WO2003023060A2 - Methode et trousse de diagnostic ou de surveillance du traitement du cancer du sein - Google Patents

Methode et trousse de diagnostic ou de surveillance du traitement du cancer du sein Download PDF

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Publication number
WO2003023060A2
WO2003023060A2 PCT/EP2002/009999 EP0209999W WO03023060A2 WO 2003023060 A2 WO2003023060 A2 WO 2003023060A2 EP 0209999 W EP0209999 W EP 0209999W WO 03023060 A2 WO03023060 A2 WO 03023060A2
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WIPO (PCT)
Prior art keywords
cells
claudin
dna
oligonucleotides
cdna
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PCT/EP2002/009999
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German (de)
English (en)
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WO2003023060A3 (fr
Inventor
Alf-Andreas Krehan
Pia Steffens
Bertha Gutierrez
Stefanie WASCHÜTZA
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Adnagen Ag
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Publication date
Priority claimed from DE10143776A external-priority patent/DE10143776A1/de
Priority claimed from PCT/EP2002/005489 external-priority patent/WO2003023057A2/fr
Application filed by Adnagen Ag filed Critical Adnagen Ag
Priority to US10/488,828 priority Critical patent/US20050014208A1/en
Priority to EP02774579A priority patent/EP1409746A2/fr
Publication of WO2003023060A2 publication Critical patent/WO2003023060A2/fr
Publication of WO2003023060A3 publication Critical patent/WO2003023060A3/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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a method and a kit for the diagnosis or treatment control of breast cancer in a human.
  • tumor markers at the protein level (immunologically or enzymatically) are determined quantitatively in the blood or in other body fluids in cancer patients.
  • tumor diagnostics or treatment control / aftercare since increased tumor marker values are also caused by non-tumor diseases (e.g. inflammation of the gastrointestinal tract). Tracts, cirrhosis of the liver, viral infections), heavy smoking or can be caused by pregnancy.
  • non-tumor diseases e.g. inflammation of the gastrointestinal tract.
  • the object of the present invention is to provide a method and a kit with which a diagnosis or treatment control of breast cancer is possible in a simple, safe and repeatable manner.
  • RNAs of the markers described are normally not expressed in the blood of healthy people, there is a direct correlation between a positive RT-PCR detection of these tumor markers and circulating tumor cells in the blood, which can lead to metastasis. Since individual markers can be expressed differently in a therapy-dependent manner and breast cancer has a pronounced heterogeneity in the expression pattern of breast cancer cells, it is advantageous to investigate a combination of tumor markers in order to detect all tumor cells circulating in the blood. In this way, tumor cells can also be recognized when the expression of a particular marker in a patient or in a disease stage is relatively low, which could otherwise lead to a supposedly negative result. However, the use of further markers usually comes up against limits if mononuclear blood cells have a background expression ("illegitimate transcription") which hinder an exact analysis.
  • GA733.2 PDGF- ⁇ , Her-2 / new, Claudin-7; GA733.2, MUCl, CEA;
  • GA733.2 PDGF- ⁇ , Claudin-7; respectively.
  • the primers given in the table below can be used for the amplification of sections of the markers.
  • an increase in specificity can be achieved by enriching the tumor cells with respect to blood cells and at the same time an increase in the sensitivity of tumor cell detection (detection rate from 1 tumor cell to 10 7 mononuclear blood cells).
  • the tumor cells are isolated using specific antibodies or an antibody mixture of mononuclear blood cells. separates. The separation can be carried out by means of magnetic particles (dynamic) to which the antibodies are bound. This is described in more detail below.
  • Eukaryotic cells have a large number of different molecules on their cell surface.
  • the combination of the expressed surface molecules differs according to the origin and the function of the individual cell, so that cell type-specific patterns arise.
