WO2002081656A2 - Potentiel diagnostique ameliore de cellules exprimant l'antigene specifique de la prostate - Google Patents

Potentiel diagnostique ameliore de cellules exprimant l'antigene specifique de la prostate Download PDF

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WO2002081656A2
WO2002081656A2 PCT/US2002/009736 US0209736W WO02081656A2 WO 2002081656 A2 WO2002081656 A2 WO 2002081656A2 US 0209736 W US0209736 W US 0209736W WO 02081656 A2 WO02081656 A2 WO 02081656A2
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pcr
psa
primers
cells
gene
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PCT/US2002/009736
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WO2002081656A3 (fr
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Chun L. Gao
Judd W. Moul
Shiv Srivastava
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The Henry M. Jackson Foundation
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Publication of WO2002081656A3 publication Critical patent/WO2002081656A3/fr
Priority to US10/677,495 priority patent/US20040241707A1/en

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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
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to sensitive and specific methods for the detection of prostatic disease such as clinically organ confined prostate cancer and potential micro- metastasis.
  • the methods relate to the use of RT-PCR (reverse-transcriptase polymerase chain reaction) to detect prostate specific antigen expression in peripheral blood derived epithelial cells.
  • RT-PCR reverse-transcriptase polymerase chain reaction
  • the invention also relates to kits and compositions that utilize these methods. Description of the Background
  • Adenocarcinoma of the prostate is the most common solid tumor in American males and is the second leading cause of cancer death in men. It is estimated that 317,100 new cases of prostate cancer (CaP) will be diagnosed this year and that 41,400 deaths will be attributed to prostate cancer. Elevated serum levels of the prostate gland-specific protein, PSA, are currently the most powerful and widely used prostate cancer screening test. However, the serum PSA test is not perfect and limited due to high false positive rates. New prostate cancer specific molecular and cellular markers are clearly needed both for early and accurate detection and prognosis of prostate cancer. While several risk factors (such as age, high fat diet and race) have been identified, the cause of the increased risk has not been identified. The identification of significant risk factors has led to global recommendations that all men over 50 be screened, and all men having a familial incidence of CaP be screened beginning at age 40. These diagnostic measures have led to greater investigation of practices for screening and staging CaP with greater detection capabilities.
  • the value of detecting circulating prostate cancer cells and their use as a staging tool is a function of the sensitivity of the test used to detect those cells.
  • PSA prostate specific antigen
  • PSA velocity is simply a measure of the change in PSA elevation over time.
  • PSA velocity is a qualitative analysis and its value is in ameliorating false positive determinations.
  • stage I cancer is found in the prostate only. It is usually found accidentally during surgery for other reasons.
  • stage ⁇ the cancer is more advanced, but has not spread outside the prostate.
  • Stage HI prostate cancer is identified by the spread of cancer beyond the outer layer of the prostate to nearby tissues. The cancer may be found in the seminal vesicles.
  • Stage IN cancer is marked by the metastasis of the cancer either to nearby tissues or to distant tissues including the lymph nodes. Metastatic cancer often spreads to the bones and once the cancer has metastasized to distant tissues the prognosis for the patient is poor.
  • mRNA when transcribed, is present in many copies, the number of which is directly correlated to the level of the gene's induction.
  • PCR polymerase chain reaction
  • the level of the expression can be statistically quantified (quantitative PCR) such that small increases in expression as measured by mRNA transcription are significant.
  • quantitative PCR quantitative PCR
  • RT-PCR is a method whereby mRNA is reverse transcribed resulting in a cDNA copy for every RNA genomic message. This is a powerful tool because, first, mRNA reflects the level of expression of a gene and second, the reverse transcribed cDNA is devoid of introns found in the genomic copy. Also, mRNA is generally shorter than genomic DNA and can be more easily distinguished.
  • the use of RT- PCR in clinical oncology was first reported in 1988 when it was used to detect the bcrlabl mRNA sequence present in the chronic myelogenous leukemia cells. The rationale for RT-PCR then and now is to identify a tumor cell's specific unique mRNA sequence in order to follow the molecular progression of the disease.
  • the tissue specific expression of the prostate specific antigen (PSA) in normal and cancerous epithelial cells makes the PSA gene expression a potentially useful marker for the detection of occult metastases of CaP.
  • the invention is directed to methods whereby patients with early stage prostate cancer, that is clinically organ confined prostate cancer, are shown to possess circulating prostatic epithelial cells. In these cells there is a significant correlation of PSA expression in peripheral blood with extracapsular disease and cancer recurrence, which has great prognostic value.
