EP1497657A2 - Procede de caracterisation de tumeurs primaires - Google Patents

Procede de caracterisation de tumeurs primaires

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Publication number
EP1497657A2
EP1497657A2 EP03725055A EP03725055A EP1497657A2 EP 1497657 A2 EP1497657 A2 EP 1497657A2 EP 03725055 A EP03725055 A EP 03725055A EP 03725055 A EP03725055 A EP 03725055A EP 1497657 A2 EP1497657 A2 EP 1497657A2
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Prior art keywords
tumor
cells
dna
cell clusters
pbs
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German (de)
English (en)
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Burkhard Hermann Brandt
Nicola Tidow
Hartmut Schmidt
Axel Semjonow
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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
    • 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
    • C12Q2600/156—Polymorphic or mutational 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 for characterizing primary tumors or individual areas thereof from peripheral blood. Such methods are required to make an assessment of the degree of malignancy, the invasiveness or the metastasis potential of primary tumors.
  • Such methods are required for all types of tumors, in particular for breast cancers, ovarian cancers, colon cancers, gastric cancers, prostate cancers and bladder cancers.
  • Prostate cancer is one of the most common causes of malignant death in the western world.
  • Three types of prostate carcinoma can be derived according to prognostic criteria: (1) the small painless carcinoma, which does not grow into a clinically conspicuous or metastatic carcinoma during the patient's life span; (2) the slowly growing carcinoma, which is regionally lymphatic at the beginning and only late in the Skeleton metastasized; (3) early metastatic carcinoma, which affects the entire prostate diffusely and metastasizes directly into the skeleton.
  • curative therapy is only possible for early, ie organ-limited tumor stages. Radical prostatectomy or radiation treatment are available for this. However, the optimal type of treatment is still under discussion.
  • prostate carcinomas removed by radical prostatectomy show characteristics similar to those found in asymptomatic autopsy and appear to be relatively benign, ie organ-limited and well differentiated with a small tumor volume. Although the natural course of these cancers is not yet well known, it is believed that these cancers may not require treatment. However, about half of all prostate preparations after radical prostatectomy show a higher proportion of poorly differentiated life-threatening carcinomas than could be predicted from the preoperative biopsies. This illustrates the poor predictability of the degree of malignancy, so that the active treatment of clinically classified as insignificant carcinomas might be the right decision. There are no parameters to distinguish between potentially life-threatening prostate carcinomas and those with a relatively benign or even asymptomatic course before treatment.
  • the clinically established tumor marker PSA has also not proven to be a metastatic marker. (Jhaveri et al. Urology 1999 Nov.; 54 (5): 884-90; Pound et al. JAMA 1999 May 5; 281 (17) .1591-7; Wolff et al. Eur Urol 1998; 33 (4): 376-81). Another serum parameter is now available with the free PSA. However, this did not improve patient staging (Lin et al. Urology 1998 Sep; 52 (3): 366-71). RT-PCR to determine PSA mRNA has become the most widely used method for the detection of circulating prostate cancer cells.
  • PSMA prostate-specific membrane antigen
  • PSM prostate-specific membrane antigen
  • a complementary parameter for the detection of PSA mRNA could be the determination of the mRNA of human glandular kallikrein (hK2).
  • the protein is expressed specifically for the prostate and has a structural homology of 80% with the PSA.
  • a third of the PSA-positive patients were also positive for hK2, while in 50% of the hK2-positive samples the PSA-RT-PCR was negative (Corey et al., Urology 50, 184-188, 1997 ).
  • the present invention has the object to provide a method for the characterization of primary tumors or individual areas of primary tumors, with which a reliable staging and a reliable prognosis of the tumors can be determined.
  • the method according to the invention is advantageously based on the analysis of short, simple, repetitive sequences, the DNA, in particular but not exclusively so-called microsatellite DNA.
  • Tumors can be characterized as to whether they are proliferative, non-proliferative or apoptotic.
  • the degree of malignancy, the invasiveness, for example with regard to organ overshoot, and metastasis can be determined on the basis of the method according to the invention by genotyping cells from cell clusters.
  • tumor cells isolated from blood samples can be assigned to individual areas of a multifocal tumor, i.e. their clonality will be determined. Such a grading enables the prognosis to be assessed and the primary tumors to be finely classified.
  • micro-satellites which is stated in claim 4, has proven to be particularly advantageous are.
