WO1998040513A1

WO1998040513A1 - Method of detecting prostate cancer metastasis

Info

Publication number: WO1998040513A1
Application number: PCT/US1998/004818
Authority: WO
Inventors: Anna C. Ferrari; Nelson N. Stone
Original assignee: Ferrari Anna C; Stone Nelson N
Priority date: 1997-03-11
Filing date: 1998-03-11
Publication date: 1998-09-17
Also published as: AU6551398A

Abstract

The present invention provides a method of detecting prostate cancer metastasis to the pelvic lymph nodes utilizing reverse transcriptase polymerase chain reaction to detect mRNA for prostate specific antigen and prostate specific membrane antigen.

Description

Method of Detecting Prostate Cancer Metastasis

Field of the Invention

The present invention relates to therapy of prostate cancer. In particular, the present invention provides a method for staging localized prostate cancer prior to definitive local therapy.

Background of the Invention The decision to pursue definitive therapy for patients with clinically localized prostate cancer (PC) rests upon the estimated probability of cure as determined by a combination of parameters including clinical tumor stage, serum prostate specific antigen (PSA) levels and Gleason score of the primary lesion. Nevertheless, in current practice, the decision to proceed with local treatment is largely determined by the pathologic assessment of pelvic lymph nodes (LN) preceding local intervention. This evaluation is performed on frozen sections of LN removed at the time of laparotomy for radical prostatectomy (RP) or on fixed LN tissues removed by laparoscopic pelvic lymph node dissection (LPLND) a few days prior to definitive radiation therapy.

In spite of the efforts to pathologically stage prostate cancer patients before local therapy, the detection of LN metastasis by routine histologic or immunohistochemical analysis does not reflect the frequency of recurrence after definitive therapy. This discordance is most outstanding in the group of patients with high risk features such as those with T > 1C or PSA ≥ 20 ng/ml or Gleason > 7. While the incidence of histologically documented LN metastasis for this group of patients ranges between 15% and 20%, the rate of biochemical failure for those assessed as metastasis free by pathology is greater than 80% within 5 years.

The discrepancy between pathologic findings and time to relapse suggests that metastatatic tumor cells escape detection by routine microscopy and that a greater number of patients are subjected to non-curative local therapies associated with frequent and permanent morbidities.

Other evidence suggests that metastasis may occur early, while the tumor is apparently confined, and remain undetectable by conventional pathology or radiographic evaluation for prolonged periods of time. For example, prostate metastasis to bone marrow and archival lymph node tissues has been detected in the absence of histologic involvement. Edelstein et al. (1996) Urology 47:370.

Accordingly, there is a need for better methods of detecting lymph node metastasis in order to more accurately stage localized prostate cancer and thereby determine appropriate treatment.

The expression of the PSA and prostate specific membrane antigen (PSM) genes almost exclusively by prostatic epithelial cells allows the identification of a metastatic prostatic epithelial cell among other cell types. A single prostatic epithelial cell expressing PSA and/or PSM can be identified from a sample of 10-100 million peripheral blood mononuclear cells (PBMNC) of patients with prostatic cancer by reverse transcriptase polymerase chain reaction (RT-PCR) for PSA and PSM. See, Q_Q_ Katz et al. (1994) Urology 43:765.

In accordance with the present invention, it has been discovered that RT-PCR can be utilized to provide a sensitive and specific method to identify prostate cancer metastasis to pelvic lymph nodes.

Summary of the Invention

The present invention provides a method of detecting prostate cancer metastasis to the pelvic lymph nodes comprising detecting mRNA for the PSA and prostate specific membrane (PSM) genes from mRNA of fresh lymph nodes by reverse-transcriptase polymerase chain reaction (RT-PCR), wherein detection of mRNA for the PSA or PSM gene is indicative of metastasis. Detailed Description of the Invention

In accordance with the present invention it has been discovered that molecular analysis of mRNA from fresh LN tissues by RT-PCR for the PSA and PSM genes is useful for the detection of metastatic prostate cells, and is more sensitive than pathological staging methods.