  • the detection of circulating cancer cells in the blood and bone marrow of prostate cancer patients has the potential to improve diagnosis, staging and follow-up of the disease. Further, and unlike conventional diagnostics which may be highly variable, the diagnostic ability of the presently claimed invention is highly reproducible between different laboratories.
  • prostate epithelial cells are isolated from blood and the RNA subjected to RT-PCR.
  • the resulting cDNA is subjected to traditional PCR amplification with primers able to distinguish between the genomic copy of the gene and the cDNA copy resulting from CaP gene expression.
  • This method provides an assay which is more sensitive, specific and reproducible as compared to conventional methods. Results disclosed herein show that there is a correlation between the presence of prostate epithelial cells in the bone marrow with cancer recurrence.
  • RT-PCR- PSA assays on enriched epithelial cells isolated from peripheral blood were performed. These results indicated that there is PSA expression in cells isolated from cancer patients which provide a diagnostic tool for the early detection of prostate disease.
  • any diagnostic method is its ability to detect a malignancy at the earliest possible time.
  • epithelial cell enrichment with RT-PCR of the PSA message (ERT-PCR/PSA)
  • ERT-PCR/PSA RT-PCR of the PSA message
  • detection of one PSA-expressing LNCaP cell per 10 7 lymphocytes is possible.
  • the ability to detect significant increases in PSA expression at lower detection limits is greatly increased by the current invention. This allows for the more accurate staging of the disease, identification of risk factors and analysis of the efficacy of radical intervention than is currently available.
  • Figure 1 shows ERT-PCR/PSA sensitivity as determined by spiking 1 ml of normal human female blood with a known number of LNCaP cells.
  • Figure 2 shows the results of the ERT-PCR/PSA assay from peripheral blood of prostate cancer patients in representative experiments.
  • Figure 3 shows the relationship of ERT-PCR/PSA and biopsy results in patients undergoing biopsy for suspicion of prostate cancer determination of sensitivity and specificity of analysis
  • the present invention provides sensitive methods for the detection of prostate cancer.
  • the invention also provides for kits and compositions used in these detection methods.
  • RT-PCR based assays to detect circulating prostatic epithelial cells face a technological challenge with respect to inter-laboratory as well as intra- laboratory variations resulting from the sensitivity limits of these assays.
  • the present invention is directed to the surprising discovery that RT-PCR-PSA assays of epithelial cells enriched from peripheral blood can provide sensitive and specific early detection of prostate cancer in at-risk individuals. Further, detection of circulating prostate cancer cells in the blood and bone marrow of CaP patients have the potential to improve the staging and follow-up of the disease.
  • Prior assays for PSA have analyzed blood for the soluble form of the PSA protein.
  • the present invention greatly improves the sensitivity of PSA detection by analyzing blood for the presence of the mRNA precursor cell from which PSA is translated. While a preferred embodiment of the instant invention uses peripheral blood, other sources of the blood samples are equally determinative. Generally, sensitivity and specificity of the method of the invention are not dependent on the origin of the sample.
  • the invention is not limited to blood samples, but may include samples of bodily fluids and/or tissues that contain or potentially contain epithelial cell such as bone marrow.
  • the present invention solves this problem by first enriching the blood sample for prostate epithelial cells. This enrichment process is most easily achieved by immuno selection although it may be achieved by other methods such as cell sorting and histologic analysis.
  • Immuno selection of epithelial cells may comprise the selection of surface markers through the use of specific antibodies which are commercially available. Such antibodies include the Ber-EP antibody which is specific to surface epithelial glycoprotein and antibodies specific to the epithelial cell membrane antigen.
  • antibodies specific to epithelial cell markers are coated on magnetic beads which are then added to the blood sample (Sambrook et al. Molecular Cloning: A Laboratory Manual (Third Edition) Cold Spring Harbor Press, 2001). After incubation for a period of time, preferably from two minutes to two hours, the beads are washed and adhered epithelial cells lysed. The nucleic acid is then isolated using conventional methods. Commercial reagents are also available for isolating RNA.
