt gtaaatagag tttgaatcaa agtatcacaa aatactgctt 2280 caagatttaa ttttaaatct gctaatttaa gggatattgg gaaaagtttt ggtgtgtttc 2340 tgttgatttc ttttttgtat gctgtgataa aagagaaatg aaaagtgcca gtcactgtgt 2400 ggtgtctagg aaaatcatat atattttttt ctccaagaaa taaattcatc ctggacattg 2460 gccatacagc tttttaaaat tattactttg tatgttcaag tgatagcagg tagccaaatt 2520 ctttgacagt gtgctctgnt ctgttaaata tctaaattac ccgtcagttg tgagtgacct 2580 cctgtgggac ttgcattcac atggggcaga gcccagaatt gcctttgact ctggctagta 2640 attttgggtt gtggctatct ggccaattgg actccttata aacccgtctt caac 2694 <210> 6 <211> 1335 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> (17) <400> 6 tcatatagta ggaaganaag cacctaggtt tgaggccagg gctggctgct gtcagaacct 60 aggccctccc ctgccttgct ccacacctgg tcaggggaga gaggggagga aagccaaggg 120 aagggaccta actgaaaaca aacaagctgg gagaagcagg aatctgcgct cgggttccgc 180 agatgcagag gttgaggtgg ctgcgggact ggaagtcatc gggcagaggt ctcacagcag 240 ccaaggaacc tggggcccgc tcctcccccc tccaggccat gaggattctg cagttaatcc 300 tgcttgctct ggcaacaggg cttgtagggg gagagaccag gatcatcaag gggttcgagt 360 gcaagcctca ctcccagccc tggcaggcag ccctgttcga gaagacgcgg ctactctgtg 920 gggcgacgct catcgccccc agatggctcc tgacagcagc ccactgcctc aagccgtggc 480 cgctacatag ttcacctggg gcagcacaac ctccagaagg aggagggctg tgagcagacc 540 cggacagcca ctgagtcctt cccccacccc ggcttcaaca acagcctccc caacaaagac 600 caccgcaatg acatcatgct ggtgaagatg gcatcgccag tctccatcac ctgggctgtg 660 cgacccctca ccctctcctc acgctgtgtc actgctggca ccagctgcct catttccggc 720 tggggcagca cgtccagccc ccagttacgc ctgcctcaca ccttgcgatg cgccaacatc 780 accatcattg agcaccagaa gtgtgagaac gcctaccccg gcaacatcac agacaccatg 840 gtgtgtgcca gcgtgcagga agggggcaag gactcctgcc agggtgactc cgggggccct 900 ctggtctgta accagtctct tcaaggcatt atctcctggg gccaggatcc gtgtgcgatc 960 acccgaaagc ctggtgtcta cacgaaagtc tgcaaatatg tggactggat ccaggagacg 1020 atgaagaaca attagactgg acccacccac cacagcccat caccctccat ttccacttgg 1080 tgtttggttc ctgttcactc tgttaataag aaaccctaag ccaagaccct ctacgaacat 1140 tctttgggcc tcctggacta caggagatgc tgtcacttaa taatcaacct ggggttcgaa 1200 atcagtgaga cctggattca aattctgcct tgaaatattg tgactctggg aatgacaaca 1260 cctggtttgt tctctgttgt atccccagcc ccaaagacag ctcctgccat atatcaagtt 1320 tcaataaata tttct 1335 <210> 7 <211> 1079 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> (268) <220>
<221> unsure <222> (688) <220>
<221> unsure <222> (700) <400> 7 tttttgaaga atgccctgca aggcatcaac tggaatgtgt ttattaccaa acaagacaga 60 agagaaceag ggcctgactt ggcagtggcc ccaggctgca tgggctcagg taggctcaga 120 ccggccccag gagtgggaga gcccagagaa gagggaaaaa gagtagtggc caggaggggt 180 ctggctggga catgccactc tgggccatca gcttctggat ccactcaaag tggtggctga 290 tattggtgta gacaccgggc cgattggncc gaccacagcc cactccccag ctcacgactc 300 caatctgata ccacagtcca ttcttgttac aggccaaggg tccacctgag tcaccgaagc 360 aggcatcctt cccgccttgg gcattgccag cacaaaccat gtctccaaag atgtccttgc 920 ggaaactgta cttgaggaag aggtggttgc acatagagtt gtttatgatg gcgacctgaa 480 cttcctggag ggtgtgggga gatggcagtg cctcatcctc tttgatgtac ccccagccag 540 tcacccagca gtctgtccgg ttctcaaact caaatgtgga ggcctggaga cagatgggct 600 ggatgtgttt agtgtaggtg acaggtgcag acagcttcac caaggcaatg tcatagggtg 660 aattccccag ttagcgaggg ctcagatnga tattcgatan gaagtaacgg gtgtagtagg 720 cctgcaggct ccagaaggat ggcatggaag tcagctggcc aaactggacc atccacccgg 780 agggatcact aaggtcacta taggtttcaa agcagtgcgc cgccgtgagt gcccagcggt 840 ggctgagcag gctcactccg catacgtggg aatcccacag gcgcaggctc ccctgccacg 900 gccaacgccc gagttcggcg tcctctccac ccacgatgcg cgacgtgatg acccgtcggc 960 cgcatggtcc tgataagggc gccgcctcct gcgactccgg cttcctgagt ccagcccgag 1020 ccagcagcag cgccagcagc agcgccccgc gcgcgcccat ggcctcctct cccgcggtg 1079

