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WO2002088388A1 - Procede et dispositif de quantification de charges de mutation - Google Patents

Procede et dispositif de quantification de charges de mutation Download PDF

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WO2002088388A1
WO2002088388A1 PCT/IB2001/000696 IB0100696W WO02088388A1 WO 2002088388 A1 WO2002088388 A1 WO 2002088388A1 IB 0100696 W IB0100696 W IB 0100696W WO 02088388 A1 WO02088388 A1 WO 02088388A1
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cells
analysis
dna
cell
rna
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Albert RÜBBEN
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of molecular diagnostics.
  • the invention relates to the quantitative analysis of mutations in cells and tissues, to the identification of the predominant form of genetic instability, and to the screening for genetic susceptibility to ma* lignant tumors.
  • the invention relates to a method, a device and a kit for the quantification of mutation loads in cells and tissues by determining mutant frequencies and genetic heterogeneity on the single-cell or oligo-cell level. Its main application is the detection and quantification of mutations in preneoplastic and neoplastic conditions in order to identify patients at risk for cancer development, to clarify diagnosis of malignant disease, to specify the mechanisms of genetic instability, to quantify the degree of genetic heterogeneity in malignant tumors as well as to screen for the presence of mutations conferring resistance to cancer treatment.
  • Tumorigenesis and tumor progression can be considered an evolutionary process in which mutant and more tumorigenic sub- populations are sequentially selected and derived from less tumorigenic or benign progenitor cells. This view of cancer first proposed by P.C.
  • Novell in 1976 is supported by genetic data demonstrating the monoclonal origin of malignant tumors and the multistep nature of tumorigenesis (Fearon & Vogel- stein, 1990, Loeb & Christians, 1996, Nowell, 1976, Whitte- more & Keller, 1978).
  • the two main driving forces of tumori- genesis are on one hand a positive growth selection which may be mediated by external influences as well as by mutations in genes responsible for cell growth and apoptosis regulation and on the other hand an enhanced mutation rate leading to genetic instability.
  • Several human diseases are associated with an enhanced mutation rate and a predisposition to cancer.
  • the genetic defects mostly reside in systems responsible for detecting or correcting genetic damage from external or internal sources. Many different genes are involved in genetic instability.
  • Hereditary conditions like ataxia teleangiectasia (ATM) , xeroderma pigmentosum (RPA, ERCC1, ERCC3, ERCC5, ERCC6, XPC) , Li- Fraumeni syndrome (P53), familial breast cancers (BRCA1, BRCA2), hereditary nonpolyposis colorectal cancer (MLH1, MSH2, MSH3, MSH6, PMSl, PMS2).
  • microsatellite instability MSI
  • the exact knowledge of the mutation rate may be replaced by the determination of the frequency of mutant cells in a specific tissue (Green et al., 1995).
  • An enhanced mutant frequency will reflect both, an enhanced mutation rate as well as the positive selection of mutant cells and will, therefore, provide a risk estimate for the development of malignant tumors. It has been demonstrated that mu- tant frequencies rise with age and show significant difference between individuals (Grist et al., 1992).
  • a second indicator of an enhanced mutation rate is genetic heterogeneity. It is an indirect evidence of the mutational pressures during tumorigenesis and tumor progression and it reflects the multistep nature of the neoplastic transformation as genetic heterogeneity may represent different steps of the early tumorigenic process. Analysis of genetic heterogeneity can, therefore, be used to estimate mutation rates in malignant and premalignant tissues. Genetic heterogeneity can be observed in many human malignancies (Boni et al. 1998, Fu- jii H et al. 1996, Mirchandani et al., 1995).
  • microsatellite DNA of small microdissected cell-clusters of malignant tissues enables the identification of the two main mechanisms of genetic instability (Boni et al. 1998, R ⁇ bben et al., 2000).
  • analysis of microdissected tissue samples of malignant melanomas showed the concurrent presence of cells with losses of either the maternal or the paternal microsatellite allele (Morita et al., 1998, R ⁇ bben et al., 2000) . Analysis of pooled tissue from these samples would have made the demonstration of genetic instability as well as its quantification impossible.