  • a multiplex PCR was developed for amplifying the DNA, as specified in claim 6, the selection of the microsatellites and the primers for the multiplex PCR, as specified in claim 8, in particular causing the microsatellites of each multiplex PCR extracts are distributed over as many chromosomes as possible and the amount of amplified fragments differs between the individual microsatellites in such a way that separation, for example by means of subsequent capillary electrophoresis, is possible without problems.
  • the PCR can be separated and evaluated, for example, on an automated system, such as the ABI Prism 310 Genetic Analyzer TM. Reproducible amplification patterns are possible in a concentration range from 100 ng down to 1 ng of used DNA.
  • the investigated genomic alterations of the microsatellite DNA concern the so-called LOH value (loss of heterozygosity) and the RER value (replication error).
  • LOH Score peak area allele 2 tumor x peak area allele 1 normal tissue / peak area allele 1 tumor x peak area allele 2 normal tissue used.
  • the formula is based on test results that were determined using an analog genetic analysis system. The ratio of the peak areas of the alleles in one run is included in the calculation. Table 1 shows, using the example of the marker D13S153, that the quotients of the peak areas with a low coefficient of variation can be determined.
  • the multiplex PCR protocols according to the invention thus allow a reproducible and sensitive determination of an LOH. are.
  • a multiplex PCR was developed for amplifying the DNA, as specified in claim 6, the selection of the microsatellites and the primers for the multiplex PCR, as specified in claim 8, in particular causing the microsatellites of each multiplex PCR extracts are distributed over as many chromosomes as possible and the amount of amplified fragments differs between the individual microsatellites in such a way that separation, for example by means of subsequent capillary electrophoresis, is possible without problems.
  • the PCR can be separated and evaluated, for example, on an automated system, such as the ABI Prism 310 Genetic Analyzer TM. Reproducible amplification patterns are possible in a concentration range from 100 ng down to 1 ng of used DNA.
  • the investigated genomic alterations of the microsatellite DNA concern the so-called LOH value (loss of heterozygosity) and the RER value (replication error).
  • LOH Score peak area allele 2 tumor x peak area allele 1 normal tissue / peak area allele 1 tumor x peak area allele 2 normal tissue used.
  • the formula is based on test results that were determined using an analog genetic analysis system. The ratio of the peak areas of the alleles in one run is included in the calculation.
  • Table 1 shows, using the example of the marker D13S153, that the quotients of the peak areas can be determined with a low coefficient of variation. The multiplex PCR protocols according to the invention thus allow a reproducible and sensitive determination of an LOH.
  • Table 1 Comparison of the quotients for alleles 1 and 2 in MCF-7 cells
  • the length of the fragments is also included in the calculation of a "replication error" (RER) using a factor which represents the center of gravity of the peak distribution.
  • RER replication error
  • the lower detection limit for multiplex PCR with three primer pairs was determined both on DNA from the cell lines SK-BR-3 and LNCaP and on patient DNA (comparison of tumor DNA and leukocyte DNA).
  • a reproducible band pattern was achieved for all polymorphic markers up to a concentration of> 1 ng DNA. This corresponds to a number of approximately 50 cells.
  • the tumor cells to be examined are advantageously isolated or enriched from a sample, for example a blood sample, in which epithelial cells are first enriched by means of density gradient centrifugation and then immunomagnetic isolation or enrichment of cytokeratin-positive and / or PSA- positive cell cluster is carried out. Magnetic beads are used for this purpose, to which corresponding antibodies are bound.
  • the density gradient centrifugation is advantageously carried out as in Brandt and Griwatz, Clin. Chem. 42, No. 11 1996, pp. 1881-1882. This publication is hereby incorporated into the present application with regard to its entire disclosure content.
  • the immunomagnetic cell isolation is advantageously carried out as in Griwatz et al. , J. Immunol. Meth.
  • the following antibodies were also used as primary antibodies: rabbit-mouse anti-PSA, mouse anti-cytokeratin biotinylated, mouse anti-cFas, mouse anti-M30, mouse anti-Mibl and mouse anti-Hl / H3 histone proteins and as Secondary antibodies the following antibodies: anti-rabbit and anti-mouse marked with Alexa-488 and -594 or FITC, Cy5, Cy3, RPE.
  • cells isolated by this method are predominantly cell clusters that are positive for the detection of PSA and cytokeratin. All patients with prostate cancer had such cell clusters, while the control samples for the detection of such cells were negative.
