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WO2012017062A2 - Procédés de criblage de cellules tumorales - Google Patents

Procédés de criblage de cellules tumorales Download PDF

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Publication number
WO2012017062A2
WO2012017062A2 PCT/EP2011/063499 EP2011063499W WO2012017062A2 WO 2012017062 A2 WO2012017062 A2 WO 2012017062A2 EP 2011063499 W EP2011063499 W EP 2011063499W WO 2012017062 A2 WO2012017062 A2 WO 2012017062A2
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tumor
cells
chromosomal
marker
markers
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WO2012017062A3 (fr
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Eva-Maria Hoffmann
Jochen Geigl
Thomas Schwarzbraun
Michael Speicher
Peter Ulz
Ellen Heitzer
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Medizinische Universitaet Graz
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Medizinische Universitaet Graz
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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
    • C12Q1/6841—In situ hybridisation
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to methods for the genetic screening of one or more cells, preferably of tumor cells, for diagnostic and research purposes.
  • the method provides for a quality control and a measure for the detection of chromosomal copy number changes for assessing the prognostic impact of the cells analyzed.
  • CTCs circulating tumor cells
  • CTCs should provide snapshots of genomic alterations in primary tumors and metastases at various stages during the course of disease. Ideally, only single CTCs are subjected to analysis in order to avoid any falsification of the results obtained due to the presence of other cells, particularly of non-tumor cells. Furthermore, the real complexity of cell dissemination and metastasis, and especially the heterogeneity of CTCs, can only be understood when considering individual cells.
  • FISH fluorescence in situ hybridization
  • array-CGH comparative genomic hybridization
  • the present invention relates to a method for the screening of one or more cells derived from a subject for the presence of a particular genotype, comprising:
  • each chromosomal region comprising at least two candidate genetic markers
  • step (c) determining in the nucleic acid amplification products obtained in step (b) for each of the candidate genetic markers in each chromosomal region at least one parameter being indicative for the allele status of the marker;
  • step (d) selecting those chromosomal regions in which the majority of the at least two genetic markers comprised in a chromosomal region shows consistent results in step (c);
  • step (e) determining for each of the candidate genetic markers in each chromosomal region selected in step (d) at least one parameter being indicative for the allele copy number of the candidate genetic marker;
  • step (f) selecting those chromosomal regions in which the majority of the at least two genetic markers comprised in a chromosomal region shows consistent results in step (e); wherein any one or more of the chromosomal regions selected in step (f) is/are indicative for the particular genotype screened for.
  • the method is performed using only one cell.
  • the one or more cells employed in the method are tumor cells, and are particularly preferably selected from the group consisting of disseminated tumor cells and circulating tumor cells.
  • the genotype for which the one or more cells are screened for is indicative for any one or more phenotype in the subject that is selected from the group consisting of the presence of a tumor, the presence of a particular tumor type, the presence of a particular tumor stage, the predisposition to develop a tumor, and response to tumor therapy.
  • the one or more chromosomal regions have a length of at least 3 Mb.
  • the method further comprises comparing the results obtained in any one or both of steps (c) and (e) with those obtained for a control.
  • control employed is a non-amplified nucleic acid sample, and is particularly preferably derived from the subject to be analyzed.
  • step (c) comprises determining at least one parameter selected from the group consisting of number of alleles and length of the alleles.
  • step (d) further comprises selecting those chromosomal regions in which for the majority of the at least two genetic markers comprised in a chromosomal region the results obtained in step (c) are preserved in the one or more cells analyzed and the control.
  • step (f) further comprises selecting those chromosomal regions in which for the majority of the at least two genetic markers comprised in a chromosomal region of the one or more cells analyzed the allele copy number is modified as compared to the control.
  • the modification of the allele copy number is selected from the group consisting of a chromosomal loss and a chromosomal gain.
  • any one or both of steps (c) and (e) are performed by means of a PCR-based technique, preferably by quantitative fluorescence PCR.
  • the method is performed in a multiplex-format, particularly preferably in a high- throughput format.
  • the present invention relates to a method for diagnosing and/or monitoring in a sample derived from a subject the presence of a tumor or a predisposition to develop a tumor, comprising:
  • step (b) determining in the one or more cells obtained in step (a) that exhibit the particular genotype at least one further parameter being indicative for the presence of a tumor or a predisposition to develop a tumor.
