WO1997039139A1

WO1997039139A1 - A method for detection of prostate specific antigen used in monitoring and diagnosis of prostate cancer

Info

Publication number: WO1997039139A1
Application number: PCT/US1997/006497
Authority: WO
Inventors: David J. Robbins
Original assignee: Smithkline Beecham Corporation
Priority date: 1996-04-16
Filing date: 1997-04-16
Publication date: 1997-10-23
Also published as: EP0904397A1; AU2676197A; EP0904397A4; ZA973231B; JP2000510331A

Abstract

Primers specific for prostate specific antigen and a method for monitoring and diagnosing prostate cancer using these primers is provided. Kits for diagnosing prostate cancer containing these primers are also provided.

Description

A METHOD FOR DETECTION OF PROSTATE SPECIFIC ANTIGEN USED IN MONITORING AND DIAGNOSIS OF PROSTATE CANCER

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.

60/015,765, filed April 16, 1996.

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 1994, it was estimated that 38,000 deaths resulted 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 early, when the cancer is still confined to the prostate, the chances of cure is significantly higher. Accordingly, there is a great need for sensitive methods for the detection of organ-confine prostate cancer.

Recently, polymerase chain reaction (PCR) and variations thereof, have been used to detect malignant cells for diagnosis and monitoring of various malignancies. For example, reverse-transcriptase PCR (RT-PCR) is a powerful technique whieh-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.

Prostate-specific antigen (PSA), a 33-kDa protein, was originally thought to be produced extensively by prostatic epithelial cells and thus was used as a tumor marker to aid in the detection and monitoring of prostate cancer. More recent studies indicate that PSA is not specific for prostatic tissue. For example, Diamandis and Yu reported the presence of PSA in 30% of female breast tumors, more rarely in other tumors, in normal breast tissue and in biological fluids such as breast milk. Clin. Chem. 1995, 41(2): 177-179. PSA has also been detected in endometrial tissue.

RT-PCR of mRNA encoding for PSA has been used in the identification of prostate cancer cells. For example, Deguchi et al. detected PSA mRNA in regional lymph nodes from patients with prostate cancer which had metastasized to the lymphatic system. Cancer Res. 1993, 53:5350-4. RT-PCR methods for detection of prostate cancer cells in the general circulation of patients with advanced prostate cancer have also been disclosed. Moreno et al. Cancer Res. 1992, 53:6110-2; Jaakkola et al. Clin. Chem. 1995, 41(2): 182- 186. However, patients with early prostate cancer and/or well differentiated or localized disease were found negative for PSA mRNA by PCR. Accordingly, it was concluded that RT-PCR for PSA mRNA would not be a useful tool for either the diagnosis or monitoring of prostate cancer. Diamandis, E.P. and Yu, H. Clin. Chem. 1995, 41(2): 177-179.

The present invention provides a new RT-PCR method with novel primers for the monitoring and diagnosis patients with prostate cancer. This method is designed to determine the presence of mRNA for PSA in the bloodstream thereby providing evidence that prostate cells are circulating in the blood and confirming a diagnosis of cancer. Circulating prostate cells also indicate that the patient is at risk of secondary tumor or bone metastatic anchoring. Samples taken after prostatectomy or radiation treatment which are reactive indicate the risk that the prostate cancer had spread and that surgery/radiation was not efficacious.

SUMMARY OF THE INVENTION

An object of the present invention is to provide primers for use in an RT-PCR method for the detection of prostatic specific antigen for the monitoring and diagnosis of patients with prostate cancer.

Another object of the present invention is to provide a method for monitoring the progression of prostate cancer which comprises detecting prostate specific antigen in patient with an RT-PCR method which uses these primers. Another object of the present invention is to provide a kit for monitoring and diagnosis of prostate cancer which comprises these primers. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides the human DNA for prostate specific antigen (PSA) (SEQ ID NOS: 1, 8, 9 and 10). Introns are abbreviated for clarity. Exons are indicated in bold. Primers previously disclosed in the art are indicated by underlining. The primers of the present invention, namely DR PSA-up (SEQ ED NO: 2), DR PSA- Down (SEQ ID NO: 3) and DR 12 mer (SEQ ID NO: 4), are indicated by underlined italics in a larger font size.