  • the size of the clusters ranges from 2 to 70 cells, with the number 10
  • the heap in 20 ml of peripheral blood was between 1 and 5400. However, about 90% of the patients have more than 100 cells (and the detection limit is therefore exceeded).
  • two classes of cell clusters could be distinguished based on the cell morphology and the nuclear staining. Heaps of dysmorphic cells were found in large numbers. In some cases, small round nucleated cells were included. In addition, 25 of the 74 patients with prostate cancer examined had clusters that consisted only of small, round and nucleated cells with a diameter of approx. 5 to 7 ⁇ m. Most of the patients (approx. 60%) had fewer than 10 such cell clusters in 20 ml of blood. In three cases, however, up to 200 such cell clusters were detectable.
  • Both groups of cell clusters differ in their detection of the apoptosis markers c-Fas and M30, as well as the proliferation markers Mib-1 and H1 / H3.
  • the dysmorphic cell clusters were positive for the cFas and M30 markers, while the group of small, round, nucleated cell clusters was negative.
  • FIG. 1 shows in partial image A the cell diameter before the cells were suspended by suspension in a hyperosmolar buffer for density gradient centrifugation. The cell diameter averages 8.02 ⁇ m.
  • FIG. 2B shows the cell diameter after being taken up in a hyperosmolar buffer such as Nycoprep or Polymorphprep. The average diameter decreased to 4.97 ⁇ m.
  • cell isolation is given as an example for different cell and tumor types.
  • Cell suspensions of the tumor are prepared as described in the procedure in V.
  • a cell suspension isolated from a breast or ovarian carcinoma as described under V. is incubated for 10 min at room temperature (RT) in PABB buffer (saturates binding sites),
  • fractions are centrifuged off and resuspended in 1% PBS / BSA
  • centrifuge epifuge, 10 min, 1500 rpm, discard supernatant, ice compartment (- 20 ° C)
  • immunomagnetic beads e.g. MACS Bead ® anti-mouse AK or, for example, also anti-rabbit, 16
  • PABB 5 ml AB serum 10% (v / v) + 50 ⁇ l BSA-C 0.1% (v / v) + 45 ml PBS
  • the ratio of tumor cells / monocytes in the first positive fraction (+) is significantly shifted, often only single monocytes are found and thus a high degree of purity of the tumor cells.
  • PBS / BSA 1 g BSA (Bovine Albumine) / 100 ml PBS
  • Antibody mouse anti-cytokeratin antibody working concentration 1:50 (in PABB) (C7, C20 or PAN) secondary antibody: anti-mouse Alexa 594 antibody working concentration 1: 100 (in PABB) isotype control in 10% AB- serum
  • Antibody Anti-PSA antibody working concentration 1:50 (in PABB) secondary antibody: Anti-Rabbit Alexa 488 antibody working concentration 1: 100 (in PABB)
  • Invasion medium (Dulbecco's Modified Eagle Medium) 1% (v / v) 2 mM L-glutamine
  • Tissue pieces from freshly operated breast carcinomas, benign breast diseases or prostate carcinomas are placed in sterile tubes with standard medium by the surgeon and placed on ice until disaggregation (at the latest 4 hours after removal). Approximately half of each piece of tissue is removed for later expression analysis and preserved in liquid nitrogen. The other half is disaggregated using a mechanical method. A medimachine (company Dako, Hamburg) is used for this. For disaggregation, the breast tissue is cut into 3 to 10 mm 2 pieces with a scalpel and placed in a Medicon together with 1.5 ml of invasive medium. The tissue is then aggregated within 2 to 3 minutes within the medimachine to form a cell suspension which contains single cells and cell aggregates (cell clusters) of up to approx. 30 cells.
  • the cells are counted microscopically in a Neubauer counting chamber and the number of living cells is determined using the trypan blue exclusion test, which is based on the fact that certain dyes (e.g. trypan blue) cannot enter the cell interior of living cells, while dead cells deal with the relevant dye stain (Kaltenbach et al., 1958; Lindl and Bauer, 1994).
  • the isolated cell clusters are genotyped for the purpose of their assignment to areas in the primary tumor by means of PCR.
  • the PSA and cytokeratin positive tumor cells and tumor cell clusters and non-positive monocytes as a negative control (or reference for the LOH calculation) are microdissected with a sterile fine needle and placed in 1.5 ml sterile reaction vessels (Eppendorf Biopure) transferred.