  • the present invention relates to the use of the methods as defined herein above for diagnosing and/or monitoring in a sample derived from a subject any one or more selected from the group consisting of the presence of a tumor, the presence of a particular tumor type, the presence of a particular tumor stage, the predisposition to develop a tumor, and response to tumor therapy.
  • FIGURE 1 Analysis of the sizes of 5 different markers on chromosome 18 (y-axis). Shown are the results of four different single cell amplifications and two amplifications with 10-cell pools (x-axis; single cell amplification products are "JG_1 ", “JG_1.3”, “JG_1.4”, and “JG_1.5", the 10-cell pools are "JG_10.4" and "JG_10.5")
  • the horizontal green bars display the standard deviation for the allele sizes as measured in all experiments together for each marker allele.
  • the vertical green bars are mainly for orientation and indicate the location of the mean size.
  • the black dots show the results obtained with non-amplified DNA the red dots the size obtained with amplified DNA.
  • a black dot left from a red dot indicates that the size obtained with the amplified DNA was larger as the size measured with non-amplified DNA.
  • a comparison with the standard deviation allows the easy identification of outliers. Here, an outlier was observed only for Marker D18S978 in cell JG_1. Otherwise, all red and black dots are close together. As marker D18S391 is uninformative, there are no dots for the second allele whereas the second allele of Marker D18S499 was not amplified in any experiment.
  • FIGURE 2 Peak areas for the same 5 different markers on chromosome 18 and the same experiments as in Figure 1 (here, the markers are indicated on the x-axis).
  • the green dot represents the mean of all peak area measurements from the amplification products; the green bar the standard deviation.
  • the black dots show the results obtained in each experiment with non-amplified DNA, the red dots the peak areas obtained with amplified DNA from the various experiments. Thus, the black dots reflect the "real" peak areas, whereas the red dots show the variability from the experiments. Note that marker D18S391 is uninformative and that the second allele of marker D18S499 was not amplified in any experiment.
  • FIGURE 3 Illustrated is the same representation as in Figure 1 but with peak areas instead of sizes.
  • FIGURE 4 Ratio values after normalization: The black line shows the normalized values for the non-amplified DNA for which all values were set to 1. Because Marker D18S391 is uninformative (i.e. there is only one allele present) the second allele has the value 0.
  • the colored lines represent the results for each of the aforementioned experiments (e.g. the red line corresponds to the values of the single cell "JG_1"). All cells have for the second allele of marker D18S391 again the value 0. As the second allele of Marker D18S499 was not amplified in any experiment all amplification products (but not the normalized DNA) have for this marker also a 0.
  • FIGURE 5 Same data set as in Figure 4 but allele 1 of each marker was duplicated in order to simulate a data set with trisomy.
  • FIGURE 6 Illustrated are experimental results of an analysis for a first mix of seven different markers.
  • FIGURE 7 Illustrated are experimental results of an analysis for a second mix of seven different markers.
  • FIGURE 8 Illustrated are experimental results of an analysis for a third mix of five different markers.
  • the present invention is based on the unexpected finding that by combining the analysis of several parameters based on the allelic status of candidate genetic markers, all of which are per se well established and can be readily determined, and using a specific evaluation algorithm in a rapid (completion within 24-48 h, or even faster) and cost-effective method for the (pre-)screening of cells could be established.
  • This pre-screening provides reliable information about how many cells are suitable for subsequent analysis, that is, represents a suitable means for quality assessment.
  • the method also provides first insight as to the genotype of the one or more cells analyzed, namely the pattern of chromosomal aberrations (i.e. changes of allelic copy numbers), which may aid in subsequent diagnosis and/or monitoring of a medical condition this genotype is indicative for.
  • the present invention relates to a method for the screening of one or more cells derived from a subject for the presence of a particular genotype, comprising:
  • each chromosomal region comprising at least two candidate genetic markers
  • step (c) determining in the nucleic acid amplification products obtained in step (b) for each of the candidate genetic markers in each chromosomal region at least one parameter being indicative for the allele status of the marker;
  • step (d) selecting those chromosomal regions in which the majority of the at least two genetic markers comprised in a chromosomal region shows consistent results in step (c);
  • step (e) determining for each of the candidate genetic markers in each chromosomal region selected in step (d) at least one parameter being indicative for the allele copy number of the candidate genetic marker;
  • step (f) selecting those chromosomal regions in which the majority of the at least two genetic markers comprised in a chromosomal region shows consistent results in step (e); wherein any one or more of the chromosomal regions selected in step (f) is/are indicative for the particular genotype screened for. ln a preferred embodiment, the method is performed using only one cell (i.e. as a single cell analysis).