DETAILED DESCRIPTION OF THE INVENTION

Expression of mRNA specific for a certain tissue in peripheral blood is believed to indicate the presence of circulating cancer cells and metastatic spread of a tumor originating from this tissue. The detection of PSA mRNA in peripheral blood has been associated with metastatic prostate cancer, however, PSA mRNA has also been detected in the blood of patients with clinically confined prostate cancer. Accordingly, clinical methods of staging prostate cancer often understage patients and underestimate the risk of metastasis.

A new method for the detection of prostate specific antigen by reverse- transcriptase polymerase chain reaction has been developed which can be used in the monitoring of prostate cancer in a patient. Primers which are used in the method of the present invention include DR PSA-Up: 5'-GTTGTCITCCTCACCCrGTCCG-3' (SEQ LO NO: 2), DR PSA-Down: S'-TCCAGCACACAGCATGAACTTG-S' (SEQ LD NO: 3), and DR 12 mer: 5'-GAATCACCCGAG-3' (SEQ LD NO: 4). The method of the present invention comprises isolation and washing of buffy coat cells, isolation of total RNA from buffy coat cells, reverse transcription (RT) reaction for PSA, PCR for PSA, and detection of the absence or presence of PSA product on a DNA sequencer. The method may further comprise RT reaction and PCR for a housekeeping gene Beta-2-microglobulin (B2G: Israeli et al. Cancer Research 1994 54:6306-6310) to momtor quality of the isolated RNA. In this method, Vacutainer Cell Preparation Tubes (CPT) (Becton Dickinson

Vacutainer Systems Cat. #362761 Sodium Citrate anticoagulant, 8 ml draw, San Jose, CA) are used to collect blood samples from patients suspected of having prostate cancer. These samples are then centrifuged in accordance with the manufacturer's instructions to separate the buffy coat from other blood components. Buffy coat cells are then washed with PBS and pelleted by centrifugation. The pelleted cells are taken up in a cell lysis buffer. Cells are homogenized by passage over a Qiagen QIA shredder microspin column. The run-through is then passed over a Qiagen RNeasy column to bind RNA. The sample is washed to remove DNA and other components, and the RNA is eluted with DEPC-treated water. The eluted sample RNA is then used as the substrate in the RT reactions. The RT reaction primer for PSA reactions is DR 12 mer (SEQ ID NO: 4) which is specific for the PSA target. An aliquot of the RT reaction is then used for the PCR reaction for PSA, using primers DR PSA Up (SEQ ID NO: 2) and DR PSA Down (SEQ ID NO: 3). In a preferred embodiment, wherein the method further comprises RT reaction and PCR for B2G to monitor quality of the isolated RNA, the RT reaction primer is 5'-AGCTTTGAGTGC-3' (SEQ ED NO: 5) and the PCR reaction primers are 5'-AGCAGAGAATGGAAAGTCAAA-3' (SEQ ED NO: 6) and 5 -TGT TGATGT TGGATAAGAGAAT-3¹ (SEQ ID NO: 7). The sizes of the products from each reaction for each patient are compared on the ABI 373 DNA Analyzer with the ABI 672 GENESCAN software. This combination provides the sensitivity required to determine the presence of as high a dilution as 1 or fewer LNCaP (PSA-producing prostate cancer tissue culture) cells per one million white blood cells (WBCs). The average 8 ml blood sample will contain approximately 40 million WBCs. The ABI 672 GENESCAN software can determine the size in bases of each product, allowing definitive identification of each product.

The specificity of the method of the present invention in detecting prostate cancer was determined. PSA levels in blood samples from normal patients and patients having benign prostatic hypertrophy (BPH), prostatitis, and varying stages of prostate cancer were measured. Results from these experiments are shown in the following Table 1.

Normal donors included males under the age of 35 and females. BPH samples included patients diagnosed with BPH but not cancer. Prostate cancer status was provided by the patient's physician.

As is demonstrated by these results, the method of the present invention is specific for determining metastatic prostate cancer, in particular prostate cancer in the progressive stage and that which has been stable for less than 5 years. By "progressive" it is meant cancer with identified metastases to the bone or other sites, with the metastases enlarged and/or more numerous than in the last examination. Patients who have been stable less than 5 years historically suffer approximately a 66% relapse rate before the end of 5 years. The results in Table 1 are consistent with this relapse rate. Tests with the assay of the present invention predict that the 2 out of 6 nonreactive patients in this group will remain stable for over 5 years while the 4 out of 6 reactive patients have a high probability of relapse. Samples from patients having stage B cancer, or organ-confined cancer, which is not metastatic were nonreactive in the method of the present invention. Further, stable patients, meaning those with metastases in the past but no further progression in 5 or more years, who are considered cured, were also nonreactive. Recent studies indicate that circulating prostate cells can also result from prostatectomy operations. Oefelein et al. J. Urol. 1996 155:238-242. If cancerous, these circulating cells caused by the surgery could result in metastases. The method of the present invention can be used in monitoring these surgical procedures to ensure that a patient is nonreactive prior to surgery as well as after the surgery.