  • RNA coated with antibodies specific to epithelial cell surface antigens can be commercially obtained. Dynal Corporation (Prod. No. 161.01). After the beads are appropriately prepared, they are incubated with the blood sample for approximately 30 minutes. The cell-adhered beads are washed, the cells lysed and the lysed cells centrifuged to remove the nuclear pellet. The supernatant is then recovered and the nucleic acid extracted using phenol/chloroform extraction followed by ethanol precipitation. This provides total RNA. mRNA can be isolated from total RNA by exploiting the polyA tail of mRNA by use of several commercially available kits. QIAGEN mRNA Midi kit (Cat. No. 70042); Promega PolyATract® mRNA Isolation Systems (Cat. No.
  • the QIAGEN kit provides a spin column using Oligotex Resin designed for the isolation of poly A mRNA from total RNA within 30 minutes.
  • the Promega system uses a biotinylated oligo dT probe to hybridize to the mRNA polyA tail and requires about 45 minutes to isolate pure mRNA.
  • RT-PCR can be carried out using commercially available methods (e.g.
  • RNA may be reversed transcribed using, preferably, random hexamers and the TaqMan Reverse Transcription Reagents Kit (Perkin Elmer) following the manufactures' protocols.
  • the cDNA is amplified using primers specific for the gene of interest.
  • RNA can be reversed transcribed using oligo(dT), which bind to the endogenous polyA tail of the mRNA. This method can be used as a universal priming means for cDNA synthesis.
  • the reverse transcription can be effected using primers specific to the gene sequence of interest.
  • RNA After the cDNA has been synthesized from the RNA traditional PCR can be used to amplify specific sequences within the cDNA. By increasing the fidelity of the PCR reaction the sensitivity of the assay is increased such that misprimed sequences, pseudogenes and genomic contaminants are not confounding factors for the identification of occult CaP metastases.
  • a modified form of Taq polymerase was chosen which is heat activated such that primer polymerization does not occur until the PCR reaction is at optimum temperatures.
  • Such polymerases are commercially available from, for example, Applied BioSystems (AmpliTaq Gold, Prod. No. 4311806).
  • HGK human glandular kallikrein
  • RNA samples were also analyzed by PCR without any RT step. PCR without any RT step did not yield any detectable product.
  • RT-PCR reactions can be run in duplicate while a third reaction comprising a separate three ⁇ l RNA aliquot without reverse translation can be run with the disclosed primers to rule out contamination with genomic DNA and pseudogene interference.
  • Sensitivity of the assay can be defined as the ability to detect PSA expressing cells in background lymphocytes.
  • sensitivity was at least as low as one PSA-expressing CaP cell per 10 7 lymphocytes which are typically found in between about 1-5 ml of blood.
  • sensitivity is one CaP cell in between about 10 6 and 10 8 lymphocytes, and more preferably, one CaP cell in between about 10 7 and 10 9 lymphocytes.
  • sensitivity of the present invention makes it possible to identify circulating prostate cancer cells in the peripheral blood of at least about 70% of prostate cancer patients, preferably at least about 80%, and more preferably at least about 90% (i.e. number of patients detected as positive by the method of the invention/number of patients with prostate cancer).
  • Specificity as determined by the number of true positives (i.e. true positives/true positives plus false positives), is at least about 65-70%, preferably at least about 75-80%, more preferably at least about 85-90%, and still more preferably at least about 95%.
  • the reproducibility of ERT-PCR PSA has been significantly improved compared to conventional methods.
  • This method may further comprise automation and quantitation such as, for example, by using ABI 7700 machine, Taqman chemistry and a single PCR step.
  • ABI 7700 machine ABI 7700 machine
  • Taqman chemistry a single PCR step.
  • ERT-PCR/PSA sensitivity was determined by spiking one ml of normal-human female blood with known numbers of LNCaP cells.
  • the detection limit of E-PSA assay was as low as one cell in one ml of blood.
  • the current assay is designed such that the sensitivity of individual assays is maintained.
  • the sensitivity is not limited to individual experiments because positive or negative scoring of each patient is based on controlled assays with two independent RT reactions and one reaction without RT for each specimen.
  • the stringency of the sensitivity criteria of the RT- PCR assay is tested with each experiment which ensures that each batch of reagents provides the sensitivity of the detection of one CaP cell/ml of blood.
  • This sensitivity is maintained by running a spiked control sample with each assay.
  • the detection limit is increased by performing gel-electrophoresis of the PCR product on 10% polyacrylamide gels with SYBR Gold® (Molecular Probes, Eugene, OR Cat. No. S-l 1494).
  • SYBR Gold® is a modified gel stain that exhibits greater than 1,000- fold fluorescence enhancement over ethidium bromide staining.