  • the one or more cells are derived from a subject to be analyzed by the present method.
  • the subject is a mammal such as a mouse, rat, hamster, rabbit, cat, dog, pig, cow, horse or monkey.
  • the subject to be diagnosed is a human.
  • the one or more cells to be employed in the present invention are purified from a sample collected from the subject to be analyzed.
  • the samples may include body tissues (e.g., biopsies or resections, bone marrow samples, placental tissue, and umbilical cord samples) and fluids, such as blood, sputum, cerebrospinal fluid, amniotic fluid, and urine.
  • the samples represent particular types of cells, such as oocytes (egg cells), adult stem cells, embryonic stem cells, and stem cell precursor cells such as blastomers.
  • the samples used in the method of the present invention should generally be collected in a clinically acceptable manner.
  • the one or more cells employed in the present invention are derived from a blood sample such as whole blood, plasma, and serum.
  • a blood sample such as whole blood, plasma, and serum.
  • whole blood refers to blood with all its constituents (i.e. both blood cells and plasma).
  • plasma denotes the blood's liquid medium.
  • serum refers to plasma from which the clotting proteins have been removed.
  • the one or more cells are tumor cells, and are particularly preferably selected from the group consisting of disseminated tumor cells and circulating tumor cells.
  • tumor also commonly referred to as “cancer”
  • cancer any type of malignant neoplasm, that is, any morphological and/or physiological alterations (based on genetic re-programming) of target cells exhibiting characteristics of a tumor as compared to unaffected (healthy) wild-type control cells.
  • alterations may relate inter alia to cell size and shape (enlargement or reduction), cell proliferation (increase in cell number), cell differentiation (change in physiological state), apoptosis (programmed cell death) or cell survival.
  • a tumor is characterized by uncontrolled division of target cells based on genetic re-programming and by the ability of the target cells to spread, either by direct growth into adjacent tissue through invasion, or by implantation into distant sites by metastasis.
  • tumors include inter alia breast cancer, colorectal cancer, prostate cancer, leukemia, lymphomas, neuroblastoma, glioblastoma, melanoma, liver cancer, and lung cancer.
  • CTCs circulating tumor cells
  • DTCs isseminated tumor cells
  • CTCs and DTCs may constitute 'seeds' for subsequent growth of metastases in different tissues.
  • the one or more cells are derived from amniotic fluid and/or chorionic villi (i.e. placental tissue).
  • the method of the present invention may be applied for pre-natal diagnosis.
  • pre-natal diagnosis it may also be possible to screen fetal cells circulating in maternal blood.
  • the one or more cells are oocytes (i.e. egg cells, often also referred to as "ova") or, more specifically, the polar bodies isolated from oocytes.
  • the egg cells of all mammals have two polar bodies. Polar bodies are produced during meiosis, contain relatively little cytoplasm and are haploid (i.e. contain 23 chromosomes).
  • oocytes i.e. egg cells, often also referred to as "ova”
  • ova i.e. egg cells, often also referred to as "ova”
  • the egg cells of all mammals have two polar bodies. Polar bodies are produced during meiosis, contain relatively little cytoplasm and are haploid (i.e. contain 23 chromosomes).
  • haploid i.e. contain 23 chromosomes
  • the one or more cells are adult stem cells, embryonic stem cells or blastomers.
  • Stem cells are generally characterized by the ability to renew themselves and to differentiate into a diverse range of specialized cell types.
  • Adult stem cells (as well as the more specific oligopotent progenitor cells) are found in adult organisms in a variety of sources such as blood, bone marrow, and umbilical cord and primarily act as a repair system for the body.
  • Adult stem cells are multipotent, that is, they can differentiate into a number of closely related cells (i.e. are lineage-restricted).
  • Embryonic stem cells are derived from the inner cell mass of blastocysts or earlier morula stage embryos.
  • Embryonic stem cells are pluripotent and can differentiate into almost all cells.