The present invention also provides kits for monitoring of progression of prostate cancer which comprise the DR PSA-Up (SEQ ED NO: 2), DR PSA-Down (SEQ ID NO: 3), and DR 12 mer (SEQ ID NO: 4) primers. In a preferred embodiment, these kits also comprise primers for RT and PCR detection of B2G such as SEQ ED NOs: 5, 6 and 7. Further, kits of the present invention may comprise tubes for collection of blood samples which are capable of separating the buffy coat from other blood components such as Vacutainer Cell Preparation Tubes. Kits of the present invention may also comprise a means for isolating RNA. For example, in one embodiment, the kit may comprise a cell lysis buffer, Qiagen QIA shredder microspin columns (Cat. #79655) for cell homogenization and Qiagen RNeasy columns (Cat. #74106) for binding of the RNA. The kits may also contain any of the following reagents including, but not limited to, Beta-Mercaptoethanol (BME), Diethyl Pyrocarbonate (DEPC), Superscript II RNase H- Reverse Transcriptase (Gibco BRL, Gaithersburg, MD), ethanol (200 Proof), 100 mM dNTPs, AmpliTaq DNA Polymerase (Perkin Elmer Cat. #N808-0153), 6% (6% T, 5%C) denaturing acrylamide gel Gibco/BRL 6% Sequencing Solution (Gel-Mix 6; Cat. #5543UA), 10 % ammonium persulfate,

Denaturing loading buffer containing Blue dextran (50 mg/ml) and 200 ml formamide and 20 ml 100 mM EDTA, IX TBE for Sequencer Gibco/BRL UltraPure Gel-Mix running mate (Cat. #15546-013) and Brij 35 (Sigma Chemical Co. St. Louis, MO, Cat. #430AG-6). Standard and controls may also be provided in the kits of the present invention. Examples include, but are not limited to Genescan ROX-2500 standard (Perkin Elmer/ABI Cat. #401100), RNA from ATCC cell lines CRL-1435 PC-3 (prostate adenocarcinoma, human) or HTB-22 MCF7 (breast adenocarcinoma, pleural effusion, human), and RNA from ATCC cell line CRL-1740 LNCaP (metastatic prostate adenocarcinoma, human). The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES EXAMPLE 1: Preparation of Reagents

Reagents are prepared in essentially RNAse-free, sterile, disposable plasticware, in glassware baked at 180°C for at least 8 hours, or in polypropylene plasticware rinsed with chloroform.

Solutions used for RNA and reverse transcription work are prepared using RNAse-free glassware, autoclaved water, and chemicals reserved for RNA work handled with baked spatulas. Water for solutions is treated with 0.1% DEPC for at least 12 hours at 37°C and then heated to 100°C for 15 minutes or autoclaved for 15 minutes at 15 lb/sq. inch on liquid cycle.

Phosphate buffered saline (PBS) used in this method is prepared by dissolving 8 grams of NaCl, 0.2 grams of KCl, 1.44 grams of Na.HPO₄, and 0.24 grams of KH-PO₄ in 800 ml of distilled ILG The pH of the buffer is adjusted to 7.4 with HCl. The volume is then adjusted to 1 liter with H.O. Prior to use, the buffer is sterilized by autoclaving for 20 minutes at 15 lb/sq. inch on liquid cycle and stored at room temperature. Lysis Buffer (BME) is prepared from 100 μl of BME per 10 ml of QIAGEN

RNeasy kit Lysis Buffer RLT. The resulting Lysis Buffer/BME is stable for 1 month.

Wash Buffer RPE is prepared for use by adding 4 volumes of 96-100% ethanol to the concentrate supplied by Qiagen.

Seventy percent Ethanol is prepared by adding 700 ml of 200 proof Ethanol to 300 ml DEPC-treated water. The solution is stored at room temperature.

Example 2: White Blood Cell Isolation

Blood samples are obtained in Vacutainer CPT tubes with sodium citrate. The samples are then centrifuged at room temperature (20 - 25°C) in a horizontal swing-out head rotor at 1500 to 1800 RCF (Relative Centrifugal Force) for 30 minutes. After centrifugation, the buffy coat appears as a whitish layer just under the plasma layer. Approximately half of the plasma is aspirated without disturbing the cell layer. The cell layer is then collected with a sterile pipette and transferred to a 15 ml conical centrifuge tube.