  • Multilineage progression may also obscure the detection of microsatellite instability, as microsatellite sequences may elongate or contract in tumor cells with defects of DNA mismatch repair genes. Thus, a continuous spectrum of elongated or contracted microsatellite sequences may not be detected in pooled tumor cells.
  • a second problem associated with the quantification of mutant frequencies and genetic heterogeneity results from the fact that tumor cells are under a strong negative selective pres- sure under in-vivo conditions:
  • the action of the immune system and of cancer treatments as well as the occurrence of mutations in essential genes lead to a constant reduction of genetic heterogeneity within the tumor cell population.
  • Evidence of negative selective pressure during tumor progression could be demonstrated in malignant melanoma (R ⁇ bben et al., 2000) .
  • Positive selection of tumor cells through a strong growth advantage basically has the same effect and will reduce overall genetic heterogeneity within the tumor cell population.
  • the biasing effect of selective pressure during tumor progression is stronger when pooled tissue is analysed, as the predominant tumor cell clone will obscure the presence of additional mutated tumor cells.
  • Multilineage progression in malignant and premalignant tissues is the reason why mutation detection using microsatel- lite analysis is greatly impaired when tissue specimens are analysed as a whole. Furthermore, as mutations represent rare events, a large number of cells or cell-clusters have to be screened in order to yield statistically significant results. The currently available techniques are too labour-intensive to routinely estimate mutant frequencies and genetic heterogeneity in clinical specimens through the analysis of micro- satellite DNA.
  • the present invention provides both a rapid and easy method and device to perform analysis of mutant frequencies and genetic heterogeneity in multiple single-cells or small cell- clusters .
  • mutant cells as well as the degree of genetic heterogeneity in premalignant and malignant cells or tissues constitute the mutation load.
  • the mutation load is dependent on the generation number of the clonal expansion as well as on the degree of the underlying genetic instability.
  • a high mutation load is a risk factor for neoplastic transformation (DePinho, 2000) , it may define the time point of neoplastic transformation, it is an indicator of tumor progression and it is a negative prognostic factor of tumor therapy.
  • the present invention has resulted from the finding that analysis of mutant frequencies and genetic heterogeneity within premalignant and malignant tissues and cells requires the separate analysis of a large number of cells on the single-cell or oligo-cells level.
  • Multilineage progression is a common feature of malignant melanoma (R ⁇ bben et al., 2000). Multilineage progression as well as strong selection for gross chromosomal rearrangements targeting the same chromosomal region but resulting in loss of one chromosomal region in some cells, and loss of the homologue chromosomal region on the other chromosome in other cells, greatly limit the application of microsatellite analysis for the quantification of mutant frequencies and genetic heterogeneity in premalignant and malignant cells or tissues. Multilineage progression may also obscure the detection of microsatellite instability, as microsatellite sequences may elongate or contract in tumor cells with defects of DNA mismatch repair genes. Thus, a continuous spectrum of elongated or contracted microsatellite sequences may not be detected in pooled tumor cells.
  • Quantification of mutant frequencies and genetic heterogeneity may also be biased by selective pressures within the tumor cell microenvironment .
  • a strong negative selective pres- sure which reduces mutant frequencies and genetic heterogeneity may result under in-vi vo conditions through the action of the immune system and by cancer treatment.
  • the occurrence of mutations in essential genes may also lead to a constant reduction of genetic heterogeneity within the tumor cell population.
  • Positive selection of tumor cells through a strong growth advantage has the same effect on overall genetic heterogeneity within the tumor cell population but will result in an overestimation of mutant frequencies.
  • the main object of the present invention is a method providing an independent mutation analysis of multiple single-cells or small cell-clusters.
  • Microsatellite DNA is a prime target for mutation analysis as both forms of genetic instability, i.e. gross chromosomal rearrangements and microsatellite instability may be detected by PCR-amplification and analysis of microsatellite DNA se- quences.
  • the principle of the invention may also be applied to other molecular techniques detecting loss of heterozygosity or mutations in mismatch repair genes such as analysis of single nucleotide polymorphisms (SNPs) or screening for mutations at repetitive DNA sequences within genomic DNA other than microsatellite DNA.
  • SNPs single nucleotide polymorphisms
  • Disease specific mutations such as codon 12 K-ras-mutations in colorectal cancer, may also represent targets for mutation analysis.
