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EP0458831A1 - Test de malignite (test de depistage du cancer) par la reaction en chaine de la polymerase - Google Patents

Test de malignite (test de depistage du cancer) par la reaction en chaine de la polymerase

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Publication number
EP0458831A1
EP0458831A1 EP90903145A EP90903145A EP0458831A1 EP 0458831 A1 EP0458831 A1 EP 0458831A1 EP 90903145 A EP90903145 A EP 90903145A EP 90903145 A EP90903145 A EP 90903145A EP 0458831 A1 EP0458831 A1 EP 0458831A1
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EP
European Patent Office
Prior art keywords
vitro
product
sequence
pcr
dna
Prior art date
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EP90903145A
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German (de)
English (en)
Inventor
Viktor Dr.Dr.Med. Balazs
Margit Dr.rer.nat. BALAZS-FRÖHLICH
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BALAZS, VIKTOR, DR. DR. MED.
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Individual
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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
    • C12Q2600/158—Expression markers

Definitions

  • Malignancy test (cancer test) using the polymerase ette ⁇ reaction.
  • the invention relates to a method for the detection of the specific mRNA sequence (target sequence) of substances of cancer cell origin from an acellular biological fluid according to the preamble of claim 1.
  • This phenomenon is actually the functional mimicry at the molecular biological and cellular level of the main features of the malicious process itself: promotion of one's own existence at the expense of the host.
  • a large spectrum of normal and malignant cells is able to take up mRNA from its environment if it is presented protected by lipid, lipoprotein or other substances.
  • the mRNA then exerts its messenger activity in the recipient cells.
  • the translation product of the mRNA taken up is produced by these cells until the replenishment from the environment reaches from the circulation.
  • the recipient cell has acquired a new phenotypic quality, a new ability to produce, which it either does not have or cannot express genetically.
  • Phenotype lending is one of the most important mechanisms by which the malignant cells achieve their main goals:
  • the influencing messages essentially go in two directions:
  • the enzyme profile of the otherwise normal liver of malignant carriers is more similar to the malignant cells than in normal humans.
  • transcripts or their fragments of cellular oncogenes which are activated by over-transcription in malignant diseases, from the blood plasma of malignancy carriers by i vitro hybridization can be detected.
  • Phenotype borrowing from these transcripts can cause phenomena similar to AI. and Bl. cause in malignant diseases, or transfer production capabilities of growth factors, enzymes or other substances in host cells which are removed from the tumor. The environment of these host cells could become attractive for the incoming metastatic cancer cells and then feed them with growth factors and other substances.
  • the inhibition of phenotypes by mRNA of malignant origin simultaneously prevents its own functions in the recipient cells of the host, which can lead to a reduction in the local and systematic defense mechanisms of the host.
  • Small fragments of transcripts from cancer cell origin also have inhibitory activity.
  • RNA transfer process is a very complicated, arduous method and is not sensitive enough to give positive results even in the early stages of malignancy.
  • the detection of the mRNA or its specific fragments of the substances of cancer cell origin in the bloodstream of malignancy carriers in accordance with the method of the invention can serve as a sensitive and well practicable test method which already provides important information about the malignant diseases in the early stages fundamental fundamental molecular-genetic changes in the malignant cells, their activities, the possible interaction can deliver between tumor and host by mRNA at a time when the cancer cell mass cannot be visualized with the usual method and cannot be reached for a histological examination.
  • the circulating mRNA transcripts or their specific fragments can be detected using very sensitive and specific methods.
  • One such technical possibility is in vitro hybridization and the other is the in vitro enzymatic multiplication of the specific target sequence of the respective mRNA by polymerase chain reaction, PCR (polymerase chain reaction).
  • the polymerase chain reaction which has been known since 1985 (Science 230: 1350-1356), is a reliable, relatively simple method for obtaining the desired sequence of low-concentration DNA from tissue, cells and even from a single hair in vitro synthesize and amplify.
  • the amplified product is then easily identifiable by a spectrum of different methods including in vitro hybridization with corresponding, labeled DNA or oligonucleotide samples.
  • PCR has been used to spots or traces of the cellular body fluids (blood, semen) or Kör ⁇ perzellen detect and identify.