  • Eppendorf Biopure 1.5 ml sterile reaction vessels
  • the cells are taken up in 10-200 ⁇ l LTE buffer (10 mM Tris / HCl, 1 mM EDTA, pH 7.5) and with 1-20 ⁇ l proteinase K (> 600mAU / ml ) incubated in a thermoblock or water bath at 50-56 ° C for 1-10 h and then placed on ice for 5 min. The samples are then centrifuged for 1 min at 10,000 rpm. The samples are then adjusted to a 70% solution with 99.8% ethanol pa (Roth). After briefly vortexing, the samples are centrifuged off at 15,000 rpm for 20 min, the supernatant is discarded and the DNA pellet is dried at room temperature. The DNA is then resuspended in 10-200 ⁇ l LTE buffer or double distilled water, incubated at room temperature for 1 h (rehydration) and frozen at -20 ° C. until the PCR is carried out. 28
  • DNA isolation from fresh and formalin-fixed or paraffin-embedded tissue from the primary tumors of one or more foci is carried out according to the protocols of the commercially available QIAmp DNA Mini Kit (from Qiagen, Hilden) or a comparable system from other manufacturers.
  • This kit contains QlAamp DNA Mini Spin Columns (columns), Proteinase K for the proteolytic digestion of the tissue, the lysis buffers AL and ATL, the ethanol-containing washing buffers AW1 and AW2 and the elution buffer AE.
  • Fresh tissue from a primary tumor is mechanically disrupted with a scalpel or tissue shredding machines (e.g. medomachine, DACO) provided for this purpose before tissue lysis.
  • a scalpel or tissue shredding machines e.g. medomachine, DACO
  • tissue sections embedded in paraffin dewaxing with 100% xylene is carried out before the actual DNA isolation.
  • the samples are first incubated in 1 ml xylene in 1.5 ml Eppendorf reaction vessels in a commercially available thermoblock at 70 ° C for 1 h. The supernatant was then centrifuged for 3 minutes and the process repeated twice. The tissue is then washed three times with 99.8% ethanol (Roth), then dried and transferred to the lysis buffer. The isolation is then carried out according to the manufacturer's protocols.
  • DNA isolation from EDTA-anticoagulated whole blood is carried out with the QIAmp DNA Blood Mini Kit (Qiagen, Hilden) according to the known protocols or comparable methods from other manufacturers.
  • the whole blood is incubated for 10 min with buffer AL and proteinase K to lyse the cells at 54 ° C in a thermoblock, then mixed with ethanol and the mixture is added to the column (QIAmp DNA Mini Spin Columns). The samples are washed with AW1 and then with AW2 and eluted with the elution buffer AE.
  • Solutions are measured photometrically at 260, 280 and 320 nm, adjusted to 10 ng / ⁇ l and frozen at -20 ° C.
  • the PSA- and cytokeratin-positive tumor cells and tumor cell clusters are microdissected with a sterile fine needle on an inverse light microscope (Leitz Diavert) and transferred to a 1.5 ml sterile reaction vessel (Eppendorf Biopure).
  • the RNA isolation is carried out strictly according to the protocols of the RNeasy Purification Kit for total RNA minipreparation (Qiagen, Hilden), consisting of: RNeasy Mini Spin Colums, Collection tubes 1.5 and 2 ml, buffer RTL, buffer RWI, buffer RPE and RNase-free water).
  • carcinoma-specific genetic changes and mRNA expressions by means of microsatellite PCR, multiplex microsatellite PCR and TaqMan TM RT-PCR is now described below by way of example.
  • a total of three multiplex PCRs are used, which consist of microsatellite combinations No. 1: D7S522 D8S258, D16S400, No. 2: NEFL, D13S153, D17S855 and No. 3 D10S541, D16S402, D16S422.
  • the principle of these PCRs is based on the co-amplification of different DNA sections in one reaction vessel.
  • the primer sequences were set in such a way that there was no overlap in the lengths of the amplification products in the capillary electrophoretic separation. All primers are labeled with fluorescent dyes that are excited at 488 nm (see Table 3). All other commercially available fluorescent labels can also be used. Furthermore 30
  • All PCRs can be in commercially available 0.2 ml or 0.5 ml reaction tubes or in 96-well plates from different manufacturers (e.g. Eppendorf, Hamburg) on an Eppendorf Mastercycler, Eppendorf Mastercycler Gradient (Eppendorf, Hamburg), a Gene AmpR PCR System 9700 (PE Applied Biosystems, Rothstadt) or a commercially available, comparable thermal cycler from other manufacturers.