  • a further benefit of the invention is the ability to record a molecular profile of circulating prostate cells which may have an impact on defining prostate cancer progression and malignancy as a whole.
  • the disclosed method provides an opportunity for the molecular characterization of circulating prostate cancer cells for genomic alterations by in situ-based assays and analysis of specific genes such as p53 and bcl-2.
  • the instant invention also provides further ability to identify gene signatures of occult metastasis because once epithelial cells are isolated there is a possibility for further characterization of the circulating prostate cancer cells.
  • cancer determinants may also include PSA, PSGR, DD3, PCGEM1 and PSMA genes, as well as variants of these genes that have been identified (e.g. Mikolajczyk et al., J. Urol. 161 :208, 1999; Mikolajczyk et al., Urol. 50:710-714, 1997).
  • the molecular profiling aspect of the invention with respect to one or a plurality of such genes may provide the identification of further genes whose expression changes, either up-regulated or down-regulated, during the course of the disease. Those changes may occur over a period of hours, days, months or even years.
  • kits for the sensitive and specific detection of early-stage prostate cancer such as, for example, a subclinical stage of prostate cancer or organ-confined prostate cancer.
  • Kits comprise reagents for conducting an RT-PCR on nucleic acid isolated from a sample of blood.
  • Reagents include a Taq polymerase enzyme and additional enzyme capable of detectably amplifying a single gene in a single cell in a background of 10 7 cells.
  • Reagents further include a set of primers such as, for example, random-hexamer primers, preferably sequences specific for exons of the PSA gene, more preferably oligo-dT primers, and still more preferably SEQ ID NO 1 and SEQ ID NO 2, and a set of PSA-specific primers, preferably a hemi -nested set of primers, and more preferably SEQ ID NO 3 and SEQ ID NO 4.
  • Reagents may further include additional solutions, buffers, and materials necessary or appropriate in conventional ERT-PCR PCR, and typical prostate cancer detection kits. Kits of the invention are capable of detecting one PSA-expressing cell in one ml of blood or 10 7 lymphocytes. Sensitivity of kits is preferably at least about 80% or greater, and specificity is preferably at least about 85% or greater.
  • Example 1 Immunomagnetic Capture of Epithelilal Cells from Blood and RNA Preparation.
  • Five milliliters of blood was collected into a sodium citrate tube and transported to the laboratory on wet ice within 2-3 hours after the blood was drawn.
  • Dynabeads coated with monoclonal antibody, Ber-EP4 specific for immunomagnetic enrichment of human epithelial cells (Dynal, product number: 116.02), were mixed thoroughly in the vial.
  • Eighty micro-liters of Dynabeads suspension was transferred to a 0.5ml Eppendorf tube, placed on a Dynal MPC rack for 2 minutes and the supernatant pipetted off.
  • RNAzol B Tel-TEST, Inc.
  • RNA was prepared according to the manufacture's recommendations and 2 ⁇ l glycogen was added as carrier prior to isopropyl alcohol precipitation.
  • RNA pellet was dissolved in lO ⁇ l DEPC water and stored at -80°C. Three ⁇ l of RNA was used for each RT-PCR reaction, performed in duplicate for each patient with a third RNA sample used as a control (e.g. Example 5).
  • RT Reverse Transcriptase
  • PCR Polymerase Chain Reaction
  • the 25 ⁇ l volume of the PCR reaction mixture included 1.0 mM MgCl 2 , 200 ⁇ M deoxy-nucleotide triphosphate (dNTP), 25ng of each primer, and 0.5 U AmpliTaq Gold Polymerase (Perkin Elmer Lot B00783).
  • PCR amplification protocol included one cycle at 95°C for 10 minutes, 25 cycle at 94°C for 30 seconds, 66°C for 1 minute and 72°C for 1 minutes, followed by one cycle at 72°C for 5 minutes.
  • Two micro liters of the first PCR reaction served as the template for the hemi-nested PCR using one internal primer and one original primer related to the first set of PCR primers in 50 ⁇ l reaction volume.
  • the PCR components and cycles for nested PCR were the same as for first PCR reaction.
  • PCR primers were designed using PrimerDetect software (Clonetech,CA) and their specificity were defined by sequence homology searches of NCBI sequence database to exclude the possibility of amplification of PSA related genes. This is an important consideration as PSA belongs to the family of kallikrein gene family.
