EP1448792A1 - Kit de diagnostic, puce adn et methodes pour etablir le diagnostic ou effectuer le controle du traitement d'un cancer du testicule - Google Patents

Kit de diagnostic, puce adn et methodes pour etablir le diagnostic ou effectuer le controle du traitement d'un cancer du testicule

Info

Publication number
EP1448792A1
EP1448792A1 EP01274739A EP01274739A EP1448792A1 EP 1448792 A1 EP1448792 A1 EP 1448792A1 EP 01274739 A EP01274739 A EP 01274739A EP 01274739 A EP01274739 A EP 01274739A EP 1448792 A1 EP1448792 A1 EP 1448792A1
Authority
EP
European Patent Office
Prior art keywords
cdna
diagnostic kit
cells
oligonucleotides
kit according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01274739A
Other languages
German (de)
English (en)
Inventor
Cengiz Tamak
Alf-Andreas Krehan
Pia Steffens
Stefanie WASCHÜTZA
Veit Zieglschmid
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alere Diagnostics GmbH
Original Assignee
Adnagen GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Adnagen GmbH filed Critical Adnagen GmbH
Publication of EP1448792A1 publication Critical patent/EP1448792A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells

Definitions

  • the present invention relates to a diagnostic kit, a DNA chip and a method for diagnosis or treatment control for testicular cancer.
  • I - In the context of cancer prevention and follow-up care, it is very important to be able to detect malignant testicular tumors or recurrent malignant testicular tumors early on using the appearance of metastatic tumor lines in the blood.
  • Testicular cancer is responsible for less than 2% of all malignant tumors in men. However, 20-30% of all cancers among men aged 40 are testicular cancer. The number of new cases each year in the Federal Republic of Germany, for example, is approximately 3600, with approximately 240 men dying from testicular cancer. The highest incidence is found between the ages of 25 and 40. By progress in oncology see therapy, over 90% of all those affected can be cured in the long term. The high survival rates are due to the pronounced effectiveness of cis-platinum-based chemotherapy.
  • tumor markers at the protein level are determined quantitatively in the blood or in other body fluids in cancer patients.
  • these detection methods are only of limited suitability for tumor diagnosis or treatment control / aftercare in testicular tumors, since increased tumor marker values in body fluids are also caused by non-tumor diseases, such as inflammation of the gastrointestinal tract, cirrhosis of the liver, viral infections or heavy smoking • can .
  • EP 0 520 794 B1 discloses such a method in which metastases in body tissues or liquids are detected. In doing so, nucleic acids are detected, for example by means of amplification by polymerase chain reaction. The method is now crucially based on the fact that the detected nucleic acid sequence is expressed in cells of the tissue of origin of a tumor, ie in tumor cells and, depending on the markec, also in the healthy cells of the tissue of origin. Another condition is that this sequence is not expressed in those cells whose tissue is being examined.
  • this method is ultimately based on the detection of cells that should not occur in the blood sample of ⁇ healthy individuals.
  • the object of the present invention is therefore to provide a method, a diagnostic kit and a nucleic acid microarray with which tumor cells in testicular tumor diseases in peripheral blood can be detected.
  • This object is achieved by the diagnostic kit according to claim 1, the microarray according to claim 20 and the method according to claim 22 and their use according to claim 45.
  • Advantageous further developments of the diagnostic kit, microarrays, method or uses according to the invention are given in the respective dependent claims.
  • the present invention is essentially based on the fact that testicular tumor markers are not detected in the blood of patients at an immunological or enzymatic level, but that the mRNA (messenger ribonucleic acid) of testicular tumor markers is detected in a blood sample, the tumor markers being associated with a tumor Represent gene expression.
  • the placenta-specific alkaline phosphatase (PLAP) and the germline-specific alkaline phosphatase (GCAP), which are expressed in testicular tumors, the epithelial glycoprotein (GA733__2 or EGP40), the high-mobility group protein isoform IC (HMGI-C) and the gastrin releasing peptide receptor (GRPR) are detected.