PBS is added to the conical tubes to bring the volume to 15 ml. The tubes are then capped and the cells and PBS mixed by inverting the tubes 5 times. The tubes are then centrifuged for 15 minutes at 300 RCF and the supernatant is aspirated without disturbing the cell pellet. PBS is again added to the cells to bring the volume to 10 ml. The tubes are capped and the cells and PBS are mixed by inverting the tubes 5 times. The tubes are then centrifuged for 10 minutes at 300 RCF. As much supernatant as possible is aspirated without disturbing the cell pellet. Lysis Buffer/BME (400 μi) is added to the pellet and the cells are resuspended by gentle vortexing. At this stage, samples may be frozen at -70°C or used immediately for RNA isolation.

Example 3: RNA Isolation The cells/Lysis Buffer/BME suspension is pipetted directly onto a QIAGEN

QIA shredder column and centrifuged at full speed for 1 minute in a microcentrifuge. The time of centrifugation may be extended if liquid remains above the column. One volume (400 μl) of 70% ethanol is added to the homogenized lysate and mixed. The sample is then applied to an RNeasy spin column and centrifuged for 15 seconds at 8000 x g (10,000 rpm). Flowthrough is discarded. Wash Buffer RWl (700 μl) is then pipetted onto the spin column. The columns are again centrifuged and the flowthrough is discarded. Following this wash, the spin column is placed in a new 2 ml collection tube. Wash Buffer RPE (500 μl) is pipetted onto the spin column and centrifuge as above. The flowthrough is discarded. A second aliquot of Wash Buffer RPE is pipetted onto the spin column and centrifuged for 2 minutes at full speed to dry the RNeasy spin column. This 2 minute spin assures that no residual ethanol will be carried over during elution. The spin column is then transferred to a new 1.5 ml collection tube and the sample RNA is eluted with 50 μl of DEPC-treated water pipetted directly onto the spin column membrane followed by centrifugation for 60 seconds at 8000 x g. Sample RNA may be used immediately in the RT reaction, or stored at -70°C until further use. Example 4: Determining the RNA concentration

The RNA concentration in each sample is determined by UV spectrophotometry. The baseline absorbance of 75 μl of distilled water is first determined at 260, 280, and 320 nm. A five microliter sample of RNA is then added and mixed. The sample absorbance is then determined at 260, 280, and 320 nm. The neat RNA concentration is calculated by: a) subtracting the A320 from the A260 to obtain the absorbance due to RNA; b) multiplying by the dilution factor (80 μ/5 μl); and c) multiplying by the conversion factor (0.04 mg/ ml). Example 5: Sample Reverse Transcription

Two microliters of 50 μM PSA RT primer (SEQ ED NO: 4) are pipetted into each PCR reaction tubes. Two microliters of 50 μM B2G RT primer (SEQ ID NO: 5) are pipetted into separate PCR reaction tubes. Tubes may be prepared in advance and stored at -20°C. RT Reaction Mix is prepared just prior to use in the following manner: The number of sample reactions (PSA and B2G) to be run is first determined as variable X. The following volumes of reagents are then mixed: 4(X+1) μl 5X First Strand Buffer; 2(X+1) μl 0.1 M DTT; 2(X+1) μl 20 mM dNTP solution; 1(X+1) μL Superscript π reverse transcriptase. The solution is then vortexed to ensure homogeneity. Two tubes per RNA sample are prepared, one tube for each reaction: PSA and

B2G. Ten microliters of sample RNA are added to each reaction tube. All RT reaction tubes are then placed in a thermal cycler programmed for 70°C for 10 minutes. Within the last minute of the program, the thermal cycler is set to PAUSE and each tube is placed on ice. Ten microliters of RT Reaction Mix are then added to each tube and the following program is run: 25°C for 5 minutes; 42°C for 15 minutes; 97°C for 15 seconds; followed by 4°C for at least 5 minutes.

Example 6: Sample Amplification

PSA PCR Reaction Mix is prepared in the following manner. The number of PSA sample reactions to be run is determined as variable X. The following volumes of reagents are then mixed: 31.95(X+1) μl H^O; 4(X+1) μl 10X PCR Buffer 0; (X+l) μl 20 mM dNTP; 0.8(X+1) μl 25 mM MgCl₂; 2(X+1) μl PSA primers (SEQ ID NOs: 2 and 3); and 0.25(X+1) μl Taq enzyme.