The improved accuracy of staging achieved by the present invention has substantial benefits. The implications of more accurate staging of pelvic LN are substantial. As opposed to circulating prostate cells, the presence of prostate cells in the pelvic lymph nodes implies metastasis to tissues that define tumor staging and outcome. Furthermore, beyond helping to define the issue of whether local therapies would be of any benefit to this high risk group of patients, a more accurate assessment of the pelvic LN helps to identify subgroups of patients with comparatively small volume disease where the role of novel treatment strategies (neoadjuvant-adjuvant hormonal therapy, and biologic modifiers) aimed at delaying progression would be appropriate. Further, the detection of both PSA and PSM mRNA increases the chances of detecting prostatic epithelial cells in the context of androgen deprivation and provides information on the degree of differentiation and hormonal dependence of PC cells. While PSA is preferentially expressed by differentiated prostatic epithelial cells and more differentiated tumors, and its expression is upregulated by androgens, PSM expression is higher in undifferentiated prostate epithelial cells and its expression is downregulated by androgen. Therefore, in the setting of prostate cancer, detection of PSM allows identification of cells that have stopped expressing PSA in response to androgen suppression, or are more undifferentiated and androgen independent. The identification of androgen dependent (AD) and independent (Al) cells is particularly attractive since the success of hormonal therapy is dependent on the ratio of AD/AI cells tumor cells present in a given tumor and these cells may differ in their metastatic potential.

The present method of detecting prostate cancer metastasis comprises obtaining fresh pelvic LN samples, extracting RNA from the samples, and performing RT-PCR using primers specific for PSA and PSM mRNA, wherein the detection of either or both of PSA and PSM mRNA is indicative of metastasis. Pelvic lymph nodes may be obtained from patients by methods known in the art, for example by laparoscopic LN dissection or laparotomy. RNA may be extracted from the LN sections by methods known to those of ordinary skill in the art, including commercially available kits for RNA extractions. For example, tissue samples may be incubated in RNAzol buffer (Biotecx Laboratories, Houston, TX), homogenized, and processed for RNA extraction according to the manufacturer's protocol.

RT-PCR is performed by reverse transcription of the LN RNA to cDNA followed by PCR. Reverse transcription of RNA to cDN A is performed by incubating randomly primed RNA with a reverse transcriptase and 2' deoxyribonucleotide triphosphates. Methods of reverse transcription of RNA are known to those of ordinary skill in the art, and can be performed using commercially available kits, for example the Superscript Preamplification System, Life Technologies, Gaithersburg, MD.

PCR is performed by standard methods using a set of primers for PSA and a set of primers for PSM. The sequences of the primers can be selected by reference to the published sequences of PSA, disclosed for example by Schulz et al. (1988) Nucleic Acids

Res. 16:6226, incorporated herein by reference, which is also available from Genebank, and PSM disclosed for example by Israeli et al (1993) Cancer Res. 51:227 incorporated herein by reference. In a preferred embodiment, sets of nested primers are used for detection of PSA and PSM. For example, nested primers for PSA may include outer primers extending from exon 3 (5'-3': 648-667 5' GATGACTCCAGCCACGACCT 3',

SEQ. ID NO:l) to exon 5 (3'-5': 1338-1357 5' CACAGACACCCCATCCTATC 3', SEQ ID NO:2) to generate an expected product of 710 base pairs, and inner primers extending from exon 4 (5'-3^*: 860-879 5' GATATGTCTCCAGGCATGGC 3^*, SEQ ID NO:3) to exon 5 (3'-5': 1296-1315 5' GCAAGTTCACCCTCAGAAGG 3', SEQ ID NO:4) to generate an expected product of 455 bp. Nested primers for PSM may include outer primers (5'-3': 1368-1390, 5'-CAGATATGTCATTCTGGGAGGTC 3', SEQ ID NO:5 and 3*-'5': 1995-2012 5' AACACCATCCCTCCTCGAACC 3', SEQ ID NO:6) to generate an expected product of 647 bp, and inner primers (5'-3': 1689-1713 5' CCTAACAAAAGAGCTGAAAAGCCC 3', SEQ ID NO:7; 3'-5': 1899-1923 5' ACTGTGATACAGTGGATAGCCGCT 3', SEQ ID NO:8) to generate an expected product of 234 bp.