  • the mRNA sequences can also be amplified by PCR in vitro if the corresponding complementary DNA (cDNA) is first synthesized by reverse transcription and this is amplified by PCR.
  • cDNA complementary DNA
  • This RNA amplification has so far only been used for cells and cellular body fluids such as blood, especially to identify RNA viruses in the blood cells.
  • two oligonucleotide primers are used which are complementary to the sequences which flank the desired specific sequence, a primer for the 3'-strand, a primer for the 5'-strand, they "designate" the sequence that synthesize and amplify the DNA polymerase enzyme.
  • the target-specific sequence can be amplified by PCR to such an extent that the direct detection of the product is possible.
  • the PCR products can be visualized by their labeling and do not have to be detected by in vitro hybridization.
  • the PCR has so many plans Share, such as high specificity, very high sensitivity, which is in the same or an order of magnitude higher than in vitro hybridization and it is easier, faster and automatable. Therefore PCR is particularly suitable for the detection of the mRNA sequence of the substances of cancer cell origin from an acellular biological fluid such as blood plasma and can serve as a sensitive malignancy test for diagnosis, early detection and monitoring of malignant diseases.
  • the sensitivity of the mRNA detection method according to the invention is significantly greater (lies in the region of 10 " 18 g) than the detection methods of the translation products, ie the substances themselves (lies in the region of pg).
  • Antibodies to the translation product either produced by the host or introduced (submitted) for therapy purposes, do not affect the test results in the mRNa transcript detection.
  • RNA transcripts or their specific fragments are omitted or excreted in detectable amounts and in a form protected from degrading enzymes only by malignant cells. That is why their detection (in the bloodstream) from the blood plasma is diagnostic, on the contrary with their translation product, which can also be produced by normal cells (such as: oncogene products; some tumor markers).
  • the half-life of the mRNA transcripts is considerably shorter (60 min for onc-fos, onc-myc; 7 hours for CEA) than their translation products (several days for oncogene products, more than one week for CEA).
  • the tumor cells present in the bloodstream cannot leave the circulation and thus cannot metastatize. This is particularly important during and after surgery as a large number of cancer cells can enter the bloodstream.
  • the lower (20 - 25%) success rate of the various forms of immunotherapy can be explained, at least in part, by the phenotype loan, because the host cells cannot participate sufficiently because of this, or the cells which have been proliferated and activated in vitro shortly after their Administration in the malignant carrier host inhibited in its function, ineffective can be.
  • the sensitivity of the malignancy test is increased by at least 4 to 5 orders of magnitude in comparison to the hybridization alone or PCR alone.
  • the activation of two or more cellular oncogenes by increased transcription or by mutation is generally considered to be a very important step in the emergence, maintenance and progression of the malignant transformation. This increase in transcription can be multiple or even 100 times that. reach normal values. Because of the peculiarities of malignant cells and the malignant tumors, the activated oncogene transcripts or their fragments can enter the bloodstream and be protected or partially protected there without further significant degradation, and they can pass through from the blood plasma of patients with an active malignant process a sensitive method such as in vitro hybridization can be detected. This method can be used as a malignancy test are and is suitable as a screening test or progress control test (monitoring) in order to detect relatively small amounts of malignant cell mass which cannot be recognized with the usual diagnostic methods in clinical practice.
  • the oncogene-specific mRNA sequences also detect such oncogenes in the blood plasma which are present there in an even lower concentration than others, such as, for example, the transcripts or transcript fragments of the oncogenes which are activated by point mutation and not by increased transcription and therefore their concentration is lower in the malignant cells and subsequently also in the blood plasma compared to oncogenes which have been activated by increased transcription and even in vivo amplification.
  • the plasma-oncogene profile oncogenes whose mRNA sequence is present in the blood plasma
  • can better reflect the cancer cell-oncogene profile oncogenes that are activated in the malignant cells).
  • the goal of cancer research and clinical oncology is the molecular kular-genetically causal, rational, meaningful therapy.
  • knowledge and especially early knowledge of which oncogenes are actually activated in the malignant cells are very important.
  • These oncogenes can serve as a good target of a molecular-genetically meaningful therapy, and can be attacked at several levels, namely (a) at the gene level: by preventing transcription; (b) at the transcript level: by preventing translation of the activated oncogenes; (c) or at the oncogene product level: by preventing the function of the OKP.