B2G PCR Reaction Mix is prepared in the following manner: The number of B2G sample reactions to be run is determined as variable X. The following volumes of reagents are then mixed: 33.15(X+1) μl H_jO; 4(X+1) μl 10X PCR Buffer JJ; (X+l) μl 20 mM dNTP; 0.8(X+1) μl 25 mM MgCL; 0.8(X+1) μl B2G primers (SEQ ED NOs: 6 and 7); and 0.25(X+1) μl Taq enzyme.

Two tubes per RNA sample are prepared, one tube for each reaction: PSA and B2G. Forty microliters of the appropriate reaction mixture are pipetted into each of the tubes. Ten microliters of the respective sample RNA or control RNA Reverse Transcription reaction are pipetted into each tube. For the negative (reaction) control, 10 ml of water are used. The tubes are spun for 10 seconds in an Eppendorf microcentrifuge to ensure all liquid is mixed together at the bottom of the tubes. The tubes are then placed in the Perkin Elmer 9600 Thermocycler and cycled as follows:

Cycle Type Temp Time # of Cycles step cycle 95°C 15 sec 30

58°C 15 sec 72°C 45 sec hold 72°C 3 minutes 1 hold 4°C indefinite 1

Example 7: Sample Detection Two 6 cm well-to-read plates (one notched, one plain) are prepared for use in the following manner. Both sides of each plate are rinsed with distilled water. One side only of each plate is then selected as the inside (gel side) of the plate. This inside is then rinsed with methanol followed by 0.5% Brij. The methanol rinse is then repeated until the plates are streak free. Spacers are then placed on the edge of the plain plate, parallel to the short side of the plates. The notched plate is placed on top of the plain plate, with the insides of the plates facing each other. The plates are then stood on their bottom edge and the spacers lined up so they are level with the bottom and sides of the gel. They are gently laid down flat with the plain plate on the bottom and clamped with 6 binder clips.

Seventy-five milliliters of ammonium persulfate is added to 20 ml of Gel-Mix 6 and swirled gently to mix. The solution is then poured between the sequencing plates. A sample comb is inserted and allowed to polymerize 1-2 hours.

After polymerization is complete, the clamps are removed and the outside of the plates are cleaned with distilled water, being careful to remove all acrylamide from the outside surfaces ofthe plates. The plates are then inserted into a 373 sequencer and a plate check is performed in accordance with the manufacturer's directions to check for interference. The top and bottom buffer reservoirs are filled with 700 ml and 300 ml of IX TBE, respectively. The sample comb is removed from between the plates, and acrylamide and urea are washed from the wells with IX TBE.

The gel is prerun for 5 minutes at 28 watts. Two microliters of PCR reaction are pipetted into a 0.2 ml thin-walled PCR reaction tube. Denaturing sample buffer (2X; 2.5 ml) and 0.3 ml of Genescan ROX-2500 standard are then added. The tubes are placed in a PE 9600 Thermocycler and run at 94 °C for 10 minutes. Within the last minute of the program, the thermal cycler is set to PAUSE and each tube is placed on ice. Four microliters of cooled sample are then loaded onto the gel. The ABI sequencer is set to run for 3.5 hours at 28 watts, 600 volts, 40 milliamps and the Genescan data collection to collect for 3 hours.

Expected values for each ofthe primer set amplifications are as follows:

Marker Size (bp) Observed Range B2G 537 529 - 540

PSA 586 585 - 588 SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: SmithKline Beecham Corporation

(ii) TITLE OF THE INVENTION: A Method for Detection of Prostate Specific Antigen used in Monitoring and Diagnosis of Prostate Cancer

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: SmithKline Beecham Corporation

(B) STREET: 709 Swedeland Road

(C) CITY: King of Prussia

(D) STATE: PA (E) COUNTRY: USA

(F) ZIP: 19406-0939

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 60/015,765

(B) FILING DATE: 16-APR-1996 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Han, William T (B) REGISTRATION NUMBER: 5219

(C) REFERENCE/DOCKET NUMBER: P50473

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 610-270-5219 (B) TELEFAX: 610-270-5090

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 92 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CCAAGCTTAC CACCTGCACC CGGAGAGCTG TGTCACCATG TGGGTCCCGG TTGTCTTCCT 60 CACCCTGTCC GTGACGTGGA TTGGTGAGAG GG 92