  • the reaction volume can be 12 ⁇ l to 100 ⁇ l.
  • the PCR reaction mixture consists of 5U / 100 ⁇ l AmpliTaq Gold TM or a qualitatively comparable polymerase and (hot start polymerases have proven successful) 1 x GeneAmp R dNTP, 2mM MgCl 2 , 30pm from each primer, 200 ⁇ M GeneAmp R buffer (all reagents from PE Applied Biosystems, Rothstadt) and 500 pg to 200 ng genomic DNA. The following temperature gradients are run for the PCR.
  • microsatellite analysis is carried out on the capillary electrophoresis devices (four color laser-induced fluorescence capillary electrophoresis system) ABI Prism 310 gene 31
  • the polymers POP4, POP5 and POP6 are used as separation medium and are suitable for the corresponding devices.
  • the length standard used is Genescan-500TM TAMRA 500 or a comparable length standard that is suitable for the capillary electrophoresis devices mentioned. The analysis and evaluation was carried out with the Genescan software.
  • Vials 0.2 ml vials from any manufacturer, suitable for PCR machines 32
  • the PSA- and cytokeratin-positive tumor cells and tumor cell clusters are microdissected with a sterile fine needle on an inverted light microscope (Leitz Diavert) and transferred to a 1.5 ml sterile reaction vessel (Eppendorf Biopure).
  • the RNA isolation is carried out strictly according to the protocols of the RNeasy purification kit for total RNA minipreparation (from Qiagen, Hilden), consisting of: RNeasy Mini Spin Colums, Collection tubes 1.5 and 2 ml, buffer RTL, buffer RW1, buffer RPE and RNase-free water).
  • the RNA is transcribed into cDNA in a two-tube reaction.
  • the reaction volume is 20 ⁇ l.
  • the reaction mixture consists of RT 1 x buffer, dNTPs (0.5 mM each) 1 ⁇ M oligo-dT primer, 10 units RNase inhibitor, 4 units Omniscript Reverse Transcriptase and RNase-free water.
  • dNTPs 0.5 mM each
  • RNase inhibitor 10 units
  • Omniscript Reverse Transcriptase 4 units
  • RNase-free water for the RT-PCR, the RNA samples are first denatured at 65 ° C for 5 min and then placed on ice.
  • RT-PCR can be performed on an Eppendorf Mastercycler, Eppendorf Mastercycler Gradient (Eppendorf, Hamburg), a Gene Amp R PCR System 9700 (PE Applied Biosystems) or a commercially available, comparable thermal cycler from other manufacturers.
  • the PCR consists of a 37 ° C incubation step for 60 min followed by a 93 ° C denaturation step.
  • the RNA isolations and the RT-PCR can furthermore be carried out using all commercially available methods which are suitable for small amounts of tissue, e.g. the ExpressDirect TM Kit For mRNA Capture And RT System For RT-PCR (Pierce Rockford).
  • Real-time PCR can be performed on an ABI Prism 9700 HT Sequence Detection System (PE Applied Biosystems, Rothstadt) in 96 or 384 well plates, sealed with an optically transparent film (ABI PRISM TM optical adhesive 36
  • the reactions are usually carried out in double or triple determinations according to the regulations for TaqMan PCR according to PE Applied Biosystems (Weiterstadt).
  • the reaction mixture consists of a TaqMan R Universal PCR Master Mix, plus each
  • the temperature gradient consists of an initial 50 ° C incubation step for 2 min followed by a 95 ° C denaturation step for 10 min; 40-60 cycles are then run, which consist of a 95 ° C denaturation step for 15 seconds and a 60 ° C amplification step for 1 minute.
  • the run and the evaluation took place with the SDS software (PE Applied Biosystems, Rothstadt). Primer and TaqMan probe as well as the TaqMan mastermix can be used by different providers.
  • RNA was isolated from cells of an LNCaP cell culture that is known to express PSA. Lymphocytes from women serve as a negative control.
  • the microscopic marker in the DNA of PSA-positive epithelial cells isolated from peripheral blood and from individual foci of the primary tumor was determined using the described method.