  • the sense primer (5' 5'
  • GCCTCTCGTGGCAGGGCAGTC-3'; SEQ ID NO 1) and the antisense primer (5'- CATCACCTGGCCTGAGGAATC-3'; SEQ ID NO 2) were selected from exon 2/3, which yield a 216-bp product from PSA RNA and a 1.8 kb product from the PSA genomic DNA. Therefore, we could easily determine the contamination of genomic DNA in RNA preparation.
  • the Nested PCR sense primer (5'CACTGCATCAGGAACAAAAGCGT-3'; SEQ ID NO 3) and the antisense primer (5'-CATCACCTGGCCTGAGGAATC-3'; SEQ ID NO 4) yield a 156-bp DNA fragment.
  • RNA aliquots from each patient sample were processed for two identical RT-PCR reactions and one control reaction with out RT (NO-RT) to rule out a false positive result with genomic DNA and pseudogene interference.
  • PCR controls also included a no template- PCR reaction as a negative control and a positive control LNCaP RNA to validate the assay.
  • the analysis of the RT-PCR-derived PSA fragment included (a) visual detection of the expected size DNA bands by SYBR GOLD staining of the TBE-10% Polyacrylamide gel from hemi-nested PCR reactions; (b) direct DNA sequencing of randomly selected PCR products confirming their identity as PSA and (c) inclusion of a positive control in each assay to define the sensitivity of the assay as one PSA- expressing LNCaP cells/ml blood.
  • Specificity of the Ampli Taq Gold DNA polymerase and reverse transcriptase enzymes was considered crucial for this experiment as we noticed that different batches of enzymes showed varying sensitivity. It was important to select the enzyme lot that reproducibly detected one PSA expressing LNCaP cell per ml of female blood.
  • Example 2 Confirmation of Assay Sensitivity.
  • ERT-PCR/PSA assay was determined by serial dilutions of total RNA derived from a known number of LNCaP cells spiked into total RNA of a known number of peripheral blood mononuclear cells (PBMC) at LNCaP/PBMC cell ratios of 1:10 2 , 1:10 3 ', 1:10 4 , 1:10 5 , 1:10 6 , 1:10 7 , 1 :10 8 , 1 :10 9 .
  • PBMC peripheral blood mononuclear cells
  • the PCR amplification product was detected by ethidium bromide staining of agarose gels.
  • the limit of ERT-PCR/PSA in this assay was 1 :10 .
  • the sensitivity of the assay from the analysis of peripheral blood was illustrated in representative experiments which are shown in Figure 2. Using primers specific for the PSA gene, sensitivity was defined as the detection of the number of prostate cancer cells (one LNCaP cell) per ten million lymphocytes (RNAzol B; RNA extraction kit; Tel-Test, Inc.). Control samples consisted of 20 healthy volunteers (2 women and 18 men). Peripheral blood samples were negative by RT-PCR in each control. Nested PCR product was analyzed on an ethidium bromide stained agarose gel.
  • ERT-PCR/PSA ERT-PCR/PSA
  • RT-PCR for PSA expressing cells in 85 pre-radical prostatectomy patients is not related to clinical stage, age, race, grade, Gleason sum, serum PSA or PAP, tumor volume, or tumor multifocality. RT-PCR positivity did not predict pathologic stage or early PSA recurrence.
  • RNA from bone marrow aspirates of patients undergoing radical prostatectomy were analyzed for PSA gene expression. Fifty-one of 116 patients (44.0%) were positive resulting in a minimum of two independent RT-PCR assays, whereas 77/116 (66%) of the patients were RT-PCR positive in at least one assay. All of these RNA specimens, whether scoring positive or negative in the RT-PCR assay, show comparable levels of glyceraldehyde phosphate dehydrogenase (GAPDH) gene expression by RT-PCPJGAPDH assay. On the basis of this blind study, ERT-PCR/PSA positivity did not correlate with age, race, acid phosphatase, grade, Gleason sum, or clinical stage categories.
  • GPDH glyceraldehyde phosphate dehydrogenase
  • RNA prepared from enriched epithelial cells fractionated on the beads was analyzed for the expression of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) and PSA using nested RT- PCR assays.