  • Independent molecular analysis of single-cells or small cell- clusters may, in its most simple form, be performed through limiting dilution of the solution containing purified single- cells. A dilution of approximately 1-2 intact cells per sample is suitable for this quantitative approach.
  • CMC cell-microarray-chips
  • Molecular analysis can be performed as described above and may be done directly on the carrier or after transfer of material onto a specific analysis device.
  • Figure 1 Analysis of genetic instability at microsatellite marker D18S65 in three microdissected regions of a malignant melanoma (electropherogram analysis, ABI
  • PRISM 373 molecular size marker: GENESCAN 400HD ROX, PE Applied Biosystems
  • Area 1 shows loss of heterozygosity (LOH) by the absence of allele A.
  • Area 2 demonstrates microsatellite instability (MSI) as allele A is shifted to the right indicating an expansion of DNA repeat sequences.
  • Area 3 does not demonstrate LOH or MSI.
  • Figure 2 Analysis of genetic instability at microsatellite marker D9S162 in eight microdissected regions of a dysplastic melanocytic nevus (electropherogram analysis, ABI PRISM 373, molecular size marker: GENESCAN 400HD ROX) . Areas 1, 2 and 7 show LOH by the absence of allele B.
  • Area 4 demonstrates partial LOH by the absence of allele A as well as MSI as allele B is shifted to the left indicating a contraction of DNA repeat sequences. Areas 3, 5, 6 and 8 also show MSI. N represents the normal tissue of the patient without LOH or MSI.
  • Figure 3 Graphic display of LOH-mutation index found in 5 normal and 5 dysplastic melanocytic nevi.
  • Figure 4 Analysis of genetic instability at marker D18S65 in tumor cell line MEL-IM (electropherogram analysis, ABI PRISM 373, molecular size marker: GENESCAN 400HD ROX) . Data were generated by performing 100 PCR reactions containing 1-2 tumor cells per reaction.
  • Figures 5-7 Schematic drawing of method for mutation detection and quantification of large number of sin- gle-cells or small cell-clusters using cell- microarray-chips .
  • Figure 8 Schematic drawing of a cell-microarray-chip for risk-assessment of melanocytic nevi .
  • mutant cells as well the degree of genetic heterogeneity in premalignant and malignant cells or tissues reflect the number of mutations which arise during the clonal expansion of a cell lineage. This accumulation of mutations as well as the accumulation of mutant cells constitute the mutation load which is dependent on the generation number of the clonal expansion as well as on the degree of the underlying genetic instability. It has been dem- onstrated that a high mutation load is a risk factor for neoplastic transformation (DePinho, 2000) .
  • the mutation load may define both, the time point of neoplastic transformation as well as the stage of tumor progression.
  • a high mutation load may increase the possibility that resistant cells are already present before a specific anti-tumor therapy is started. Therefore, the mutation load may be regarded as a general negative prognostic factor of any anti-tumor therapy.
  • the present invention is a result from the recent finding that quantification of mutant frequencies and genetic heterogeneity within premalignant and malignant tissues and cells does require the analysis of a large number of cells on the single-cell or oligo-cells level: Firstly, it has been confirmed that multilineage progression is a feature of malignant melanoma by quantitative analysis of mutations affecting microsatellite DNA (LOH and MSI) at different progression stages of a malignant melanoma (R ⁇ bben et al., 2000). Multilineage progression had already been shown in other human cancers (Tsao et al .
  • the inventor has then analysed mutations of microsatellite DNA in multiple microdissected areas of primary malignant melanomas and has been able to demonstrate a very high degree of genetic heterogeneity with different tumor cell clones displaying loss of heterozygosity (LOH) of either alleles. Mutation loads in these tumors may not be correctly quantified if pooled tumor cells are taken for analysis. It also has been demonstrated that a primary malignant melanoma may harbour both classes of genetic instability in different tu- mor areas, i.e. gross chromosomal rearrangements detected by LOH at microsatellite loci as well as microsatellite instability (R ⁇ bben et al., 2000). Figure 1 shows an example.