  • the microsatellite markers were determined according to the protocol as described under 4. from DNA, which was obtained according to protocols 1 and 2.
  • the prostate preparations were isolated before DNA isolation according to the Schmid et al. (Schmid HP et 37
  • the method described in large area sections at a distance of 3 to 4 mm was systematically processed and a detailed cartographic recording of the carcinoma extension was created.
  • the tumor size, the position of the tumor in relation to the pseudocapsule of the prostate (infiltration or penetration of the capsule) and the reaching or exceeding of the surgical margins in the carcinoma were documented. Histologically confirmed tumor tissue was then obtained from the paraffin-embedded material of the primary tumor. 7 shows a so-called tumor map in which the removal sites are marked.
  • Table 5 Comparison of the genetic alterations between different foci of a primary tumor and the circulating tumor cell clusters isolated from blood
  • FIG. 5 The cluster with the marker D10S541, which is associated with early metastasis, is also preferably found in the cell clusters of the blood. In contrast, changes in the marker D8S258 prevent the cells from being sown into the peripheral blood (FIG. 6). The evaluation of the disease-free interval shows that a change in this polymorphic DNA marker is associated with a good prognosis (FIG. 6).
  • PCa prostate cancer
  • BPH benign prostate tissue

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Abstract

L'invention concerne un procédé permettant de déceler et de caractériser des tumeurs primaires ou des zones isolées de tumeurs primaires. Ce procédé consiste à isoler ou enrichir les amas cellulaires de cellules tumorales contenus dans une matière échantillon puis à analyser les modifications génétiques des amas cellulaires.
EP03725055A 2002-04-17 2003-04-17 Procede de caracterisation de tumeurs primaires Withdrawn EP1497657A2 (fr)

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DE10217102 2002-04-17
DE10217102A DE10217102B4 (de) 2002-04-17 2002-04-17 Verfahren zur Charakterisierung von Primärtumoren
PCT/EP2003/004037 WO2003087405A2 (fr) 2002-04-17 2003-04-17 Procede de caracterisation de tumeurs primaires

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DE102010060964A1 (de) 2010-12-02 2012-06-06 Universitätsklinikum Hamburg-Eppendorf Verfahren zur Prädikation der therapeutischen Wirksamkeit von EGFR-Inhibitoren
CN112921097A (zh) * 2021-04-20 2021-06-08 四川大学华西医院 用于纤维/肌纤维母细胞肿瘤的基因检测试剂盒及应用
JP7677690B2 (ja) * 2022-03-03 2025-05-15 ソウル ナショナル ユニヴァーシティ アール アンド ディービー ファウンデーション Ｆａｍ１６７ａを含むｂｃｒ－ａｂｌ非依存性チロシンキナーゼ阻害剤耐性を診断するためのバイオマーカーおよびｆａｍ１６７ａをターゲットとする慢性骨髄性白血病の予防または治療用組成物
KR102769507B1 (ko) * 2024-07-16 2025-02-18 서울대학교산학협력단 마우스 fam167a 에피토프 및 이에 특이적으로 결합하는 단일클론항체 또는 이의 항원 결합 단편

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000047998A1 (fr) * 1999-02-10 2000-08-17 Cell Works Inc. Caracterisation de classes de cellules cancereuses circulantes isolees a partir de fluides corporels et methodes d'utilisation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA00009654A (es) * 1994-08-31 2005-09-08 Univ Johns Hopkins Med Deteccion de secuencias de acidos nucleicos hipermutables en tejidos.
US5925519A (en) * 1996-06-03 1999-07-20 The Regents Of The University Of California Genetic alterations associated with prostate cancer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000047998A1 (fr) * 1999-02-10 2000-08-17 Cell Works Inc. Caracterisation de classes de cellules cancereuses circulantes isolees a partir de fluides corporels et methodes d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WEISS LEONARD: "Heterogeneity of cancer cell populations and metastasis", CANCER AND METASTASIS REVIEWS, vol. 19, no. 3-4, 2000, pages 351 - 379, ISSN: 0167-7659 *

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DE10217102A1 (de) 2003-11-13
AU2003227645A8 (en) 2003-10-27
AU2003227645A1 (en) 2003-10-27
WO2003087405A3 (fr) 2004-06-17
WO2003087405A2 (fr) 2003-10-23
US20090035774A1 (en) 2009-02-05
DE10217102B4 (de) 2005-04-14

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