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

GTTGTCTTCC TCACCCTGTC CG 22

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3;

TCCAGCACAC AGCATGAACT TG 22

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GAATCACCCG AG 12

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

AGCTTTGAGT GC 12

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

AGCAGAGAAT GGAAAGTCAA A 21

(2) INFORMATION FOR SEQ ID NO:7

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

TGTTGATGTT GGATAAGAGA AT 22

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 208 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

TGATCTAGCA CCCCCTCTGC AGGCGCTGCG CCCCTCATCC TGTCTCGGAT TGTGGGAGGC 60

TGGGAGTGCG AGAAGCATTC CCAACCCTGG CAGGTGCTTG TGGCCTCTCG TGGCAGGGCA 120 GTCTGCGGCG GTGTTCTGGT GCACCCCCAG TGGGTCCTCA CAGCTGCCCA CTGCATCAGG 180

AAGTGAGTAG GGGCCTGGGG TCTGGGGA 208

(2) INFORMATION FOR SEQ ID NO:9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 639 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

ATCATCCTCG CTCCTCATTC CTGCGTCTGC TTCCTCCCCA GCAAAAGCGT GATCTTGCTG 60

GGTCGGCACA GCCTGTTTCA TCCTGAAGAC ACAGGCCAGG TATTTCAGGT CAGCCACAGC 120

TTCCCACACC CGCTCTACGA TATGAGCCTC CTGAAGAATC GATTCCTCAG GCCAGGTGAT 180

GACTCCAGCC ACGACCTCAT GCTGCTCCGC CTGTCAGAGC CTGCCGAGCT CACGGATGCT 240

GTGAAGGTCA TGGACCTGCC CACCCAGGAG CCAGCACTGG GGACCACCTG CTACGCCTCA 300 GGCTGGGGCA GCATTGAACC AGAGGAGTGT ACGCCTGGGC CAGATGGTGC AGCCGGGAGC 360

CCAGATGCCT GGGTCTGAGG GAGGAGGGGA CAGGACTCCT GGGTCTGAGG GAGGAGGGCC 420

AAGGAACCAG GTGGGGTCCA GCCCACAACA GTGTTTTTGC CTGGCCCGTA GTCTTGACCC 480

CAAAGAAACT TCAGTGTGTG GACCTCCATG TTATTTCCAA TGACGTGTGT GCGCAAGTTC 540

ACCCTCAGAA GGTGACCAAG TTCATGCTGT GTGCTGGACG CTGGACAGGG GGCAAAAGCA 600 CCTGCTCGGT GAGTCATCCC TACTCCCAAG ATCTTGAGG 639 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 491 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

ATCTCACTCT CTCCCTGCTT TTACCCTTAG GGTGATTCTG GGGGCCCACT TGTCTGTAAT 60

GGTGTGCTTC AAGGTATCAC GTCATGGGGC AGTGAACCAT GTGCCCTGCC CGAAAGGCCT 120 TCCCTGTACA CCAAGGTGGT GCATTACCGG AAGTGGATCA AGGACACCAT CGTGGCCAAC 180

CCCTGAGCAC CCCTATCAAG TCCCTATTGT AGTAAACTTG GAACCTTGGA AATGACCAGG 240

CCAAGACTCA AGCCTCCCCA GTTCTACTGA CCTTTGTCCT TAGGTGTGAG GTCCAGGGTT 300

GCTAGGAAAA GAAATCAGCA GACACAGGTG TAGACCAGAG TGTTTCTTAA ATGGTGTAAT 360

TTTGTCCTCT CTGTGTCCTG GGGAATACTG GCCATGCCTG GAGACATATC ACTCAATTTC 420 TCTGAGGACA CAGTTAGGAT GGGGTGTCTG TGTTATTTGT GGGATACAGA GATGAAAGAG 480

GGGTGGGATC C 491

Claims

What is claimed is:

1. A method of monitoring and diagnosing prostate cancer in a patient comprising detecting prostate specific antigen mRNA in a sample from a patient by

> reverse transcriptase-polymerase chain reaction with primers having SEQ ED NO: 2, SEQ ED NO: 3 and SEQ ED NO: 4.

2. Primers for detecting prostate specific antigen mRNA having SEQ ID NO: 2, SEQ ED NO: 3 and SEQ ED NO: 4.

3. A kit for monitoring and diagnosing prostate cancer comprising primers having SEQ ED NO: 2, SEQ ED NO: 3 and SEQ ED NO: 4.