PCR may be performed with the outer primers only, or nested PCR may be performed using outer and inner primers, by methods known to those of ordinary skill in the art. In an example of a PCR reaction, a cDNA equivalent to 1 μg of test RNA is added to a 50 μl final volume reaction containing IX PCR buffer, 1.5 mM MgCl₂, 0.2 niM each dNTP, 0.1 μM each 3' and 5' outer primers, and 1.25 units Thermus aquaticus polymerase. The mixture is subjected to temperature cycling as follows: step 1, 95° C for 10 minutes; step 2, one cycle at 95°C for 50 seconds, 57°C for 2 minutes and 72°C for 2 minutes; step 3: 29 cycles at 95°C for 30 seconds, 57°C for 1 minutes, 72°C for 1 minute; step 4, 49 cycles at 95°C for 30 seconds, 57°C for 1 minute, 72°C for 1 minute; step 4, 49 cycles at 95°C for 30 seconds, 57°C for 1 minute, 72°C for 10 minutes. For nested PCR, identical reaction mixtures are used but also contain 0.1 μm each of the 3' and 5' inner primers, and are used to amplify 4 μl of a 1/100 dilution of the product of the first PCR reaction, under cycling conditions that are the same except that only one cycle is used in Step 4. All PCR assays include negative and positive controls as well as controls for RNA integrity.

PCR products are identified by methods known in the art. In a preferred embodiment, PCR products are identified by size after agarose gel electrophoresis and ethidium bromide staining.

The identification of a PCR product having the expected size of a fragment of PSA or PSM cDNA based upon the selected primers is indicative of metastasis to LN. Cases that are negative but show adequate cDNA/RNA integrity in control reactions are re-evaluated. The following examples serve to further illustrate the present invention.

Example 1 Materials and Methods PATIENT STAGING

The size of the primary prostate tumor (T) was determined for all patients by digital rectal examination performed by the same urologist. TISSUE SOURCE PROCESSING AND RNA EXTRACTION

Pelvic LN were obtained by laparoscopic dissection of the right and left external iliac and obturator areas. Approximately 6-10 frozen LN sections from each tissue block were cut with a microtome and directly dropped in 1 ml. RNAzol buffer (Biotecx Laboratories). The tissues were homogenized for one minute, left for 5 minutes on ice and then processed for RNA extraction following the manufacturers protocol (Biotecx Laboratories). The RNA pellets were washed once in 75% ethanol and dissolved in autoclaved DEPC treated water.

REVERSE TRANSCRIPTION Approximately 5 μg of RNA were mixed with 50 ng random hexamers and heated at 70 °C for 10 minutes according to the Superscript Preamplification System (Life Technologies, Bethesda, MD) instruction manual. The mixture was placed directly on ice for one minute and incubated in a 20 μl solution containing lx Buffer, 2.5 mM MgCl₂, 10 mM DTT and 0.5 mM each 2' deoxyribonucleotide triphosphates (dNTP's: 2' deoxadenosine 5' triphosphate (dATP), 2' deoxycytidine 5' triphosphate (dCTP), 2' deoxyguanosine 5' triphosphate (dGTP), 2' deoxythymidine 5' triphosphate (dTTP). The mixture was heated at 25 °C for 5 minutes. Superscript II murine Moloney leukemia virus reverse transcriptase (200 units) were added and the mixture heated at 42 °C for 50 minutes. To terminate the reaction the sample was heated at 70 °C for 15 minutes and cooled on ice for at least 5 minutes. In the final step, 2 units of RNAse H were added and activated at 37° C for 20 minutes to destroy residual RNA.