  • very stimulating results with cancer cell cultures are known in vitro.
  • An oligonucleotide with a specific sequence for the initiation of protein synthesis is preferably taken up by the cancer cells in larger amounts than by normal cells and inhibits the translation of the targeted oncogene RNA transcripts, with the result that the cancer cells lose their properties and are no longer malignant multiples ⁇ adorn. This possibility is a good candidate to develop a causal therapy in practice.
  • the anti-cancer cell antibodies are bound with chemotherapeutic, toxic or radioactive substances, therapeutic effects can be achieved in animals and patients, but the success rate is generally not very high.
  • chemotherapeutic, toxic or radioactive substances therapeutic effects can be achieved in animals and patients, but the success rate is generally not very high.
  • Several factors are responsible for this, one of which is the difference in the expression of the targeted antigen on the different cancer cells of the same tumors because of their diversification.
  • Better results can be achieved by targeting several antigens on the cell surface at the same time immunologically attacks.
  • the oncogene products of the activated oncogenes would be particularly suitable, in particular those which have their oncogene product in the cell membrane and those which do not occur in normal cells at all, such as the oncogene products altered by point mutation or some tumor markers.
  • Another advantage is that the oncogene transcripts or their sequences can be detected as early as is not possible with the most sensitive immune method.
  • the immunological detection of products of the oncogenes as a malignancy test is not suitable because the normal and the pathological values (in malignancies) overlap, and some abnormal but non-aligning states produce higher values than the malignancies, apart from that much lower sensitivity of the immunological methods compared to the present invention.
  • Another particular advantage is that changes in the activation of the oncogenes during the follow-up can be more reliably and earlier reflected by the plasma oncogene profile than in previous methods.
  • oncogene changes associated with a deterioration in the prognosis due to diversification of the cancer cells, or with an amplification of one of the activated oncogenes can be detected at an early stage and the appropriate therapy can be initiated in good time.
  • the disappearance of an existing oncogene transcript or the appearance of a new oncogene transcript not yet present in the blood plasma or its sequence is also a sign of the deterioration in progressiveness.
  • the possibility of therapy with mRNA requires careful monitoring of the mRNA used or its specific sequence in the bloodstream.
  • the method of the invention is very suitable for these purposes because of its very high sensitivity and reliable specificity.
  • the invention has for its object to detect specific mRNA sequences of the substances of cancer cell origin from an acellular biological fluid, even if they or their fragments are present in a very low concentration and degraded to a certain extent, but have the specific sequence and for in vitro Amplifizie ⁇ tion are suitable, and this process so as Malignticianstest, be ⁇ to use Sonder as a very sensitive screening test.
  • RNA or its fragments are prevented by using an effective and reliable RNase inhibitor, which does not cause RNA to emerge from the cells, even when the cellular biological fluid is being sampled. Since the chosen RNase inhibitor does not cause RNA to escape from the cells before and during their removal, it is important because this sensitive method can detect even small contamination.
  • the PCR products can be radioactive or non-radioactive already during the PCR by incorporating marked components or primers be marked and visualized in this way.
  • the unlabelled PCR products can be identified by direct detection after ethidium bromide staining, or by their size, or according to their sequence either by sequence analysis or especially with in vitro hybridization.
  • the method is suitable as a malignancy test, especially for early detection.
  • the cellular biological fluid (such as blood, exudates, etc.) is mixed with a reliable RNase inhibitor which does not cause RNA cell leakage when the sample is taken and the cells are removed.
  • the total RNA is mixed with an aqueous medium under the constant action of the RNase inhibitor and in the presence of a detergent and a proteolytic enzyme such as Proteinase K and at 37 degrees C for 5 to 8 h in one Incubated buffer solution.
  • yeast tRNA is added as a carrier (50 ⁇ g / ml) and extracted with phenol and chloroform at 60 ° C and precipitated with alcohol at -20 ° C.
  • the extract can be further purified quickly using ion exchange column chromatography.