  • the screening of said cells may also be used in the context of pre-fertilization diagnosis as well as for assessing stem cell quality for therapeutic purposes.
  • genotype refers to the inherited instructions of an organism it carries within its genetic code.
  • genotype denotes the genetic constitution of a cell or an organism (i.e. the specific allele makeup) usually with reference to a specific character under consideration. Said character is commonly referred to as "phenotype” which denotes any observable feature or trait of a cell or an organism, such as its morphology, developmental status, biochemical or physiological properties, etc. under a particular set of environmental conditions. Phenotypes result from the expression of an organism's genes as well as the influence of environmental factors and the interactions between the two.
  • the method of the invention may be employed for the screening of any cellular genotype.
  • the genotype screened for is indicative for a specific phenotype of the cells analyzed, particularly for the presence of a medical condition such as cancer or a hereditary disease
  • the genotype is indicative for any one or more phenotype in the subject that is selected from the group consisting of the presence of a tumor, the presence of a particular tumor type (i.e. in order to discriminate between different tumors), the presence of a particular tumor stage (i.e. in order to monitor tumor progression), the predisposition to develop a tumor, and response to tumor therapy.
  • pre-cancerous state i.e. an intermediate state in the transformation of a normal cell into a tumor cell.
  • pre-cancerous state i.e. an intermediate state in the transformation of a normal cell into a tumor cell.
  • pre-cancerous state is a benign adenoma, which may progress into a malignant adenocarcinoma.
  • the identification (or selection) of the chromosomal regions will depend inter alia on the application of the method, the type of cell(s) employed, the parameters to be analyzed, and the like.
  • the skilled person is well aware of methods how to identify one or more such chromosomal regions for a given setting. Typically, the selection is accomplished based on information retrieved from databases or the scientific literature. For example, for multiple tumors ample information is available with regard to the genetic re-programming of the tumor cells as compared to healthy controls (i.e. the occurrence of chromosomal aberrations, gene mutations, etc.). Accordingly, based on this information the skilled person may readily select one or more chromosomal regions that - at least with a reasonable likelihood - have the potency to be indicative for that tumor.
  • the one or more chromosomal regions have a length of at least 3 Mb, such as, e.g., at least 3.2 Mb, at least 3.4 Mb, at least 3.6 Mb, at least 3.8 Mb, at least 4 Mb, at least 4.5 Mb, at least 5 Mb, or even larger.
  • candidate genetic marker denotes a gene or a DNA sequence with a known location on a chromosome that can be used to identify a cell or an organism (i.e. that - at least with a reasonable likelihood - is characteristic for the genotype of the one or more cells analyzed).
  • Such marker can be described as a variation (which may arise due to mutation or alteration in the genetic locus on the chromosome) that can be observed (for example, by means of an altered sequence or gene expression level).
  • candidate genetic markers include inter alia single nucleotide polymorphisms, restriction fragment length polymorphisms, short tandem repeats, mini-satellites, and entire (mutated) genes).
  • each of the one or more chromosomal regions comprises at least two candidate genetic markers.
  • each chromosomal region comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or even more candidate genetic loci.
  • the candidate genetic loci comprised in a chromosomal region may be located in close proximity to each other or may be distributed over the whole length of the chromosomal region. It may also be possible that only part of the candidate genetic loci comprised in a chromosomal region may be clustered, whereas the remaining ones are located at different positions.
  • the one or more cells are subjected to nucleic acid amplification, which is typically accomplished by a PCR-based technique well known in the art (cf., e.g., Sambrook, J., and Russel, D.W. (2001), supra; Ausubel, F.M. et al. (2001) supra).
  • At least one parameter being indicative for the allele status of the marker is determined.
  • this determination step is performed by means of a PCR-based technique, preferably by quantitative fluorescence PCR (QF-PCR; Peril , B. et al. (1999) Mol. Hum. Reprod. 5, 1 176-1 179; see also below).
  • QF-PCR quantitative fluorescence PCR
  • the selection of a particular method may depend inter alia on the at least one parameter analyzed, the type of cells employed, and the genotype to be screened for. The skilled person is well aware how to select a specific technique for a given application.
