WO1998040513A1 - Procede de detection de la metastase du cancer de la prostate - Google Patents

Procede de detection de la metastase du cancer de la prostate Download PDF

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Publication number
WO1998040513A1
WO1998040513A1 PCT/US1998/004818 US9804818W WO9840513A1 WO 1998040513 A1 WO1998040513 A1 WO 1998040513A1 US 9804818 W US9804818 W US 9804818W WO 9840513 A1 WO9840513 A1 WO 9840513A1
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Prior art keywords
seq
psa
psm
primers
pcr
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PCT/US1998/004818
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English (en)
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Anna C. Ferrari
Nelson N. Stone
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Ferrari Anna C
Stone Nelson N
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Application filed by Ferrari Anna C, Stone Nelson N filed Critical Ferrari Anna C
Priority to AU65513/98A priority Critical patent/AU6551398A/en
Publication of WO1998040513A1 publication Critical patent/WO1998040513A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates to therapy of prostate cancer.
  • the present invention provides a method for staging localized prostate cancer prior to definitive local therapy.
  • metastasis may occur early, while the tumor is apparently confined, and remain undetectable by conventional pathology or radiographic evaluation for prolonged periods of time.
  • 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.
  • 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.
  • RT-PCR can be utilized to provide a sensitive and specific method to identify prostate cancer metastasis to pelvic lymph nodes.
  • 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.
  • PSM prostate specific membrane
  • the improved accuracy of staging achieved by the present invention has substantial benefits.
  • the implications of more accurate staging of pelvic LN are substantial.
  • the presence of prostate cells in the pelvic lymph nodes implies metastasis to tissues that define tumor staging and outcome.
  • 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.
  • 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.
  • AD androgen dependent
  • Al independent
  • 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
  • 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.
  • a cDNA equivalent to 1 ⁇ g of test RNA is added to a 50 ⁇ l final volume reaction containing IX PCR buffer, 1.5 mM MgCl 2 , 0.2 niM each dNTP, 0.1 ⁇ M each 3' and 5' outer primers, and 1.25 units Thermus aquaticus polymerase.
  • 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.
  • 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.
  • T 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
  • 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.
  • RNA 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 ⁇ l solution containing lx Buffer, 2.5 mM MgCl 2 , 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).
  • dNTP's 2' deoxadenosine 5' triphosphate (dATP), 2' deoxycytidine 5' triphosphate (dCTP), 2' deoxyguanosine 5
  • RNAse H were added and activated at 37° C for 20 minutes to destroy residual RNA.
  • POLYMERASE CHAIN REACTION a 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.
  • G3PDH housekeeping gene gluteraldehyde 3 phosphate dehydrogenase
  • ACCCTCCCCGCTATCTTGTTA 3' SEQ ID NO: 11 and 3'5': 1585-1566 5' GTGCCCCTGCCCATTTCTGT 3', SEQ ID NO: 12
  • 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.
  • RNA integrity was established in a competitive assay using a fixed amount (0.2 ⁇ g) 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.
  • cDNA equivalent to 0.2 ⁇ g 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 ⁇ g 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 MgCl 2 , 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.
  • 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.
  • 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 ⁇ g of test RNA was added to a 50 ⁇ l final volume reaction containing IX PCR Buffer, 1.5 mM MgCl 2 , 0.2 mM each DTNP, 0.1 ⁇ M 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.
  • a 46 ⁇ l reaction mixture identical to the one used for the first round of amplification but containing 0.1 ⁇ M each of 3' and 5' Nested Primers was used to amplify 4 ⁇ l of a 4/400 dilution of the outer primers PCR product.
  • the cycling conditions were the same as those described for G3PDH MIMIC.
  • 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
  • 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 32 P end-labeled 22 oligomer probe that would not be recognized by any of the primer sets utilized in the reaction.
  • 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.
  • 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
  • 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).