POLYMERASE CHAIN REACTION a. PRIMERS: Two sets of primers were synthesized for PS A and PSM genes. The sequences for the PSA cDNA primers was obtained from the gene bank and selected for the lowest homology with human Kallikrein. The outer primers extend from exon 3 (5'-3': 648-667, SEQ ID NO: 1) to exon 5 (3'-5*: 1338-1357, SEQ ID NO:2). The size of the expected cDNA fragment is 710 bp. The nested primers extend from exon 4 (5'-3': 860-879, SEQ ID NO:3) to exon 5 (3'-5': 1296-1315, SEQ ID NO:4), the size for the expected cDNA fragment is 455 bp. The sequence for PSM cDNA (2653 bp) and the two set of primers were obtained from the literature [Israeli et al. (1993) Cancer Res. 53:227]. The outer primers (5'-3': 1396-1390, SEQ ID NO:5, 3'-5': 1995-2015, SEQ ID NO:6) amplify a fragment of 647 bp. The nested primers (5'-3': 1689-1713, SEQ ID NO:7; 3'-5': 1899-1923, SEQ ID NO: 8) amplify a fragment of 234 bp. To test for RNA integrity, a set of 20-mers for the housekeeping gene gluteraldehyde 3 phosphate dehydrogenase (G3PDH) primers (5'-3': 416-435 5'-CATCTCTGCCCCCTCTGCTG 3', SEQ ID NO:9; 3'-5': 859-840 5' CCCTCCGACGCCTGCTTCAC 3', SEQ ID NO:10) to amplify a fragment of 444 bp was used. A G3PDH MIMIC was derived from a fragment ofV-erbB cDNA attached to G3PDH primers. The primers (5'-3': 1135-1154 5'

ACCCTCCCCGCTATCTTGTTA 3', SEQ ID NO: 11 and 3'5': 1585-1566 5' GTGCCCCTGCCCATTTCTGT 3', SEQ ID NO: 12) were designed to amplify the 229 bp V-erb cDNA fragment attached to 40 bp derived from the G3PDH gene primers with a final fragment size of 289 bp [Gallagher]. b. CONTROLS: Each PCR reaction included negative and positive controls.

The negative controls included: one control for non-specific amplification containing the reaction mixture, primers, TAQ Gold and human female peripheral blood mononuclear cells (PBMNC) cDNA; the second contained the reaction mixture, primers and TAQ Gold without cDNA template. The positive controls utilized cDNA from a mixture of one LNCaP prostatic carcinoma cell in one million human female PBMNC which contains one log higher concentration than the limit of detection.

COMPETITIVE G3PDH PCR

To control for RNA integrity and to determine the significance of a negative PCR assay, an aliquot of each cDNA sample was amplified for G3PDH in conjunction with a synthetic G3PDH cDNA (MIMIC) used as a competitor. The standard for cDNA/RNA integrity with G3PDH was established in a competitive assay using a fixed amount (0.2 μg) of control cDNA prepared from one LNCaP cell mixed with one million female PBMNC and increasing concentrations of MIMIC cDNA (from 0.01-5 attomoles). The amount of control cDNA and MIMIC that achieved equal G3PDH amplification in a competitive assay were considered the optimum ratio and representative of adequate RNA/cDNA integrity. In these experimental conditions, the cDNA equivalent to 0.2 μg of control RNA (1 LNCaP/1 million PBMNC) yielded equal G3PDH amplification to 0.1 attomole of G3PDH MIMIC. A cDNA equivalent of 0.2 μg of RNA was used for all test cases to determine RNA integrity and mixed in a 50 reaction with lx PCR Buffer, 1.5 mM MgCl₂, 0.2 mM each dNTP's, 0.1 pM G3DPH 5' and 3' Primers, 0.1 attomoles G3DPH MIMIC, 1.25 units Ampli TAQ Gold, 2 drops mineral oil. The mixtures were subjected to temperature cycling in an aluminum block programmed as follows: step 1 : 95 °C for 10 minutes; step 2: one cycle at 95 °C for 50 seconds, 57 °C for 2 minutes and 72°C for 2 minutes; step 3: 29 cycles at 95°C for 30 seconds, 57°C for 1 minute, 72°C for 1 minute; step 4: one at 95°C for 30 seconds, 57°C for 1 minute, 72°C for 10 min.