  • Antibodies are used to recognize these cell type-specific patterns.
  • Antibodies bind to their antigen with high specificity, here to selected surface molecules. This property is used to recognize and differentiate cells by means of specific antibody binding based on their cell type-specific patterns.
  • the expression of special surface proteins distinguishes tumor cells from untransformed cells of this cell type. Since this special pattern of surface antigens in tumor cells also differs from the typical blood cell patterns, tumor cells in the blood can be distinguished. To identify tumor cells, antibodies that specifically recognize these special surface proteins are used as tools. The specific antibody binding is used for various analysis and separation methods.
  • Antibodies are coupled with fluorescent dyes for flow cytometric analysis. Scattered cells are individually guided past a light source (laser) in a constant liquid flow. When the cells are illuminated, the fluorescent dyes bound to the antibodies are excited and emit light of specific wavelengths. The emitted light is detected and the measured signal is stored digitally. The light signal can be assigned to individual cells.
  • Cell is recognized in this way and can now be separated from other cells.
  • the cells are separated into tiny drops for separation. After detection of the antibody-labeled cell, the corresponding drop is directed into a collecting container.
  • Antibodies are coupled to pseudomagnetic particles for magnetic separation. After the pseudomagnetic particles have been introduced into a magnetic field, the particles migrate in the magnetic field. When moving in this magnetic field, cells to which these coupled antibodies are bound are entrained and separated from other cells.
  • pseudomagnetic particles are consequently attached to the one have a defined number of chemically activated sites on their surface, antibodies are covalently coupled.
  • the specificity of the separation is determined via the specificity of the antibodies.
  • a blood sample containing tumor cells is mixed with antibody-coupled magnetic particles; then particles and blood are moved relative to each other. Those (tumor) cells that are recognized by the solid-phase-bound antibodies and thus firmly bound follow the movement of the particles. This makes it possible, when a magnetic field is applied, to pull the particles with the cells attached to them out of the blood (eg on the wall of the separation vessel).
  • the blood thus depleted of tumor cells can be exchanged for other solutions, the cells separated via magnetic particles remaining on site until the magnetic field is switched off / removed and are available for further applications.
  • specific antibody mixtures can be used for the detection of the tumor cells, which are either optimized generally for tumor cells or also specifically for breast cancer cell detection.
  • a combination of the antibodies MOC-31 (Novocastra) and Ber-EP-4 (DAKO) is suitable for the detection of tumor cells in the blood.
  • a detection that is specifically aimed at breast cancer cells can be achieved by a further optimized antibody mixture according to the following table:
  • Antibody mixtures of this type show an increased sensitivity in comparison to the separately used antibodies in cell recognition and cell separation, regardless of the method used.
  • FIGS. 2A to 2C a tumor marker detection using light cycler
  • FIGS. 4 to 8 show further examples for the detection of breast cancer cells by means of several tumor markers.
  • RNA processing was carried out from 1 ml of EDTA whole blood using the QIAamp RNA Blood Mini Kit (from Qiagen, Hilden). Contamination with genomic DNA was caused by an additional DNA digestion on the column using RNase-free DNase Set, from Quiagen, Hilden) avoided.
  • RNA processing from 1 ml of EDTA whole blood was verified photometrically using the quotient 260: 280 nm.
  • 1 ⁇ l of the batch can be analyzed by electrophoretic separation on an RNA 6000 chip using the Agilent Bioanalyzer 2100.
  • oligo (dT) 15 primers from Promega, Mannheim
  • the cDNA synthesis was carried out using the Sensiscript TM reverse transcriptase kit (from Qiagen, Hilden) in a 20 ⁇ l reaction mixture according to Table 1 at 37 ° C. for 1 hour with subsequent inactivation of the reverse transcriptase for 5 minutes at 95 ° C and then cooling on ice.