  • the blood plasma RNA is mixed with a buffer solution, which is composed according to the optimal mode of action of the selected reverse transcriptase (Tris.Cl. ph 8.3, KC1, MgCl 2 ). It also contains RNasin, reverse transcriptase enzyme, each of the four deoxyribonucleoside triphosphates, the 3'primer all in sufficient concentration. The total reaction mixture is then incubated for a total of 20 microliters at 37 degrees C for 30 minutes.
  • a buffer solution which is composed according to the optimal mode of action of the selected reverse transcriptase (Tris.Cl. ph 8.3, KC1, MgCl 2 ). It also contains RNasin, reverse transcriptase enzyme, each of the four deoxyribonucleoside triphosphates, the 3'primer all in sufficient concentration.
  • the total reaction mixture is then incubated for a total of 20 microliters at 37 degrees C for 30 minutes.
  • the reaction mixture is then mixed with 80 microliters of PCR buffer and 100 micrograms of bovine serum albumin, with the 3 'primer and the 5' primer and the thermostable Taq DNA poly erase enzyme, each in a suitable concentration.
  • the reaction mixture is then covered with mineral oil to prevent evaporation.
  • the primers are extended at 72 degrees C for 1 min. The cycle is started again by heating and repeated 40 to 50 times.
  • reaction mixture is left at 72 degrees C for 10 minutes and then cooled in ice.
  • the multiplication during PCR is detected by ethidium bromide staining in comparison to the negative control.
  • the size of the product or products can be identified by electrophoresis in gel or by chromatography or according to their sequence.
  • the products can be labeled with radioisotope, biotin, enzymes, fluorescent dyes or other substances during the amplification by labeled components or primers and then recognized according to the labeling.
  • each reaction mixture is applied to nylon or nitrocellulose filters and immobilized by baking at 80 degrees C for 60 ml, prehybridized at 60 degrees C in a suitable buffer and then in the presence of 2 x 10 6 cqm of end-labeled oncogene specific oligonucleotide samples per ml hybridized for 5 to 7 h at 60 degrees C. Washing is carried out at 60 degrees C in 0.75 M NaCl / 0.075 Na citrate, 0.% SDS. The result of the hybridization is visualized by autoradiography.
  • the products of the simultaneous pooled PCR can be identified at the same time.
  • the unlabeled corresponding samples are immobilized on a solid support, such as a nylon filter, and brought into contact with the (bio tin) labeled PCR products in order to hybridize with one another.
  • the results are visualized by a color reaction due to enzyme affinity.
  • a series of purified molecular-cloned DNA containing the sequence as the code of the oncogenes is labeled either radioisotopically or non-radioisotopically (J. Mol Biol. 113: 237-251 1977).
  • the non-radioisotopic labeling can be done with biotin.
  • end labeling is used to achieve high specific activity.
  • DNA samples and oncogene-specific oligonucleotide samples labeled or unlabeled are commercially available (ONCOR, Inc. Gaithersburgh, Ma. 20877, USA; ONCOGENE SCIENCE Inc. Mineola, NY 11501, USA; Amersham, Little Chalfont, England).
  • a biotin labeling and visualization kit is commercially available (Amersham, Little Chalfont, England ONCOR, Gaithersburgh, USA). Oligonucleotides with the desired sequences can be ordered and obtained from the Custom Service in Europe and the USA.
  • stages a to c The risk of admixing the ubiquitous, highly resistant RNase enzyme from exogenous sources is particularly evident in all stages of the RNA phase of the process (stages a to c) through the use of baked glassware, autoclaved solutions, baked spatulas, tools, dry chemical preparations, distilled from glass, reduced with autoclave-sterilized water, and wearing gloves.
  • yeast tRNA 50 ⁇ g / ml
  • an equal volume of phenol and chloroform-isoamyl alcohol 24: 1
  • the interphase is additionally extracted once as above and the combined aqueous phases are treated once with chloroform-isoamyl alcohol as above but at room temperature.
  • the nucleic acid is precipitated by alcohol in the presence of Na acetate (0.3 M, pH 5.2) at -20 degrees C and centrifuged. The sediment is dissolved and subjected to RNase-free DNase enzyme treatment.
  • RNase-free DNase enzyme and MgCl 2 are mixed to the plasma RNA solution (50 mM Tris.Cl, pH 7.5 in 1 mM EDTA) and in the presence of RNase inhibitors at 37 degrees C. incubated for 30 min, then phenol-chloroform is extracted and in the presence of Na acetate (pH 5.2, 0.3 M) the RNA is precipitated with 70% ethanol at -20 ° C.