  • the detection can be carried out for only one of the markers or for any number of these four testicular tumor markers in combination with one another, but at least the mRNA of the markers GA733.2 and / or GCAP / PLAP is detected.
  • the kit according to the invention can therefore Contain oligonucleotide pairs for only one or any selection or all of the four testicular tumor markers.
  • testicular tumor markers are also expressed in the original tissue and as a protein in the bloodstream. long.
  • only cells are detected that are on the one hand in the blood sample and on the other hand express the respective testicular tumor marker. Consequently, these are tumor cells that originate from their original testicular tumor tissue and have been carried over into the patient's blood. Since the mRNA of the markers examined is not expressed in the blood of a testicular tumor patient, there is a direct correlation between the occurrence of the associated mRNA and metastasis even in the early stage of the metastasis process.
  • testicular tumor marker not only is the mRNA of an individual testicular tumor marker detected, but a combination of markers is examined.
  • This makes it possible, for example, to detect all types of testicular cancer via their cells metastasizing in the blood.
  • seminous and non-seminous testicular cancer forms or mixed tumors with a semino fraction and thus 90-95% of all malignant tumors of the testis, namely all germ cell tumors, are detected.
  • 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.
  • additional markers mostly comes up against limits because mononuclear blood cells express background expression
  • the specificity is a critical point due to the very high amplification rate.
  • the slightest contamination, such as from foreign RNA or atypical transcription, can falsify the result.
  • 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 from the blood sample used. Various methods are available for this:
  • tumor cell detection rate from 1 tumor cell to 10 7 mononuclear blood cells.
  • the tumor cells are separated from mononuclear blood cells by means of specific antibodies or an antibody mixture.
  • the separation can be carried out by means of magnetic particles (Dynal) 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.
  • For ' detection antibodies are used in this cell type-specific pattern.
  • 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.
  • tumor 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 can be differentiated in the blood. To identify tumor cells, antibodies that specifically recognize these special surface proteins are used as tools. The specific antibody binding is used for different analysis and separation methods.
  • Antibodies are coupled with fluorescent dyes for flow cytometric analysis. Scattered cells become constant
  • Laser light source
  • the fluorescent dyes bound to the antibodies are excited and emit light of specific wavelengths.
  • the emitted light is detected and the measured one
  • Digitized signal stored The light signal can be assigned to individual cells.
  • the antibody-labeled cell is thus recognized and can now be separated from other cells. For separation, the cells are made in the smallest
  • Such an enrichment can be done, for example, by FACS flow cytometry.
  • the mononuclear cells from the fraction enriched under b) are incubated with fluorescence-labeled mononuclear antibodies against tumor-specific surface proteins.
  • the labeled cells are washed twice with PBS and then 10 7 cells are resuspended in 1 ml PBS.
  • a FACS Vantage SE flow cytometer (Becton Dickinson) is used to isolate the tumor cells.
  • the CellQuest program is used for data acquisition, instrument control and data evaluation.
  • the sorted cells are transferred to a 1.5 l reaction vessel (filled with 1 ml PBS).
  • the RNA can then be isolated as described later.
  • antibodies are consequently 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 mixed with antibody-coupled magnetic particles; then particles and blood are moved relative to each other, for example by “over-
  • tumor cells are detected in general, but with high specificity. This is due to the selective expression of certain surface proteins that differentiate cancer cells from other cells.
  • 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.
  • a blood sample is taken from a patient.
  • RNS processing from a milliliter of this whole blood is carried out using the QIAampRNA-Blood Mini Kit TM (from Qiagen, Hilden). Contamination with genomic DNA is avoided by an additional DNA digestion with the RNase-Free DNase Set TM (company Qiagen, Hilden) on the column.