  • a pair of primers was used for each tumor marker, as shown in Table 3 below.
  • the PCR was carried out under the PCR conditions given in Table 5 and with the marker-specific melting temperatures and number of cycles given in Table 6.
  • FIG. 1 1 ⁇ l of the PCR product thus generated was separated in an Agilent Bioanalyzer 2100 on a DNA chip (500) and the separation result was documented electronically.
  • track 1 shows a 100 kb conductor and tracks 2-13 the results of the corresponding samples.
  • lane 5 shows a PCR product for the tumor marker stanniocalcin
  • lane 9 shows a PCR product for the tumor marker EGF-R
  • lane 13 shows a PCR product for the tumor marker CEA
  • all samples with biological material show lanes 4 , 5, 8, 9, 12, 13 PCR products for the internal control contain ⁇ -actin.
  • Lanes 2, 3, 6, 7, 10, 11 contain no biological material, so that there are no corresponding PCR products.
  • the so-called cDNA control is an approach without RNA
  • the so-called PCR control is an approach without cDNA
  • the negative control is an approach with RNA from a healthy control person in FIG. 1.
  • CEA stands for carcinoembryonic antigen
  • STC for stanniocalcin
  • EGF-R epidermal growth factor receptor
  • this tumor marker detection can also be carried out using a light cycler (from Röche, Basel).
  • the reverse transcription of the mRNA is carried out as described above.
  • the PCR was then carried out using the Light Cycler-DNA Master Sybr Green I Kit (Röche, Basel) according to the manufacturer's instructions under the conditions optimized for each tumor marker.
  • Table 3 The oligonucleotides given in Table 3 were used as primers.
  • Table 7 and Table 8 show the approach for the PCR and the PCR conditions in the light cycler.
  • FIGS. 2A to 2C The result of this PCR and evaluation by light cycler technology is shown in FIGS. 2A to 2C. posed.
  • the control curve is denoted by 2, while the curve which was recorded for the sample is denoted by 1.
  • the melting curve of the PCR products is analyzed by Sybr Green I detection.
  • the respective graph in FIGS. 2A to 2C represents the measured fluorescence as a function of the temperature.
  • the fluorescence peaks occurring in the control batches are due to primer dimers.
  • 2A shows the melting curve analysis of the Stanniocalcin PCR product.
  • the melting point of the main product is 89.2 ° C. and the melting point of the by-product is 85.3 ° C. Such fluorescence peaks cannot be seen in the control sample.
  • 2B shows the melting curve analysis of the EGFR-PCR product with a melting point at 84.6 ° C.
  • 2C shows the melting curve analysis of the CEA-PCR product with a melting point at 89.06 ° C.
  • agarose gel electrophoresis can of course also be used, in which, for example, 25 ⁇ l of the PCR product shown above are separated using a 2.5% agarose gel and the DNA bands are subsequently stained and visible with ethidium bromide be made. Documentation can be carried out, for example, with the help of the DUO Store System from Intas.
  • a fragment analysis using the ABI Prism 310 Genetic Analyzer can also be used for the evaluation.
  • a PCR with fluorescence-labeled primers is carried out and then, for example, 1 ⁇ l of the respective PCR product is used in a dilution of 1:50.
  • RNA isolated as a detection basis and the cDNA synthesized from it Central to the quality of the RNA isolated as a detection basis and the cDNA synthesized from it is the enrichment of the cell fraction used for this purpose from the blood sample used. Four different methods are available for this:
  • Erythrocyte lysis buffer ("QIAmp Blood Kit", Qiagen; Hilden) lysed on ice for 20 minutes. After removing the plasma / lysate from the pelleted cells and resuspension, centrifugation is carried out again at 3000 x g for 20 minutes. After removing the supernatant, the pelleted leukocyte fraction is available for the RNA preparation.
  • Hypaque gradients (Pharmacia, Uppsala, Sweden) separated and then washed twice with PBS / 1% FCS.
  • the mononuclear cells from the fraction enriched under b) are labeled with fluorescence-labeled mononuclear antibodies against tumor-specific
  • RNA can then be isolated as described above.
  • the isolated fraction of the mononuclear blood cells isolated by one of the above methods can also be isolated in trizole