  • RNA fraction which preferably has poly (A) + RNA.
  • the RNA-liproprotein fraction can be separated from the serum of malignancy carriers by flotation in a KBr density gradient (discontinuous: 1,006-1,221 g / ml) after 16 hours of flotation in an ultracentrifuge at 105,000 xg; it is separated as an opal band visible between the HDL and LDL fractions.
  • the eluate can be used directly for in vitro hygridization or for PCR amplification, or after alcohol precipitation and DNase treatment.
  • the sediment is then in a buffer solution which corresponds to the optimal effectiveness of the reverse transcriptase enzyme used (and the regulations of the respective manufacturer) (Bethesda Research Laboratories; promega Biotec, USA) (50 mM Tris.Cl, pH 8.3, 6mM MgCl 2 , 40 mM KC1, 1 nM dithiorheitol, 1 U RNasin).
  • the solution is supplemented with 100 mM of each deoxyribonucleoside triphosphate and 1000 U reverse transcriptase, 10 pM of the respective 3'PCR primer.
  • PCR buffer 50 mM Tris.Cl, 50 mM KCl, 2.5 mM MgCl 2 , 100 micrograms bovine serum albumin / ml, pH 8,440 pM 3'primer, 50 pM 5'primer and 1 U thermostabi
  • PCR buffer 50 mM Tris.Cl, 50 mM KCl, 2.5 mM MgCl 2 , 100 micrograms bovine serum albumin / ml, pH 8,440 pM 3'primer, 50 pM 5'primer and 1 U thermostabi
  • ⁇ Tag DNA polymerase (Perkin-Elmer-Cetus, USA; New England Biolab, (USA)) is mixed in.
  • Mineral oil is layered over the reaction mixture to prevent evaporation.
  • the RNa-cDNA complex is dissociated by heating to 95 degrees C for 20 seconds and the primers are anealed at 55 degrees C for 15 seconds, the extension of the primers takes place at 72 degrees C for 1 min. The cycle is started again by heating and repeated 40 to 50 times.
  • Thermal Cycler instrument Perkin-Elmer-Cetus, USA significantly facilitates the reliable and fast execution of the PCR.
  • Primer pairs to synthesize PCR products of different sizes An example:
  • Labeling of the PCR product is carried out during the amplification by incorporating either labeled deoxyribonucleoside triphosphate or labeled primers.
  • the label can be radioactive or non-radioactive such as: biotin, enzymes, dyes and other substances. Fluorescent dyes have found an advantageous use as a label for primers: these dyes, a different color for each amplifying sequence, who conjugate to the oligonucleotide primers (Applied Biosystem, Foster City, Ca, USA).
  • Labeled PCR products are identified by their labeling. In this case, the unincorporated marked elements must be removed (as above).
  • Radioactive-labeled PCR products are determined by measuring the radioactivity compared to the negative control, or visualized by agarose gel electrophoresis without prior treatment by autoradiography and even identified by the size of the product.
  • Biotin-labeled PCR products are recognized after the elimination of the unincorporated, labeled elements due to a color reaction due to enzyme affinity. Visualization kits are commercially available (ONCOR, Gaithersburgh, MD 20884 U.S.A.; Amersham, Little Chalfont, England).
  • PCR products labeled with fluorescent dyes are identified by their color using a fluorometer (Perkin-Elmer LS-5).
  • Electrophoresis in agarose or acrylic amide gel according to the size of the products. Visualization either by EB fluorescence or according to the marking. The size of the products is determined by comparison with markers of a known size running in parallel.
  • Ion exchange chromatography High resolution separation: The reaction mixture after amplification is placed on a Mono Q HR 5/5 column (Parmacia Uppsala, Sweden) and passed through the specified gradient of buffer solutions A and B at a flow rate of 0. 15 ml / min eluted. Buffer solution A: 20 mM Tris.Cl. pH 8.3, 0.4% M NaCl; Buffer solution B: A + 1.0 M NaCl. Gradient: 40 to 60% B in 2 h and 60 to 80% B in 6 h. Simultaneously multi-target sequence PCR (pooled PCR).