  • microsatellite sequences may elongate or contract in tumor cells with defects of DNA mismatch repair genes, a continuous spectrum of elongated or contracted microsatellite sequences may also not be detected in pooled tumor cells. Therefore, multilineage progression may obscure the detection of microsatellite instability as well.
  • mutant frequencies and genetic heterogeneity may also be biased by selective pressures within the tumor cell microenvironment .
  • a strong negative selective pressure which reduces mutant frequencies and genetic heterogeneity may be the consequence of the action of the immune system and or of a specific cancer treatment.
  • the occurrence of mutations in essential genes may also lead to a constant reduction of genetic heterogeneity within the tumor cell population.
  • Positive selection of tumor cells through a strong growth advantage has the same sup- pressing effect on overall genetic heterogeneity within the tumor cell population, but will result in an increase of mutant frequencies.
  • Evidenvce of negative as well as positive selective pressures during tumor progression in malignant melanoma R ⁇ bben et al., 2000
  • strong selective pressure is present in most human cancers.
  • the biasing effect of selective pressure during tumor progression is stronger when pooled tissue is analysed as the predominant tumor cell clone will obscure the presence of additional mutated tumor cells.
  • the preferred embodiment of the invention consists of a separate mutation analysis of multiple single-cells or small cell-clusters.
  • the method of diagnosing comprises the steps of:
  • RT-PCR reverse transcription PCR
  • DNA-DNA/RNA-DNA/RNA-RNA-hybridisation performed in small scale volumes on all extracted DNA or RNA samples in order to detect microsatellite instability, gross chromosomal rearrangements or disease specific mutations and changes in gene expression;
  • Solid tissues are analysed after enzymatic or mechanical dissociation of the tissue into single-cells or small cell- clusters .
  • Microdissection of formalin-fixed tissue sections of routine histology slides at multiple regions may be used to analyse archival formalin-fixed tissue specimen that may not be transformed into a single-cell solution due to crosslinking of cells.
  • the number of analysed cells per reaction has to be sufficiently large in order to avoid false positive results through unbalanced degradation of the analysed alleles.
  • melanocytic nevi molecular nevi
  • early melanomas is performed by analysis of microsatellite markers on the short arm of chromosome 9 and the long arm of chromosome 10 and 14 such as D9S259, D9S171, D9S942, D9S1748, D9S162, D10S215, D10S209, D14S267 and D14S53.
  • Markers on the short arm of chromosome 18 such as D18S65 are suitable for analysis of mutation loads in advanced malignant melanomas.
  • microsatellite markers D6S260, D6S261, D9S162 and D18S65 are particularly suitable for the analysis of cu- taneous T-cell lymphomas.
  • Markers on the short arm of chromosome 3 such as D3S1300, D3S4103 and D3S1283 are used for the analysis of cervical scrapes, cervical cancer as well as epithelial head and neck cancers.
  • Analysis of sputum for lung cancer screening can be performed with markers on 9p and 9q as well as with microsatellites on the short arm of chromosome 8 such as D8S133 and D8S137.
  • An alternative system for demonstration of gross chromosomal rearrangements would be the detection of loss of heterozygos- ity by analysis of single nucleotide polymorphisms (SNPs) .
  • SNPs single nucleotide polymorphisms
  • Allele specific PCR amplification or hybridisation with sequence specific DNA or RNA probes labelled with enzymes or fluorescent dyes can be used to discriminate between alleles.
  • Measurement of melting curves of DNA-DNA or RNA-RNA or DNA- RNA-complexes by laser scanning represents an alternative method to distinguish between alleles.
  • PCR may also be used to detect gross chromosomal rearrangements leading to homozygous loss of tumor suppressor genes such as pl6 ⁇ nk4 . Loss of chromosomal material can also be demonstrated by hybridization with specific DNA or RNA probes.
  • Analysis for mutations at repetitive DNA sequences within genomic DNA other than microsatellite DNA may also be performed in order to detect defects of DNA mismatch repair.
  • Disease specific mutations such as codon 12 K-ras-mutations in colorectal cancer, may also represent molecular targets for determination of mutation loads. They may be demonstrated by sequence-specific polymerase chain reaction (SSP-PCR) or by hybridisation with sequence-specific DNA probes labelled with enzymes or fluorescent dyes.