Claims (6)

What is Claimed is:

1. A method for diagnosing the presence of prostate cancer in a patient comprising:
(a) measuring levels of PSG in a sample of cells, tissue or bodily fluid obtained from the patient; and (b) comparing the measured levels of PSG with levels of PSG in a sample of cells, tissue or bodily fluid obtained from a control, wherein an increase in measured levels of PSG
in the patient versus the PSG levels in the control is associated with the presence of prostate cancer.

2. A method of diagnosing metastatic prostate cancer in a patient comprising:
(a) measuring levels of PSG in a sample of cells, tissue, or bodily fluid obtained from the patient; and (b) comparing the measured levels of PSG with levels of PSG in a sample of cells, tissue, or bodily fluid obtained from a control, wherein an increase in measured PSG levels in the patient versus the PSG levels in the control is associated with a cancer which has metastasized.

3. A method of staging prostate cancer in a patient comprising:
(a) identifying a patient suffering from prostate cancer;
(b) measuring levels of PSG in a sample of cells, tissue, or bodily fluid obtained from the patient; and (c) comparing the measured levels of PSG with levels of PSG in a sample of cells, tissue, or bodily fluid obtained from a control, wherein an increase in the measured levels of PSG versus the levels of PSG in the control is associated with a cancer which is progressing and a decrease in the measured levels of PSG versus the levels of PSG in the control is associated with a cancer which is regressing or in remission.

4. A method of monitoring prostate cancer in a patient for the onset of metastasis comprising:
(a) identifying a patient having prostate cancer that is not known to have metastasized;
(b) periodically measuring PSG levels in samples of cells, tissue, or bodily fluid obtained from the patient; and (c) comparing the periodically measured levels of PSG
with levels of PSG in cells, tissue, or bodily fluid obtained from a control, wherein an increase in any one of the periodically measured levels of PSG in the patient versus the levels of PSG in the control is associated with a cancer which has metastasized.

5. A method of monitoring changes in a stage of prostate cancer in a patient comprising:
(a) identifying a patient having prostate cancer;
(b) periodically measuring levels of PSG in samples of cells, tissue, or bodily fluid obtained from the patient;
and (c) comparing the measured levels of PSG with levels of PSG in a sample of the same cells, tissue, or bodily .fluid of a control, wherein an increase in any one of the periodically measured levels of PSG versus levels of PSG in the control is associated with a cancer which is progressing in stage and a decrease in any one of the periodically measured levels of PSG versus the levels of PSG in the control is associated with a cancer which is regressing in stage or in remission.

6. The method of claim 1, 2, 3, 4 or 5 wherein the PSG comprises SEQ ID NO:1.