  • EB fluorescence is used to determine whether amplification has taken place or not, but which sequence (s) has been amplified must be determined with special additional measures.
  • identification is achieved by the product size.
  • the size of the different PCR products is programmed beforehand by selecting and inserting the corresponding primer pairs so that the product of each amplifying target sequence is synthesized and amplified in an individual, characteristic, different size during the PR (see example) . It is thus achieved that the PCR products are identified by their size with gel electrophoresis or by ion exchange chromatography
  • the third possibility for the differentiation of simultaneously amplified products is the identification by their sequence.
  • Sequence analysis is particularly suitable for checking the authenticity of enzyme activity.
  • scanning-tuning microscopy can be used to identify the PCR products. It is currently possible to differentiate only the purine and pyrimidine bases from each other with this microscopy. By improving the resolution capability of the microscope, it will be possible in the near future to identify the different bases, even from a concentrated RNA or DNA extract.
  • In vitro hybridization is the most practiced method for identifying nucleic acids according to their sequence, in their conventional form such as dot, slot or Southern blot.
  • the PCR product is immobilized on a solid substrate and is brought into contact with labeled specific oligonucleotide or DNa samples which are freely available in solution in order to hybridize with one another if there is a complementary sequence. The non-specifically bound samples are removed by washing.
  • the buffer in which the prehybridization took place is removed and with the buffer of the same composition, which also contains 2 ⁇ 10 6 cpm / ml of 5'-end-labeled, denatured oligonucleotides with an oncogene-specific sequence, or with added to the sequence containing the mutant codon of an oncogene (such as ras oncogenes) and incubated at 60 degrees C for 6 to 8 h to take place the hybridization.
  • the filters are then washed in a solution of 0.75 M NaCl, 0.075 M Na citrate, 0.1% SDS at 60 degrees C.
  • the visualization of the occurrence of the hybridization is achieved by autoradiography with the aid of an intensifying plate.
  • the buffer solution for prehybridization, hybrid and washing still contains tetramenthylammonium chloride (3 M tetramethyl ammonium chloride, 50 M Tris.Cl, pH 7.5, 2 mM EDTA, 0.3% SDS, 100 micrograms / ml sonicated Sa__mon sperm DNA, 5 x Denhardt's solution ( 1 x Denhardt's solution: Ficoll, polyvinylpirrolidone, bovine serum-alb ⁇ _min each 0.02%), in which case the filters are then washed twice in 2 x SSPE (1 x SSPE: 10 M Na-phosphate, p 7.2, 0.18 M NaCl, 1 mM EDTA, 0.1% SDS
  • the following known, 18 molecularly cloned human oncogene or its oncogene-specific sequences can be used as samples.
  • the oncogenes are grouped below according to their frequency of increased activity in human malignancies:
  • Group A fos, myc, Ha-ras, Ki-ras
  • Group B fes, yb, fms, N-ras, src, abl, raf, Ertf, N-mye
  • Group C Erb ⁇ , mos, sis, alu, bcr.
  • the oncogene samples are usually radioisotopically with P 32 (Spezifi ⁇ specific activity: 10 8-10 9 cpm / microgram) or labeled with biotin.
  • Okogen oligonucleotide samples including those which have a point mutation-modified sequence of oncogenes activated by point mutation, are radioisotopically labeled by end labeling in order to achieve high specific activity. If new oncogenes were discovered, additional oncogene oligonucleotide samples might be necessary, which can be used according to the diagnostic goal.
  • nylon filter membrane
  • UV light or by baking then wash with 200 ml of 5 x SSPE and 0.5% for 30 min at 55 degrees C to remove the unbound oligonucleotide.
  • a filter has several different oligonucleotide samples.
  • Each filter with the various specific oligonucleotide samples immobilized on it is composed in 4 ml of hybridization solution of 20 ul of the amplified, labeled (biotin) DNA and 5 x SSPE, 0.5% SDS.
  • the amplified DNA was mixed by mixing 400 iriM NaOH, 10 mM EDTA and was added quickly to the hybridization solution. Then incubate at 55 degrees C for 4 to 6 h. They are quickly with 2 x SSPE, 0.1% SDS at room temperature, once with the same solution at 55 degrees C for 10 min.