  • SSP-PCR sequence-specific polymerase chain reaction
  • RNA transcription due to specific mutations may be detected after reverse transcription and PCR (RT-PCR) with specific primers.
  • RT-PCR reverse transcription and PCR
  • mutation loads are analysed by determining mutant frequencies and the frequency of individual cell clones (genetic heterogeneity) separately.
  • mutation loads are determined by a mutation index integrating mutant frequencies (m) and the frequency of individual cell clones (n) by the formula: Vmxn.
  • the identification of individual cell clones is enhanced by analysing a set of multiple microsatellite markers on each single-cell or on a small cell-cluster. This procedure enhances the probability of detecting different cell clones by the pattern of chromosomal rearrangement or by the presence of mutated alleles.
  • Primary fields of application of the present invention are the detection and quantification of mutations: in preneoplastic conditions in order to identify patients at risk for cancer development; - in order to clarify diagnosis and progression state of malignant disease; in order to specify the mechanisms of genetic instability and the degree of genetic heterogeneity in malignant tumors; and - in order to screen for the presence of mutations conferring resistance to cancer treatment.
  • CMC cell-microarray-chips
  • CMC CMC for the analysis of mutant frequencies and genetic heterogeneity
  • a large number of cells are immobilised in a specified geomet- ric order, primarily as a grid array, onto a miniaturised carrier which then enables the independent analysis of single-cells or small cell-clusters in separate reaction and analysis spaces (Fig. 5) .
  • Said approach provides information on the number of modified cells as well as on the nature of the molecular alteration.
  • solid tissues are analysed after enzymatic or mechanical dissociation of tissue into a single-cell suspension while blood cells, cells of effusions or smears may be analysed directly as single-cell suspensions.
  • the single-cell suspension then interacts with a carrier that bears multiple binding areas for target cells present within the cell suspension (Fig. 6) .
  • the binding areas on the carrier are organised in a defined geometric order that may form a two dimensional grid array, a more complex two dimensional array or even a three dimensional organisation depending on the requirements of the subsequent analysis procedure.
  • the binding areas define separate reaction and analysis spaces.
  • the number of individual analysis spaces depends on the scope of the diagnostic procedure.
  • the analysis spaces are densely packed.
  • the carrier bearing the binding areas forms a miniaturised device in order to enable simultaneous analysis of multiple single-cells or multiple small cell- clusters.
  • the preferred number of analysis spaces lies in the range of 50 to 20000.
  • selective analysis of specific cells is ensured by interaction of the binding area on the carrier with target molecules on the surface of these cells.
  • antibodies, integrins, lectins, recep- tor-ligand complexes, bioengineered molecules or related mechanisms enable binding (Fig. 6).
  • the number of cells bound to each analysis area is determined by the surface area of the molecules mediating specific binding on the carrier (Fig. 6) . This approach enables to modify the number of cells that are analysed together in one analysis space according to requirements of the inventive method.
  • Procedures related to the analysis of the bound cells are performed di- rectly on the carrier.
  • Molecular detection procedures are performed after providing separate reaction volumes filled with reaction and detection reagents.
  • separate small scale reaction volumes are provided by a cover yielding concave depressions.
  • the carrier may bear concave depressions as well (Fig. 7).
  • Molecular analysis may be performed using antibody-antigen binding, DNA or RNA hybridisation, polymerase chain reaction (PCR) or computer-assisted image processing of specific mor- phological changes. Analysis may be performed directly on the carrier or after transfer of material onto a specific analysis device.
  • PCR polymerase chain reaction
  • fluorescent probes are used that can be detected by laser scanning through a transparent cover (Fig. 8) .
  • Simultaneous analysis of melting or dissociation curves may provide information on base composition and the presence of mutation within the ana- lysed DNA or RNA molecules.
  • material from the separate reaction and analysis spaces can be transferred to an external analysis device after piercing of a thin membrane on the outer surface of the cover (Fig. 8).
  • a transverse 0.2 cm thick tissue strip is obtained from the freshly excised nevus and cut into 0.1 x 0.1 cm cubes and placed in vortex at 37 °C for 60 min in 1 ml of trypsin/EDTA-solution (Sigma) containing 10 mg/ml dispase (Sigma) .