  • those chromosomal regions are selected in which the majority of the at least two genetic markers comprised in a chromosomal region shows consistent (i.e. identical or at least similar) results in the above analysis. For example, if a chromosomal region comprises five genetic markers it is selected for further analysis provided that at least three of the markers display identical results with respect to the at least one parameter analyzed (e.g., the number of alleles). Such consistent results provides a measure for the quality of the amplification product used, and thus for the usefulness to include such chromosomal regions in further analyses. On the other hand, multiple differences within the results obtained for the genetic markers comprised in a chromosomal region indicate a low quality.
  • the likelihood to obtain consistent results will depend inter alia on the distance of the genetic markers on the chromosome (i.e. their respective locations to each other). The smaller the distance, the higher is the likelihood to obtain consistent results.
  • the method further comprises comparing the results obtained for the "test sample" with those obtained for a control.
  • control employed is a non-amplified nucleic acid sample (e.g., genomic cellular DNA not subjected to any amplification step), and is particularly preferably derived from the subject to be analyzed (that is, test sample and control are derived from the same subject).
  • control also refers to reference values derived from databases or published in the scientific literature.
  • comparing the results also includes normalizing the results obtained for the test sample relative to the control (for example, by setting the control to 100% and calculating the corresponding value for the test sample). In one preferred embodiment, such normalization is performed by means of using the algorithm described in the experimental section below.
  • the method further comprises selecting those chromosomal regions in which for the majority of the at least two genetic markers comprised in a chromosomal region the results obtained are preserved in the one or more cells analyzed and the control. For example, if the parameter being indicative for the allele status to be analyzed is allele length, then obtaining identical results in the test sample and the control (e.g., non-amplified DNA) indicates a correct size and provides an additional measure for the quality of the amplification products used.
  • the method of the present invention comprises determining for each of the candidate genetic markers in each chromosomal region selected in the previous step at least one parameter being indicative for the allele copy number of the candidate genetic marker. As used herein, determining the allele status of a genetic marker (as described above) may also include determining of the allele copy number.
  • the allele copy number can be seen as a measure for the occurrence of chromosomal aberrations (i.e. abnormalities of the normal chromosomal structure).
  • chromosomal aberration detection procedures many of them PCR-based, are known in the art such as inter alia multiplex ligation-dependent probe amplification (MLPA; Schouten, B. et al. (2002) Nucl. Acids Res. 30, e57) and quantitative fluorescence PCR (QF-PCR; Peril , B. et al. (1999), supra), with the latter one being particularly preferred herein.
  • this determination step is performed by means of a PCR- based technique, preferably by quantitative fluorescence PCR.
  • PCR-based techniques employed herein are performed according to established protocols well known in the art.
  • any one of the following basic experimental approaches may be used: primer extension pre-amplification PCR (Zhang, L. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 5847-5851), multiple displacement amplification (Dean, F.B. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 5261-5266), linker adapter PCR (Ludecke, H.J. et al. (1989) Nature 338, 348-350), and degenerate oligonucleotide primed PCR (Telenius, H. et al. (1992) Genomics 13, 718-725). Kits for performing such PCR- techniques for whole genome amplification are also available from various manufacturers.
  • the method again further comprises comparing the results obtained for the "test sample” with those obtained for a control.
  • control employed is a non-amplified nucleic acid sample, and is particularly preferably derived from the subject to be analyzed.
  • control also refers to reference values derived from databases or published in the scientific literature.
  • comparing the results also includes normalizing the results obtained for the test sample relative to the control. In one preferred embodiment, such normalization is performed by means of using the algorithm described in the experimental section below.
  • the method further comprises selecting those chromosomal regions in which for the majority of the at least two genetic markers comprised in a chromosomal region of the one or more cells analyzed the allele copy number is modified (i.e. altered) as compared to the control.
  • the modification of the allele copy number is selected from the group consisting of a chromosomal loss and a chromosomal gain.
  • chromosomal gain denotes an increase in copy number due to a single, double or triple duplication of chromosomal regions.
  • the duplicated or gained regions may be derived from the same chromosome or from different chromosomes. Preferably, they are from the same chromosome.
  • chromosomal loss denotes a decrease in copy number due to a deletion of chromosomal regions.
  • any one or more of the chromosomal regions selected in the final step of the method is/are indicative for the presence of a particular genotype.
  • the identification of a single chromosomal region may be sufficient to indicate the presence of a particular phenotype.
  • the selection of more than one chromosomal region will be required.
  • the one or more of the chromosomal regions selected may also have direct diagnostic and/or prognostic value.