  • RNA-containing aqueous phase is precipitated in isopropanol at -80 ° C. After washing twice in 80% ethanol, the pellet is on the
  • RNA isolation of the RNA is then followed by reverse transcription and mRNA detection as described above.
  • the expression of special surface proteins distinguishes tumor cells from untransformed cells of this cell type. Since this special pattern of surface antigens in tumor cells also differs from the typical blood cell patterns, tumor cells in the blood can be distinguished. To identify tumor cells, antibodies that specifically recognize these special surface proteins are used as tools. The specific antibody binding is made usable for the method according to the invention. Antibodies are covalently coupled to pseudomagnetic particles that have a defined number of chemically activated sites on their surface. The specificity of the separation is determined via the specificity of the antibodies. A blood sample containing tumor cells is coupled with antibody
  • tumor cells are generally recorded with high specificity. This is due to the selective expression of certain surface proteins that differentiate cancer cells from other cells.
  • the tracks la to 4a, 1b to 4b and 1c to 4c then show the detection of RNA after RNA preparation and RT-PCR with tumor marker-specific primers as described above for samples with a volume of 1 ⁇ l each. 3 was determined by means of electrophoretic separation in an Agilent TM bioanalyser 2100 according to the manufacturer's instructions.
  • FIGS. 3A and 3B When using magnetic particles labeled only with an antibody, as shown in FIGS. 3A and 3B, positive detection was only possible with a content of 1000 cells. When using an antibody mixture as in FIG. 3C, detection was already provided with only 100 cells and thus by a factor of 10 more sensitive.
  • FIG. 4 shows the detection of breast cancer cells by the simultaneous detection of the tumor cell markers GA733.2, MUCl, Her-2 and Claudin-7.
  • Breast tumor cells were inoculated into a sample, various Zeil lines, Zeil line 1 and Zeil line 2 being introduced. Before the markers were determined, the tumor cells were enriched by means of antibody-coupled magnetic particles, the antibodies BerEp4, HMTV.2, GP1.4 being used.
  • FIG. 4 shows that a reliable detection down to two cells per 5 ml can be detected for both cell lines by such a combination of the tumor markers to be detected. There was no unspecific reaction.
  • FIG. 5 also shows the detection of breast tumor cells, the combinations of the markers GA733.2, PDGF- ⁇ , Her-2 and Claudin-7 (FIG. 5A) or GA733.2, MUCl and CEA were detected. Again, a very sensitive detection down to two cells per 5 ml sample takes place, while in a control blood sample no unspecific detection reactions occurred.
  • FIG. 6 shows the use of the marker combination GA733.2, PDGF- ⁇ and Claudin-7 (FIG. 6A) or GA733.2, PDGF- ⁇ and Claudin-7 (FIG. 6B) for a first cell line ( Fig. 6A) and a second cell line (Fig. 6B) shown.
  • a highly specific detection takes place without unspecific reactions, with cell line 2 being able to be detected better than cell line 1.
  • FIG. 7 again shows detection using the combination of markers GA733.2, MUCl and Claudin-7 , There is highly specific detection for cell line 2 down to two cells per 5 ml for each of the markers and in particular for the combination of the markers without non-specific detection reactions.
  • the diagnostic kit according to the invention and the method according to the invention also make it possible to subsequently continue to use the sorted and separated cells as desired.
  • these can be inserted into a suitable cell culture medium and cultivated in situ.
  • chromosome analyzes Since the cells are intact after separation, the properties of the cell membrane and cell preserved core. This opens up the possibility of microscopically examining the expression of further surface markers or of carrying out chromosome analyzes.
  • the sorted cells are applied to slides. Additional surface markers can be detected cytochemically or using fluorescence microscopy. Genetic analyzes can also be carried out, such as chromosome analyzes using FISH (Flurorescence in situ hybridization) or karyogram preparation.
  • FISH Fluorescence in situ hybridization