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Abstract

La présente invention concerne un procédé de détection de la métastase du cancer de la prostate sur les ganglions lymphatiques pelviens, au moyen d'une réaction en chaîne de la polymérase par transcriptase inverse, dans le but de détecter un ARN messager d'un antigène prostatique spécifique et d'un antigène prostatique spécifique d'enveloppe.
PCT/US1998/004818 1997-03-11 1998-03-11 Procede de detection de la metastase du cancer de la prostate WO1998040513A1 (fr)

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AU65513/98A AU6551398A (en) 1997-03-11 1998-03-11 Method of detecting prostate cancer metastasis

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000044940A2 (fr) * 1999-01-28 2000-08-03 Gen-Probe Incorporated Sequences d'acide nucleique permettant de detecter des marqueurs genetiques pour le cancer dans un echantillon biologique
EP1390523A2 (fr) * 2000-11-20 2004-02-25 Eastern Virginia Medical School Procedes et dispositifs permettant la detection quantitative des antigenes de membrane specifiques de la prostate (psma) et d'autres marqueurs prostatiques

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5506106A (en) * 1992-10-29 1996-04-09 Thomas Jefferson University Methods of detecting micrometastasis of prostate cancer

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US5506106A (en) * 1992-10-29 1996-04-09 Thomas Jefferson University Methods of detecting micrometastasis of prostate cancer

Non-Patent Citations (5)

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Title
DEGUCHI T., ET AL.: "DETECTION OF MICROMETASTATIC PROSTATE CANCER CELLS IN LYMPH NODES BY REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 53., no. 22., 15 November 1993 (1993-11-15), US, pages 5350 - 5354., XP002910108, ISSN: 0008-5472 *
MILLER W. H., ET AL.: "SENSITIVE NESTED REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION DETECTION OF CIRCULATING PROSTATIC TUMOR CELLS: COMPARISON OF PROSTATE-SPECIFIC MEMBRANE ANTIGEN AND PROSTATE-SPECIFIC ANTIGEN-BASED ASSAYS.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 54., no. 24., 15 December 1994 (1994-12-15), US, pages 6306 - 6310., XP002910107, ISSN: 0008-5472 *
STONE N., ET AL.: "DETECTION OF CIRCULATING METASTATIC TUMOR CELLS BY PSA AND PSM MAY BE USEFUL TO IDENTIFY SUBGROUPS OF ADVANCED PROSTATE CANCER PATIENTS.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 37., 1 March 1996 (1996-03-01), US, pages 247., XP002910105, ISSN: 0008-5472 *
STRAY J., ET AL.: "REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) DETECTS METASTATIC PROSTATE CANCER CELLS IN LYMPH NODES, BLOOD AND POTENTIALLY IN BONE MARROW USING PSA-MRNA AS TEMPLATE.", JOURNAL OF UROLOGY., LIPPINCOTT WILLIAMS & WILKINS, BALTIMORE, MD, US, vol. 151., no. SUPPL. 05., 1 January 1994 (1994-01-01), BALTIMORE, MD, US, pages 412A., XP002910106, ISSN: 0022-5347 *
VERKAIK N. S., ET AL.: "CLINICAL USEFULNESS OF RT-PCR DETECTION OF HEMATOGENOUS PROSTATE CANCER SPREAD.", UROLOGICAL RESEARCH, SPRINGER VERLAG, BERLIN, DE, vol. 25., no. 06., 1 December 1997 (1997-12-01), DE, pages 373 - 384., XP002910109, ISSN: 0300-5623, DOI: 10.1007/BF01268851 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000044940A2 (fr) * 1999-01-28 2000-08-03 Gen-Probe Incorporated Sequences d'acide nucleique permettant de detecter des marqueurs genetiques pour le cancer dans un echantillon biologique
WO2000044940A3 (fr) * 1999-01-28 2000-12-07 Gen Probe Inc Sequences d'acide nucleique permettant de detecter des marqueurs genetiques pour le cancer dans un echantillon biologique
US6551778B1 (en) 1999-01-28 2003-04-22 Gen-Probe Incorporated Nucleic acid sequences for detecting genetic markers for cancer in a biological sample
US6811985B2 (en) 1999-01-28 2004-11-02 Gen-Probe Incorporated Nucleic acid sequences for detecting genetic markers for cancer in a biological sample
US7267956B2 (en) 1999-01-28 2007-09-11 Gen-Probe Incorporated Nucleic acid sequences for detecting genetic markers for cancer in a biological sample
EP1390523A2 (fr) * 2000-11-20 2004-02-25 Eastern Virginia Medical School Procedes et dispositifs permettant la detection quantitative des antigenes de membrane specifiques de la prostate (psma) et d'autres marqueurs prostatiques
EP1390523A4 (fr) * 2000-11-20 2004-03-31 Eastern Virginia Med School Procedes et dispositifs permettant la detection quantitative des antigenes de membrane specifiques de la prostate (psma) et d'autres marqueurs prostatiques

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