  • Oligonucleotides coupled to a poly-dT tail (several hundred BP long): This tail is then used to immobilize the samples on a piece of nylon filter by UV light so that the specific sequence or sequences of the fragment are free in Is a solution and so the hybridization takes place quickly.
  • the point mutation possibilities of some oncogenes are numerous and require quick examination procedures.
  • One possibility is the simultaneous, pooled sequence PCR and the differentiation of the different products by different fluorescent dye labeling, by using labeled primer pairs, a primer of which is labeled with a different dye and complementary to the mutated sequence.
  • Each 5 to 6 mutated sequence is drawn with its own dyes, its own color. The amplification of these mutated sequences can be identified quickly and easily in a pooled PCR system.
  • PCR can be used for substances whose nucleotide sequence is not is known not to be carried out. In these cases, in vitro hybridization with the corresponding DNA samples is the method of choice.
  • the plasma RNA concentrate is denatured by incubation at 65 degrees C for 15 min and then by rapid cooling. Then the RNA is on a solid support such as nitrocellulose paper that was previously with 20 x NaCl / Cit. was equilibrated and then dried by a dotting apparatus (Schleicher & Schuell, Keene, New Hampshire USA). The nitrocellulose paper treated in this way is then baked in a vacuum oven at 80 ° C.
  • Prehybridization buffer Formamide (50% (Vol / V ⁇ l) 5 x NaCl / Cit; mM sodium phosphate, pH 6.5; sonicated denatured Salmon Sperma DN (250 micrograms / ml) and 0.2% per Bovine Serum Albumin (BSA); Ficoll and Polyinylpyrrolidon incubated for 6-8 hr at 55 degrees C.
  • the biotin-labeled oncogene and other samples are first denatured and become the prehybridization solution which contains the nitrocellulose paper with the plasma RNA immobilized thereon mixes.
  • the concentration of the sample or samples (pooled samples) is high: 600 ng / ml. In the case of pooled samples of 5 components, each 120 ng / ml. Hybridization at 55 degrees C for 7 to 10 h.
  • the hybridization is detected with the visualization kit according to the manufacturer's instructions (ONCOR, Gaithersburgh USA) after the prescribed washes.
  • biotin-labeled samples and visualization kits from ONCO enables rehybridization after removal of the hybridized samples.
  • the pooled samples (each 5) the plasma RNA can be tested relatively quickly and get a negative or a positive result with the Tester ⁇ ih * menschli chen malignancies frequently activated oncogenes.
  • the positive pool is then further tested with its components (the individual samples) by hybridization and / or by re-hybridization in order to determine the plasma oncogene profile. men.
  • the sensitivity of this method is in the range of 0.1 pg. Numerous other modifications are possible.
  • the intensity of the black spot after autoradiography due to the intensity of the color can be measured quantitatively and quantitative comparisons can thus be made.
  • a serial dilution is made from the plasma RNa with 10 microgram / ml yeast tRNa as diluent carrier RNA, then with each dilution first the reverse transcription, then the PCR, and the product by in vitro hybridization with corresponding specific oligonucleotide samples identified by other methods.
  • the highest dilution from which there is still a positive signal is de titer.
  • quantitative monitoring can be carried out during the monitoring of a patient during the observation period. In this way, one can observe, for example, amplification of one of the activated oncogenes. If the titer of one oncogene is increased disproportionately compared to the other oncogenes detectable in the plasma during the follow-up, this can increase the in vivo amplification of this oncogene, among other things. mean.
  • This method is particularly suitable for screening people who are particularly at risk of malignancy, such as people who are exposed to X-rays or radioactive radiation, workers in chemical or asbestos plants, patients with impaired immune systems (after chemotherapy) or members of families with genetic -conditional tendency for various malignant diseases.
  • Each mutated sequence in the pool is drawn with its own color by means of fluorescent dye-labeled primers, which is complementary to the mutated codon region.
  • the most sensitive test method is when the PCR products are detected by in vitro hybridization.
  • the reaction mixture is hybridized with the appropriately labeled oligonucleotide or pooled DNA samples after amplification.
  • the positive pooled PCR mixture is then further hybridized with labeled individual oligonucleotide or DNA samples in order to know which sequence was amplified in the pool.