G3PDH/MIMIC PCR PRODUCT IDENTIFICATION

The G3PDH products were identified by size after agarose gel electrophoresis and ethidium bromide staining. Partial or complete RNA degradation was established in all cases showing less than equal or absent G3PDH cDNA amplification in a competitive assay. The increment in cDNA required to compensate for partial RNA degradation was estimated for each case by comparing the intensity of the G3PDH cDNA bands with a standard G3PDH competitive reaction in each experiment.

PCR FOR PSA AND PSM WITH OUTER PRIMERS A cDNA equivalent of 1 μg of test RNA was added to a 50 μl final volume reaction containing IX PCR Buffer, 1.5 mM MgCl₂, 0.2 mM each DTNP, 0.1 μM each 3' and 5' outer primers, 1.25 units Ampli TAQ Gold. The mixture was subjected to temperature cycling steps similar to those described for G3PDH. The number of cycles in the 4th step was increased to 49.

NESTED PCR for PSA and PSM

A 46 μl reaction mixture identical to the one used for the first round of amplification but containing 0.1 μM each of 3' and 5' Nested Primers was used to amplify 4 μl of a 4/400 dilution of the outer primers PCR product. The cycling conditions were the same as those described for G3PDH MIMIC.

PSA and PSM PCR PRODUCT IDENTIFICATION

The PCR products were identified by size after agarose gel electrophoresis and ethidium bromide staining. All samples with a negative, nested PSA and PSM RT-PCR assay that had shown adequate RNA integrity in the G3PDH/MIMIC competitive reaction were subjected to a second round of amplification using the same conditions and processed for Southern transfer and hybridization with an oligo-end labeled PSA and

PSM probe. Example 2

SENSITIVE AND SPECIFICITY OF THE RT-PCR ASSAY FOR PSA AND PSM

The sensitivity of the assay was established by cell-cell dilution experiments of

LNCaP cells and human female PBMNC in ratios of one LNCaP cell in ten thousand to one hundred million female PBMNC. Normal female PBMNC served as negative controls, the LNCaP cells as positive controls. It was found that 50 cycles of amplification with the outer primers and 30 cycles of amplification with nested primers increased the sensitivity of the assay from one LNCaP cell in one hundred thousand

PBMNC to one LNCaP cell in ten million PBMNC. The sensitivity of detection was determined to be one prostate cell expressing PSA or PSM among 10 million non- prostatic cells provided there was equal competitive amplification between G3PDH cDNA and MIMIC.

The specificity of the band was determined by the size of the amplified product, restriction endonuclease pattern and by Southern transfer of the nested product and hybridization with a ³²P end-labeled 22 oligomer probe that would not be recognized by any of the primer sets utilized in the reaction.

Example 3

RT-PCR of Clinical Samples

Sixty LN tissues were prospectively procured from the right and left pelvic area of 33 patients with clinically localized, high risk prostate cancer (T > IC or PSA > 10 ng/ml or Gleason > 7) that were staged with seminal vesical biopsies and LPLND in preparation for radioactive seed implants. Twenty seven patients had bilateral LN samples removed (54 specimens) while six patients had unilateral LN samples (6 specimens) available for molecular analysis. All patients signed informed consent approved by the Mount Sinai Institutional Review Board for molecular analysis of tissue specimens procured during a staging LPLND.

Based on the clinical staging of the prostate primary (T) there were: 4 TIC, 7 T2A, 12 T2B, 6 T2C, 4 T3A cases. In addition to their T status, all cases had either a PSA > 10 ng/ml or a Gleason score > 7. There were 4 (12%>) patients with pathologically documented LN metastasis. This number falls within the expected range of LN metastasis according to Partin's nomogram [Partin et al. (1993) J. Urol. 150: 110] for prediction of final pathological stage based on clinical stage of the prostate primary, serum PSA levels and Gleason score. Overall, 5 (15%) patients had seminal vesical (SV) involvement on pathological sampling, 1 T2A, 1 T2B, 2 T2C and one T3 case which also had LN metastasis. RNA integrity and cDNA quality were determined by performing an

RT-PCR G3PDH/MIMIC competition assays in the sixty mRNA's extracted from LN tissues. After evaluating each patient's cDNA and adjusting for equivalent amounts with the standard, 120 nested RT-PCR assays for PSA and PSM were performed.