  • RNA isolation As an alternative to the RNA isolation described above, it is also possible to take up the isolated fraction of mononuclear blood cells, as described above, in TRIzol reagent from Gibco BRL, NY, USA, to lyse them and to homogenize them using a pipette. The RNA-containing aqueous phase is then precipitated from a chloroform extraction in isopropanol at -80 ° C. After washing twice in 80% ethanol, the pellet is air-dried and then resuspended in RNase-free water.
  • RNA was then denatured in an appropriate volume of water together with oligo (dT) ⁇ 5 primers from Promega, Mannheim for 5 minutes at 65 ° C. and then incubated directly on ice. This was followed by cDNA synthesis at 37 ° C for one hour and a subsequent inactivation of the added reverse transcriptase for 5 minutes at 93 ° C and Cooling down on ice.
  • the entire reverse transcription was carried out using the Sensiscript TM reverse transcriptase kit from Qiagen, Hilden.
  • the PCR synthesis was carried out in a 20 ⁇ l reaction mixture
  • the multiplex PCR was carried out under the conditions given in Table 5 and at the marker-specific annealing temperatures and number of cycles given in Table 6.
  • the annealing temperature is indicated by x, whereby the corresponding annealing temperature from table 6 was used in each case.
  • Lane 1 shows the result of a detection by means of electrophoresis using a DNA 500 chip (Agilent) and an Agilent Bioanalyzer 2100.
  • Lane 1 shows a 100 bp ladder
  • lane 2 shows a cDNA control without RNA in the approach
  • lane 3 one PCR control without cDNA in the approach
  • lane 4 a negative control for GCAP / PLAP with RNA from a healthy control person in the approach
  • lane 5 the sample from a diseased person.
  • control measurement of ⁇ -actin is only positive in the two real blood samples of lanes 4 and 5, while only lane 5 has the expected band for GCAP / PLAP as testicular tumor marker.
  • FIG. 2 Another result is shown in FIG. 2.
  • the detection was also carried out using an Agilent Bioanalyzer 2100 and a DNA 500 assay chip from Agilent.
  • Lanes 1 to 6 show a multiplex PCR of the cDNA from GCAP and GA733.2 and lanes 7 to 13 show a multiplex PCR of the cDNA from GCAP, GA733.2, GRPR and HMGI-C.
  • the designations cDNA- Control, PCR control and negative control refer to samples without RNA, without cDNA or with RNA from a healthy control person. It can be seen in lanes 5, 9 and 13, respectively, that only the testicular tumor samples in the detection of the cDNA and therefore the mRNA are positive for GCAP / PLAP, GA733.2, GRPR and HMGI-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.
  • the documentation can be carried out, for example, using the Inta DUO Store System.
  • a fragment analysis using the ABI Prism 310 Genetic Analyzer can also be used for 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.
  • tumor marker detection can also be carried out using real-time PCR using DANN-intercalating fluorescent dyes (eg LightCycler TM and CybrGreen TM from Hoffmann-Röche). For this purpose, for example, quantification is carried out using fluorescence-based real-time PCR.
  • DANN-intercalating fluorescent dyes eg LightCycler TM and CybrGreen TM from Hoffmann-Röche.
  • quantification is carried out using fluorescence-based real-time PCR.
  • a sequence-specific fluorescence-labeled hybridization sample is added to the PCR approach, by means of which the product development, ie the amplification, can be followed after each cycle of the PCR by means of the fluorescence emitted by it.
  • Special standards can then be used to draw conclusions about the quantities of starting RNA.
  • This also makes it possible to quantify the testicular tumor-associated RNA present in the blood sample and, consequently, to make a direct statement about the response to a selected therapy method.
  • This method can be carried out, for example, with a Light-Cycler TM from Röche, Basel or a Taq-Man TM from PE Applied Bio-Systems, Wieterstadt, as is already well known from the literature.
  • the device according to the invention and the method according to the invention also make it possible to continue using the sorted and separated cells as described above as desired.
  • these can be inserted into a suitable cell culture medium and cultivated in situ.
  • the sorted cells are carrier applied. Additional surface markers can be detected chytochemically or using fluorescence microscopy. Genetic analyzes can also be carried out, such as chromosome analyzes using FISH (fluorescence in situ hybridization) or karyogram generation.
  • FISH fluorescence in situ hybridization