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Abstract

La présente invention concerne une méthode et une trousse de diagnostic ou de surveillance du traitement du cancer du sein chez un être humain. Cette méthode consiste à détecter la présence ou l'absence d'au moins deux ARNm différents dans un prélèvement sanguin humain, lesdits ARNm codant pour différents marqueurs tumoraux parmi EGF-R, CEA, stanniocalcine, CK20, MAGE-3, GA733.2, MUC1, Her-2/neu, claudine-7 et/ou PDGF-β, ainsi que la présence de cellules cancéreuses mammaires dans ledit prélèvement sanguin, de manière à diagnostiquer d'éventuelles métastases.
PCT/EP2002/009999 2001-09-06 2002-09-06 Methode et trousse de diagnostic ou de surveillance du traitement du cancer du sein WO2003023060A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/488,828 US20050014208A1 (en) 2001-09-06 2002-09-06 Method and kit for diagnosing or controlling the treatment of breast cancer
EP02774579A EP1409746A2 (fr) 2001-09-06 2002-09-06 Methode et trousse de diagnostic ou de surveillance du traitement du cancer du sein

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE10143776A DE10143776A1 (de) 2001-09-06 2001-09-06 Verfahren und Kit zur Diagnostik oder Behandlungskontrolle von Brustkrebs
DE10143776.5 2001-09-06
EPPCT/EP02/05489 2002-05-17
PCT/EP2002/005489 WO2003023057A2 (fr) 2001-09-06 2002-05-17 Procede et kit de diagnostic destines a la selection et/ou detection qualitative et/ou quantitative de cellules

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WO2003023060A3 WO2003023060A3 (fr) 2004-01-29

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Cited By (9)

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EP1693467A1 (fr) * 2005-02-17 2006-08-23 Tosoh Corporation Methode d'analyse d' ARNm de la Stanniocalcin (STC1)
EP1694865A1 (fr) * 2003-12-12 2006-08-30 Bayer Pharmaceuticals Corporation Procede de prediction et de pronostic du cancer, et surveillance de traitement anticancereux
WO2007048978A2 (fr) * 2005-10-28 2007-05-03 Biomerieux Sa Procede de detection du cancer
FR2892730A1 (fr) * 2005-10-28 2007-05-04 Biomerieux Sa Methode pour detecter la presence ou le risque de developper un cancer
FR2899239A1 (fr) * 2006-03-31 2007-10-05 Biomerieux Sa Procede de detection du cancer
US20080138805A1 (en) * 2004-08-11 2008-06-12 Condeelis John S Isolation, Gene Expression, and Chemotherapeutic Resistance of Motile Cancer Cells
US7507528B2 (en) 2001-09-06 2009-03-24 Adnagen Ag Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells
EP2088204A1 (fr) * 2008-02-05 2009-08-12 Adnagen AG Procédé et kit pour diagnostiquer ou contrôler le traitement du cancer ovarien
WO2011086174A3 (fr) * 2010-01-15 2011-10-06 Diagenic Asa Plateforme d'expression de gènes diagnostiques

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7507528B2 (en) 2001-09-06 2009-03-24 Adnagen Ag Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells
EP1694865A1 (fr) * 2003-12-12 2006-08-30 Bayer Pharmaceuticals Corporation Procede de prediction et de pronostic du cancer, et surveillance de traitement anticancereux
EP1694865A4 (fr) * 2003-12-12 2007-06-06 Bayer Pharmaceuticals Corp Procede de prediction et de pronostic du cancer, et surveillance de traitement anticancereux
US8298756B2 (en) * 2004-08-11 2012-10-30 Albert Einstein College Of Medicine Of Yeshiva University Isolation, gene expression, and chemotherapeutic resistance of motile cancer cells
US20080138805A1 (en) * 2004-08-11 2008-06-12 Condeelis John S Isolation, Gene Expression, and Chemotherapeutic Resistance of Motile Cancer Cells
EP1693467A1 (fr) * 2005-02-17 2006-08-23 Tosoh Corporation Methode d'analyse d' ARNm de la Stanniocalcin (STC1)
WO2007048978A2 (fr) * 2005-10-28 2007-05-03 Biomerieux Sa Procede de detection du cancer
FR2892730A1 (fr) * 2005-10-28 2007-05-04 Biomerieux Sa Methode pour detecter la presence ou le risque de developper un cancer
WO2007048978A3 (fr) * 2005-10-28 2007-09-07 Biomerieux Sa Procede de detection du cancer
FR2899239A1 (fr) * 2006-03-31 2007-10-05 Biomerieux Sa Procede de detection du cancer
WO2009098045A1 (fr) * 2008-02-05 2009-08-13 Adnagen Ag Procédé et coffret pour la détection, le diagnostic ou le contrôle du traitement du cancer de l'ovaire
EP2088204A1 (fr) * 2008-02-05 2009-08-12 Adnagen AG Procédé et kit pour diagnostiquer ou contrôler le traitement du cancer ovarien
WO2011086174A3 (fr) * 2010-01-15 2011-10-06 Diagenic Asa Plateforme d'expression de gènes diagnostiques

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