The 4 patients with 4 pathologically documented LN metastasis were also RT- PCR positive for both markers. Therefore 100% of the metastatic LN were identified by molecular analysis and both PSA and PSM markers were expressed. In addition, two of the 4 patients had molecular evidence of metastasis to the contralateral LN which was undetected by pathology.

Overall, 27 (82%) of 33 patients had evidence of pelvic LN metastasis by PSA and/or PSM mRNA expression in contrast to 4 (12%) patients with pathologically documented LN metastasis (p<.0001). Twenty three (70%) patients demonstrated micrometastasis by RT-PCR only.

There were 57 (47.5%) total RT-PCR positive assays corresponding to 41 (68.3%) LN and 63 (52%) negative PSA and PSM assays corresponding to 19 (31.6%) LN. Only six (18.2%>) patients were free of micrometastasis by both PSA and PSM RT-PCR. Nineteen (57%) patients had 23 (38.3%) PSA RT-PCR positive LN while 25 (75%) patients had 34 (56%) PSM RT-PCR positive LN. Compared to 4 metastatic LN detected on routine pathology, the sensitivity of RT-PCR with either marker alone was significantly higher (p<.001). When the sensitivity of each marker to detect micrometastasis was compared, it was found that seventeen (51 >) of 33 patients were positive for both PSA and PSM, 6 patients were negative for both markers and 10 patients were positive for either PSA or PSM, 8 (24%) patients for PSM RT-PCR and 2 (6%>) patients for PSA RT-PCR only. The ability of either marker alone to detect micrometastasis did not reach statistically significant difference (p < 0.11) in spite of a higher number of PSM positive cases. It cannot be concluded that PSM is more sensitive than PSA to detect micrometastasis by RT-PCR. Therefore, both markers were necessary to achieve high sensitivity.

Five (15%>) of 33 patients had seminal vesical (SV) involvement. All 5 patients had micrometastasis to the LN by both PSA and PSM RT-PCR; only one patient had pathologically documented LN metastasis. Molecular assessment of LN was far more sensitive than SV pathology to detect metastasis (p < .0001).

The detection of LN micrometastasis was not significantly different (83-100%) among patients with different clinical stage except for the 7 patients with T2A disease which had 4 (57%>) LN metastasis by RT-PCR, in one case also documented by pathology.

A Gleason score between 7 and 9 was not associated with differences in detection of molecular metastasis and did not affect the detection of PSM versus PSA expressing cells.

Serum PSA levels at the time of diagnosis did not separate patients with molecular LN metastasis whether it remained below 10 ng/ml (13 patients), between

10-20 ng/ml (12 patients) or above 20 ng/ml (8).

Fifteen of 33 patients received combined androgen suppressive therapy for variable periods of time before LN sampling while 18 patients did not receive hormonal therapy prior to LN procurement. Overall, molecular metastasis by RT-PCR was equally frequent among the treated (12 of 15) and non-treated (15 of 18) group. In the hormonally treated group, a slightly lower number of cases (8 of 15, 53%) were PSA RT- PCR positive compared to the non-treated group (1 1 of 18, 61%>). PSM expression was equal (-70%) in both groups (11/15 and 14/18 respectively). Because the range of duration of hormonal therapy was very variable (1-44 weeks, median 6 weeks) and less than half of the patients received hormonal therapy for more than 6 weeks, it was not possible to precisely determine the effect of androgen suppression on the expression of these markers and in particular on the suppression of PSA. However, the expression of PSA is likely to be decreased under hormonal therapy. With regard to PSM expression, it appears to be equal in both groups and independent of androgen suppression. The foregoing molecular analysis of pelvic LN tissue by RT-PCR for PSA and