  • chromosomal aberrations for example, colorectal cancer/adenocarcinoma and breast cancer.
  • the detection of such chromosomal aberrations in tumor cells or cells suspected to be derived from a tumor may directly indicate or confirm the actual presence of a tumor.
  • the method of the present invention may also be performed using a computer-based approach employing various algorithms for evaluating the experimental data obtained.
  • the method is performed using the algorithm described in the experimental section below.
  • the method is performed in a multiplex-format.
  • multiplex-format refers to the parallel analysis of multiple (i.e two or more) samples, each sample comprising one or more cells.
  • term also relates to (automated) high-throughput analyses of hundreds or thousands of samples, for example by employing array technology.
  • the present invention relates to a method for diagnosing and/or monitoring in a sample derived from a subject the presence of a tumor or a predisposition to develop a tumor, comprising:
  • step (b) determining in the one or more cells obtained in step (a) that exhibit the particular genotype at least one further parameter being indicative for the presence of a tumor or a predisposition to develop a tumor.
  • the method is performed as an in vitro method.
  • the terms "diagnosing” and “monitoring” are intended to encompass predictions and likelihood analysis (based on both the qualitative and quantitative measurements).
  • the present method is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, disease staging, and disease monitoring and surveillance.
  • an intermediate result for examining the condition of a subject may be provided. Such intermediate result may be combined with additional information to assist a physician, nurse, or other practitioner to diagnose that a subject suffers from the disease.
  • the present invention may be used to detect tumor cells in a subject-derived sample, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
  • the results may optionally be corroborated and/or supplemented by performing an additional (more sophisticated or specific) analysis, for example, in order to extend the result of the pre-screening that in fact a tumor is present to the determination of stage of tumor progression. Accordingly, the screening method per se may be sufficient to also enable the diagnosis of a tumor. However, an accurate diagnosis may typically require performing further analyses.
  • the present invention relates to the use of the methods as defined herein above for diagnosing and/or monitoring in a sample derived from a subject any one or more selected from the group consisting of the presence of a tumor, the presence of a particular tumor type, the presence of a particular tumor stage, the predisposition to develop a tumor, and response to tumor therapy.
  • Example 1 Pre-screening of the whole-genome amplification product of single cells
  • the pre-screening method developed employs a quantitative fluorescence (QF)-PCR assay, which provides information on both the quality of the single-cell PCR product and on copy- number changes.
  • QF quantitative fluorescence
  • the analyses focus on copy number changes frequently reported in the literature for certain tumor entities. However, in principle any region in the genome can be tested.
  • the method involves determination of the allele size, number of alleles and ratio values corresponding to the copy number of certain microsatellite markers located in close proximity at defined regions with frequent copy number changes in breast and colorectal cancer.
  • the aim is to use sets of primers, which are multiplexed together, as a "one-tube" test.
  • a set of 21 chromosomal markers (STRs, short tandem repeats) is used for the analysis of circulating tumor cells (CTCs) derived from breast and colorectal cancers.
  • CTCs circulating tumor cells
  • the marker panel shown in Table 1 covers seven chromosomal regions that are frequently gained or lost in breast and colorectal tumors.
  • Control 2q23.5 The present strategy includes enrichment of the STR marker panel for those markers located in regions often involved in copy number changes in the respective tumor entity. This significantly increases the likelihood to detect alterations in the analyzed CTCs. The following characteristics are determined for each marker: chromosomal location, heterozygosity, and size range (bp).
  • PCR analyses were performed following established protocols known in the art (see, e.g., Sambrook, J., and Russel, D.W. (2001), supra; Ausubel, F.M. et al. (2001), supra), preferably by quantitative fluorescence PCR (QF-PCR; PertI, B. et al. (1999), supra).
  • QF-PCR quantitative fluorescence PCR
  • any one of the following basic experimental approaches may be used: primer extension pre-amplification PCR (Zhang, L. et al. (1992), supra), multiple displacement amplification (Dean, F.B. et al. (2002), supra), linker adapter PCR (Ludecke, H.J. et al.
  • PCR-techniques for whole genome amplification are also available from various manufacturers.
  • the PCR products obtained were analyzed on 3000 and 3100 capillary-based genetic analyzers (Applied Biosystems, Carlsbad, CA, USA).