  • the cells are then pelleted through centrifugation at 500 rpm for 3 min at RT, washed with phosphate-buffered saline (PBS) and then resuspended in PBS.
  • PBS phosphate-buffered saline
  • concentration of cells is first determined by cell count and the solution is diluted with PBS to 1-2 cells per ⁇ l .
  • Markers D9S259, D9S171 and D9S162 labelled 5'prime with the dyes 6FAM, HEX and NED (Microsynth) are suitable for analysis of mutation load of melanocytic nevi.
  • PCR is done in a total volume of 20 ⁇ l with 0.5 U Taq- polymerase (AmpliTaq Gold, PE Applied Biosystems, Foster City) . 1 ⁇ l of the diluted cell solution is used for every PCR amplification.
  • Final primer concentration is 1.5 ⁇ M and Mg 2+ concentration is 2 mM.
  • 384 samples are prepared on a 384-well microtiter plate with each primer for every analysed nevus.
  • PCR is done on a DNA Thermal Cycler Dual 384-Well GeneAmp® PCR System 9700 (PE Ap- plied Biosystems) using the following temperature profiles: 8 min at 95°C; 2 cycles of 1 min at 58°C, 15 sec at 72 °C, 30 sec at 94.5 °C; 5 cycles of 1 min at 55°C, 15 sec at 72 °C, 30 sec at 94 °C, 22 cycles of 1 min at 55°C, 20 sec at 72 °C, 20 sec at 92 °C; a final step of 25 sec at 55°C and 5 min at 72 °C.
  • c) Analysis of amplified microsatellite DNA is done on a DNA Thermal Cycler Dual 384-Well GeneAmp® PCR System 9700 (PE Ap- plied Biosystems) using the following temperature profiles: 8 min at 95°C; 2 cycles of 1 min at 58°C, 15 sec at 72 °C, 30 sec at 94.5 °C; 5 cycles of 1 min at 55°C, 15 sec
  • the carrier consists of a FastTM slide (Schleicher & Schuell) .
  • the array contains 50 to 20*000, preferably 1000 analysis spaces.
  • Each binding area contains a spot of 50 ⁇ m diameter that contains 10 nl of biotinylated mouse IgG antibodies (50 ⁇ g/1) against the S100 protein expressed predominantly on melanocytic cells.
  • One cell can be bound in each analysis space.
  • the analysis and reaction volume is approximately 0.01 ⁇ l .
  • When analysing small cell-clusters 2 to 1000 cells may be immobilized in each space.
  • the reaction volume will increase ac- cordingly.
  • a silicone coating ensures tightness between carrier and cover.
  • the cover yields a 2 ⁇ m thick transparent polyester film that can be pierced by a capillary in order to take up the reaction fluid.
  • the carrier is covered and incubated with the non-diluted single-cell solution at 37 °C for 15 min. Then, the carrier is washed twice with PBS. The carrier is covered with the PCR reagents containing primers for microsatellite markers and hot-start Taq-polymerase.
  • the CMC is placed on a thermal cycler for PCR. The initial denaturation step is performed for 15 minutes at 95°C in order to disrupt the cells and to activate enzyme. PCR amplification is done as described by exam- pie lb. After PCR-amplification, fluid is taken from every reaction space with a glass capillary which pierces through the polyester film. This DNA solution is analysed with an automated DNA-sequencer as described by example lc.
  • Ghadimi BM Sackett DL, Difilippantonio MJ, Schrock E, Neu- mann T, Jauho A, Auer G, Ried T: Centrosome amplification and instability occurs exclusively in aneuploid, but not in dip- loid colorectal cancer cell lines, and correlates with numerical chromosomal aberrations. Genes Chromosomes Cancer 2000; 27:183-190. Glaab WE, Tindall KR: Mutation rate at the hprt locus in cancer cell lines with specific mismatch repair-gene defects. Carcinogenesis 1997; 18:1-8.
  • Grist SA McCarron M, Kutlaca A, Turner DR, Morley AA: In vivo human somatic mutation: frequency and spectrum with age. Mutat. Res. 1992; 266:189-196. Hussain SP, Raja K, Amstad PA, Sawyer M, Trudel LJ, Wogan GN, Hofseth LJ, Shields PG, Billiar TR, Trautwein: Increased p53 mutation load in nontumorous human liver of Wilson disease and hemochromatosis oxyradical overload diseases. Proc. Natl. Acad. Sci. 2000; 97:12770-12775.