  • a sensitivity can be achieved which is in the range of ag (10 -18 g). Theoretically, this means the detectability of some target sequences (3-5 target sequence pieces) in the plasma pattern.
  • This test is able to reflect not only the fundamental molecular-genetic changes, mainly responsible for the malignant transformation, its maintenance and progression, but also one of the most important ways of influencing the host organism by the malignant cancer cell population.
  • the activated oncogenes drive up the "motor” of the malignant cells, which have to be “switched back” in order for the host to be able to save the patient.
  • this method of the invention creates the most important prerequisites: the early detection of these events in each patient individually.

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Abstract

Dans un test de malignité (test de dépistage du cancer), on concentre l'ARN sous l'effet constant d'un inhibiteur fiable de la RNase par multiplication enzymatique in vitro de la séquence spécifique d'ARNm des substances originaires de cellules cancéreuses contenues dans un liquide biologique acellulaire. On soumet le produit ainsi obtenu à une transcription inverse in vitro, afin de synthétiser de l'ADN complémentaire (ADNc), puis on utilise le produit ainsi obtenu afin de synthétiser enzymatiquement de façon répétée in vitro la séquence spécifique cible de la substance voulue originaire de cellules cancéreuses, la multipliant ainsi. Le produit ainsi obtenu est détecté par détermination de la multiplication des acides nucléiques.
EP90903145A 1989-02-16 1990-02-16 Test de malignite (test de depistage du cancer) par la reaction en chaine de la polymerase Withdrawn EP0458831A1 (fr)

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US5618670A (en) * 1988-08-26 1997-04-08 The United States Of America As Represented By The Department Of Health & Human Services Detection method for c-raf-1 genes
AU630001B2 (en) * 1989-10-02 1992-10-15 United States of America, as represented by the Secretary, U.S. Department of Commerce, The A sensitive method for measurement of chimeric transcripts of dna containing translocations
EP0612353B1 (fr) * 1991-09-16 1999-03-31 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Procede de detection de genes c-raf-1
EP0534640B1 (fr) * 1991-09-23 1996-10-02 Pfizer Inc. Procédé pour détecter ARNm et ADN spécifiques dans les cellules
GB2260811B (en) * 1991-10-23 1995-07-05 Yorkshire Cancer Research Camp Detection of malignant tumours
ATE397097T1 (de) * 1995-03-17 2008-06-15 Wayne John Cancer Inst Nachweis von brustmetastasen unter verwendung eines mehrfachmarker-tests
US7785842B2 (en) 1996-03-26 2010-08-31 Oncomedx, Inc. Comparative analysis of extracellular RNA species
US6607898B1 (en) 1996-03-26 2003-08-19 Oncomedx, Inc. Method for detection of hTR and hTERT telomerase-associated RNA in plasma or serum
ES2347854T5 (es) 1996-03-26 2014-07-24 Michael S. Kopreski Método que permite el uso de RNA extracelular extraído de plasma o suero para detectar, monitorizar o evaluar un cáncer
US6759217B2 (en) 1996-03-26 2004-07-06 Oncomedx, Inc. Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer
US8043835B1 (en) 1996-03-26 2011-10-25 Oncomedx, Inc. Methods for detecting and monitoring cancer using extracellular RNA
US8440396B2 (en) 1997-03-14 2013-05-14 Oncomedx, Inc. Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer
US8163524B2 (en) 1998-09-22 2012-04-24 Oncomedx, Inc. Comparative analysis of extracellular RNA species
US7767422B2 (en) 1998-09-22 2010-08-03 Oncomedx, Inc. Detection of 5T4 RNA in plasma and serum
EP1158055A1 (fr) 2000-05-26 2001-11-28 Xu Qi University of Teaxs Laboratoire de Leucémie Chen Méthode pour le diagnostic de cancers
CA2467629A1 (fr) 2001-11-20 2003-05-30 Oncomedx, Inc. Procedes pour evaluer une expression genique de resistance aux medicaments chez un patient atteint d'un cancer

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EP0272098A3 (fr) * 1986-12-15 1990-06-06 City Of Hope National Medical Center Procédé d'amplification et de détection de séquences ARN
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