Abstract

L'invention concerne un kit de diagnostic, une puce ADN et des méthodes pour établir le diagnostic ou effectuer le contrôle du traitement d'un cancer du testicule, deux éléments extrêmement importants dans le cadre de la prévention et du suivi post-opératoire dans ce domaine. L'invention est caractérisée en ce que la présence d'ARNm de marqueurs de tumeurs du testicule est mise en évidence dans un échantillon sanguin, les marqueurs tumoraux représentant une expression génétique associée à une tumeur. A cet effet, on utilise en particulier les marqueurs suivants : βbeta-hCG, AFP, PLAP ou GCAP. La mise en évidence de l'ARNm s'effectue par transcription inverse dans l'ADNc, suivie de l'amplification de segments sélectionnés de l'ADNc au moyen d'une PCR.
EP01274739A 2001-11-22 2001-11-22 Kit de diagnostic, puce adn et methodes pour etablir le diagnostic ou effectuer le controle du traitement d'un cancer du testicule Withdrawn EP1448792A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2001/013606 WO2003044224A1 (fr) 2001-11-22 2001-11-22 Kit de diagnostic, puce adn et methodes pour etablir le diagnostic ou effectuer le controle du traitement d'un cancer du testicule

Publications (1)

Publication Number Publication Date
EP1448792A1 true EP1448792A1 (fr) 2004-08-25

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Application Number Title Priority Date Filing Date
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Country Status (4)

Country Link
US (1) US20050118591A1 (fr)
EP (1) EP1448792A1 (fr)
AU (1) AU2002219137A1 (fr)
WO (1) WO2003044224A1 (fr)

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WO2003023057A2 (fr) 2001-09-06 2003-03-20 Adnagen Ag Procede et kit de diagnostic destines a la selection et/ou detection qualitative et/ou quantitative de cellules
EP2107113B1 (fr) * 2002-02-20 2012-04-18 Sysmex Corporation Amorces pour amplification d'acide nucléique pour la détection du gène ARNm domestique et procédé de test utilisant ces amorces
WO2004029221A2 (fr) 2002-09-27 2004-04-08 The General Hospital Corporation Dispositif microfluidique pour la separation de cellules et utilisations de ce dispositif
AU2005218622A1 (en) * 2004-03-03 2005-09-15 Living Microsystems Magnetic device for isolation of cells and biomolecules in a microfluidic environment
JP2008538282A (ja) * 2005-04-05 2008-10-23 セルポイント ダイアグノスティクス， インコーポレイテッド 装置および循環腫瘍細胞および他の粒子の濃縮および変更のための方法
US20070196820A1 (en) 2005-04-05 2007-08-23 Ravi Kapur Devices and methods for enrichment and alteration of cells and other particles
US8921102B2 (en) 2005-07-29 2014-12-30 Gpb Scientific, Llc Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070059716A1 (en) * 2005-09-15 2007-03-15 Ulysses Balis Methods for detecting fetal abnormality
US20080070792A1 (en) 2006-06-14 2008-03-20 Roland Stoughton Use of highly parallel snp genotyping for fetal diagnosis
US20080050739A1 (en) 2006-06-14 2008-02-28 Roland Stoughton Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
US8372584B2 (en) 2006-06-14 2013-02-12 The General Hospital Corporation Rare cell analysis using sample splitting and DNA tags
US8137912B2 (en) 2006-06-14 2012-03-20 The General Hospital Corporation Methods for the diagnosis of fetal abnormalities
LT2334812T (lt) 2008-09-20 2017-04-25 The Board Of Trustees Of The Leland Stanford Junior University Neinvazinis fetalinės aneuploidijos diagnozavimas sekvenavimu

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AU662906B2 (en) * 1991-06-26 1995-09-21 F. Hoffmann-La Roche Ag Methods for detection of carcinoma metastases by nucleic acid amplification
US6165467A (en) * 1991-07-20 2000-12-26 Yoshihide Hagiwara Stabilized human monoclonal antibody preparation
ATE172890T1 (de) * 1995-02-21 1998-11-15 Iqbal W Dr Siddiqi Apparat und verfahren zum mischen und trennen durch verwendung von magnetischen teilchen
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WO2003044224A1 (fr) 2003-05-30
AU2002219137A1 (en) 2003-06-10
US20050118591A1 (en) 2005-06-02

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