PSM indicated that over 80% of high risk prostate cancer patients have micrometastasis to the pelvic LN at the time of local therapy. This high prevalence of LN metastasis is in sharp contrast with the 15% average (range 7 to 50%>) expected on the basis of conventional pathology for the same group of patients. Both markers, used alone or combined, were far more sensitive than routine pathology (p < .0001) or other parameters routinely used to establish risk of metastasis, such as size of the primary lesion, the serum PSA levels or a Gleason score equal to or higher than 7 (p < .0001). Considering that 80%) of the high risk, localized prostate cancer patients have recurrence within 5 years of local therapy, an 83-100%) detection of metastasis by RT-PCR analysis indicates that the present molecular estimate of left over tumor burden after removal of the primary is likely to be closer to reality than standard pathology, serum PSA, T stage or tumor grade.

The foregoing results demonstrate that the RT-PCR assay is reproducible and highly sensitive for the detection of metastatic prostate cells, and that it is superior to routine conventional pathology for detection of metastasis. SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: Ferrari, Anna C. Stone, Nelson N.

(ii) TITLE OF THE INVENTION: METHOD OF DETECTING PROSTATE CANCER METASTASIS

(iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Baker & Botts, L.L.P.

(B) STREET: 30 Rockefeller Plaza

(C) CITY: New York

(D) STATE: NY

(E) COUNTRY: U.S.A.

(F) ZIP: 10112

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Not Yet Assign

(B) FILING DATE: ll-MAR-1998

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/040,175

(B) FILING DATE: ll-MAR-1997

(viii) ATTORNE /AGENT INFORMATION:

(A) NAME: Richard S. Clark

(B) REGISTRATION NUMBER: 26,154

(C) REFERENCE/DOCKET NtJMBER: 30973-PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212-705-5000

(B) TELEFAX: 212-705-5020

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GATGACTCCA GCCACGACCT 20

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : CACAGACACC CCATCCTATC 20

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : GATATGTCTC CAGGCATGGC 20

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : GCAAGTTCAC CCTCAGAAGG 20

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 : CAGATATGTC ATTCTGGGAG GTC 23

(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 : AACACCATCC CTCCTCGAAC C 21

(2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : CCTAACAAAA GAGCTGAAAA GCCC 24

(2) INFORMATION FOR SEQ ID NO : 8 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 : ACTGTGATAC AGTGGATAGC CGCT 24

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : CATCTCTGCC CCCTCTGCTG 20

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CCCTCCGACG CCTGCTTCAC 20

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ACCCTCCCCG CTATCTTGTT A 21

(2) INFORMATION FOR SEQ ID NO : 12 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 : GTGCCCCTGC CCATTTCTGT 20

Claims

1. A method of detecting prostate cancer metastasis in a patient comprising detecting mRNA for the prostate specific antigen (PSA) and prostate specific membrane (PSM) genes from mRNA of pelvic lymph nodes by reverse-transcriptase polymerase chain reaction (RT-PCR) wherein detection of mRNA for the PSA or PSM gene is indicative of metastasis.

2. The method of Claim 1 wherein said mRNA for the PSA and PSM genes is detected by obtaining fresh pelvic lymph node samples from said patient, extracting RNA from said samples, and performing RT-PCR using primers specific for PSA and PSM mRNA.

3. The method of Claim 2 wherein said primers comprise sets of nested primers for PSA mRNA and PSM mRNA.

4. The method of Claim 3 wherein said nested primers for PSA mRNA comprise outer primers having the sequences of SEQ ID NO: 1 and SEQ ID NO:2, and inner primers having the sequences of SEQ ID NO: 3 and SEQ ID NO:4.

5. The method of Claim 3 wherein said nested primers for PSM mRNA comprise outer primers having the sequences of SEQ ID NO: 5 and SEQ ID NO: 6 and inner primers having the sequences of SEQ ID NO:7 and SEQ ID NO:8.