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Abstract

L'invention concerne un nouveau procédé, un dispositif spécialisé et une trousse d'analyse quantitative de charges de mutation dans les cellules et les tissus. Le procédé de cette invention consiste à analyser les mutations, essentiellement au sein d'un ADN microsatellite, dans un grand nombre de cellules simples ou de petits groupes cellulaires indépendamment. Cette approche est particulièrement adaptée lorsque la détection d'une modification moléculaire spécifique est difficile à détecter en raison de la présence de molécules normales ou en raison de la présence de modifications moléculaires à orientation opposée. En outre, elle permet la quantification concurrente de fréquences mutantes et d'hétérogénéité génétique. Le dispositif spécialisé de cette invention vise à immobiliser un grand nombre de cellules dans un ordre géométrique spécifié, premièrement sous forme de matrice en treillis, sur un support miniaturisé, qui permet, par la suite, l'analyse moléculaire de cellules simples ou de petits groupes cellulaires dans des espaces de réaction et d'analyse séparés, et par conséquent l'apport d'informations relatives au nombre de cellules modifiées et à la nature de l'altération moléculaire.
PCT/IB2001/000696 2001-04-26 2001-04-26 Procede et dispositif de quantification de charges de mutation Ceased WO2002088388A1 (fr)

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Cited By (8)

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WO2004044209A1 (fr) * 2002-11-13 2004-05-27 Monoquant Pty Ltd Methode de detection
JP2013055922A (ja) * 2011-09-09 2013-03-28 Norihiko Ito 生体に対する負荷を評価する方法
WO2015017125A3 (fr) * 2013-07-30 2015-06-04 Redpath Integrated Pathology Inc. Procédé de traitement du syndrome de barrett et de l'adénocarcinome œsophagien
CN106282016A (zh) * 2016-08-10 2017-01-04 中国科学院电子学研究所 二维细胞划痕芯片及其制备方法、应用
US9873913B2 (en) 2013-03-08 2018-01-23 Roche Molecular Systems, Inc. Mutation testing
US9914975B2 (en) 2013-03-08 2018-03-13 Roche Molecular Systems, Inc. EGFR blood monitoring
US10131942B2 (en) 2011-12-01 2018-11-20 Interpace Diagnostics Corporation Methods for treating barrett's metaplasia and esophageal adenocarcinoma
US10255410B2 (en) 2011-12-01 2019-04-09 Interpace Diagnostics Corporation Methods for treating Barrett's metaplasia and esophageal adenocarcinoma

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004044209A1 (fr) * 2002-11-13 2004-05-27 Monoquant Pty Ltd Methode de detection
JP2013055922A (ja) * 2011-09-09 2013-03-28 Norihiko Ito 生体に対する負荷を評価する方法
US10131942B2 (en) 2011-12-01 2018-11-20 Interpace Diagnostics Corporation Methods for treating barrett's metaplasia and esophageal adenocarcinoma
US10255410B2 (en) 2011-12-01 2019-04-09 Interpace Diagnostics Corporation Methods for treating Barrett's metaplasia and esophageal adenocarcinoma
US12205676B2 (en) 2011-12-01 2025-01-21 Interpace Diagnostics Corporation Methods for treating Barrett's metaplasia and esophageal adenocarcinoma
US9873913B2 (en) 2013-03-08 2018-01-23 Roche Molecular Systems, Inc. Mutation testing
US9914975B2 (en) 2013-03-08 2018-03-13 Roche Molecular Systems, Inc. EGFR blood monitoring
WO2015017125A3 (fr) * 2013-07-30 2015-06-04 Redpath Integrated Pathology Inc. Procédé de traitement du syndrome de barrett et de l'adénocarcinome œsophagien
CN106282016A (zh) * 2016-08-10 2017-01-04 中国科学院电子学研究所 二维细胞划痕芯片及其制备方法、应用
CN106282016B (zh) * 2016-08-10 2020-08-07 中国科学院电子学研究所 二维细胞划痕芯片及其制备方法、应用

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