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • Peripheral blood specimens of 111 of 137 (81.6%) (pre-surgery) prostate cancer patients who underwent radical prostatectomy were positive in ERT- PCR/PSA assay. Reproducibility of ERT-PCR/PSA assay was 83.8% (93 of 111) in specimens of cancer patients in two independent RT-PCR assays. Peripheral blood specimens from 13 of 79 patients who had biopsy for suspicion of prostate cancer were positive in ERT-PCR/PSA assay. Thirteen of 15 patients (87%) with biopsy proven prostate cancer and 58 of 64 patients with biopsy negative for prostate cancer (90%) were negative in ERT-PCR/PSA assay. These results are illustrated in figure 3.

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Abstract

L'invention concerne des méthodes et des kits sensibles et spécifiques utilisés dans le dépistage du cancer de la prostate chez un patient. L'invention utilise la technique de RT-PCR pour détecter l'expression ou le changement d'expression de l'antigène prostatique spécifique (PSA) dans les cellules épithéliales enrichies en sang entier. Ces méthodes et ces kits sont nettement améliorés par rapport aux méthodes antérieures de diagnostic du cancer de la prostate (CaP). Selon un premier mode de réalisation, les cellules épithéliales de la prostate sont isolées du sang et l'ARN soumis à la technique de RT-PCR. L'ADNc obtenu est soumis à l'amplification PCR via des amorces capables de distinguer entre la copie génomique du gène et la copie d'ADNc résultant de l'expression génique du CaP. L'invention concerne en outre un test plus sensible, plus spécifique et plus reproductible que les méthodes classiques. Les résultats de l'invention démontrent l'existence d'une corrélation entre la présence de cellules épithéliales de la prostate exprimant le PSA et la récurrence du cancer ; on obtient ainsi un outil diagnostique pour la détection précoce des maladies prostatiques.
PCT/US2002/009736 2001-04-05 2002-04-01 Potentiel diagnostique ameliore de cellules exprimant l'antigene specifique de la prostate WO2002081656A2 (fr)

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Cited By (4)

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EP1540002A2 (fr) * 2002-09-12 2005-06-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procede de detection de micro-metastases
JP2006072656A (ja) * 2004-09-01 2006-03-16 Hitachi Software Eng Co Ltd リアルタイムｐｃｒのプライマー設計方法
EP2407554A1 (fr) * 2010-07-14 2012-01-18 Fundacio Institut de Recerca de l'Hospital Universitari Vall d'Hebron Procédés et kits pour le diagnostic du cancer de la prostate
EP2407555A1 (fr) * 2010-07-14 2012-01-18 Fundació Institut de Recerca Hospital Universitari Vall d'Hebron, Fundació Privada Procédés et kits pour le diagnostic du cancer de la prostate

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US5858673A (en) * 1996-06-24 1999-01-12 Charlotte-Mecklenburg Hospital Authority Method for detecting prostate cells
US6365362B1 (en) * 1998-02-12 2002-04-02 Immunivest Corporation Methods and reagents for the rapid and efficient isolation of circulating cancer cells

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1540002A2 (fr) * 2002-09-12 2005-06-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procede de detection de micro-metastases
JP2006072656A (ja) * 2004-09-01 2006-03-16 Hitachi Software Eng Co Ltd リアルタイムｐｃｒのプライマー設計方法
EP1640887A1 (fr) * 2004-09-01 2006-03-29 Hitachi Software Engineering Co., Ltd. Procédé de conception d'amorce pour PCR en temps réel
US7698069B2 (en) 2004-09-01 2010-04-13 Hitachi Software Engineering Co., Ltd. Method for designing primer for realtime PCR
EP2407554A1 (fr) * 2010-07-14 2012-01-18 Fundacio Institut de Recerca de l'Hospital Universitari Vall d'Hebron Procédés et kits pour le diagnostic du cancer de la prostate
EP2407555A1 (fr) * 2010-07-14 2012-01-18 Fundació Institut de Recerca Hospital Universitari Vall d'Hebron, Fundació Privada Procédés et kits pour le diagnostic du cancer de la prostate
WO2012007545A1 (fr) * 2010-07-14 2012-01-19 Fundació Institut De Recerca Hospital Universitari Vall D'hebron, Fundació Privada Procédés et kits pour le diagnostic du cancer de la prostate
WO2012007546A1 (fr) * 2010-07-14 2012-01-19 Fundació Institut De Recerca Hospital Universitari Vall D'hebron, Fundació Privada Procédés et kits pour le diagnostic du cancer de la prostate
CN103221553A (zh) * 2010-07-14 2013-07-24 瓦尔德希伯伦大学医院发展研究院非公募基金会 诊断前列腺癌的方法和试剂盒

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