  • D18S386 located at 18q22.1 : 65,794,580-65,794,944
  • D18S391 located at 18p11.31 : 5,781 ,224-5,781 ,405)
  • D18S499 located at 18q21.32- q21.33
  • D18S535 located at 18q12.3: 38, 148,789-38, 148,934
  • D18S978 located at 18q12.3: 38,338, 136-38,338,382).
  • Each marker provides information about the following characteristics: 1. number of alleles (i.e. one or two alleles), marker having a high rate of heterozygosity are selected according to data bases in order to increase the likelihood that for each marker two alleles will be observed;
  • the quality criteria are based on the following parameters:
  • a high number of preserved alleles in the amplified DNA having the correct size suggests good quality (e.g. if 5 markers specific for a chromosomal region are used, 10 alleles should be present in the amplification product; the size of at least 8-9 alleles should be correctly reflected in the amplification product) (cf. Fig. 1).
  • the number of alleles and their peak size is used for estimation of the copy number as described in detail below.
  • the distance of the markers is chosen so that the majority of markers should show similar if not identical results. Multiple differences (i.e. the first marker indicates a loss, the second marker a gain, the third marker a normal copy number, the fourth marker again a loss, and so on) indicate a poor quality.
  • a lost region is characterized by loss of heterozygosity (LOH).
  • LOV heterozygosity
  • the presence of only one allele at a single locus is not sufficient to determine that the respective region is indeed lost as such losses may also be due to other factors, such as chance (i.e. the person has in this locus two identical alleles, in this case the person would have only a single peak and such a marker is described as uninformative) or by artifact ("allelic drop out" due to the whole-genome amplification). Therefore, the present results are not based on only a single locus and for this reason the marker panel was selected so that several markers were in close proximity. Only if these markers show concordantly only one allele instead of two is this region defined as lost.
  • the detection of lost regions can be greatly facilitated if the marker analysis of non-amplified DNA is done in parallel, which provides, for each marker, very accurate information about the number of alleles (i.e. one or two alleles), the size of the markers and the peak areas:
  • a region is classified as lost if the majority or all markers show only one allele.
  • the diagnosis will be more accurate if non-amplified DNA is available and shows the presence of two alleles so that the presence of a LOH can be easily confirmed.
  • Identification of gained regions represents a particular challenge. For example, in prenatal applications the diagnosis of a trisomy is based either on the observation of three alleles (i.e. three peaks) or on two alleles in a 2: 1 or a 1 :2 ratio. In the experimental settings used herein three alleles should not occur.
  • Figures 2 and 3 demonstrate the variability of peak areas in the amplification products analyzed. There are a few patterns, which appear to occur relatively frequently (e.g. peak areas of amplification products are more likely to be smaller as compared to peak areas with non-amplified DNA; the variability tends to increase with the size of the peak areas), however, these patterns are insufficient for a reliable peak area ratio calculation. The most accurate peak area ratio calculations can be achieved by normalizing the peak areas.
  • Each informative marker has two alleles, a paternal and a maternal allele.
  • the respective peak areas could be designated as:
  • Pe A (yWi j ) and Pe A (Pi j ) / Peu(Mi j ) and ⁇ ( ⁇ ,)
  • Pe A is the peak area obtained with the amplified DNA
  • Peu is the peak area obtained with the non-amplified DNA
  • y is the maternal allele for chromosomal region / ' and marker y
  • i j is the paternal allele for chromosomal region / ' and marker j
  • the peak areas of the non-amplified DNA are set to the value 1 :
  • r is the number of regions tested in the assay ;m, is the number of markers for each region / ' ; and NPeu is the normalized peak area obtained with the unamplified DNA.
  • the peak areas of the amplified DNA are normalized via dividing their values by the respective value of the non-amplified DNA.
  • NPe A is the normalized peak area obtained with the amplified DNA.
  • NPe A will have a value between 0 and 1 ; otherwise, if NPe A is larger than Peu then NPe A is larger than 1.
  • Figure 4 depicts the data after the normalization procedure.
  • peak areas show substantial variability after amplification the two alleles of a given marker often (but not always) have comparable peak areas after normalization (cf. Fig. 4). In the present case, similar peak areas are expected as the experiments were done with normal diploid cells.
  • RPe A is the ratio between normalized peak areas obtained with the amplified DNA and-i j indicates the location for chromosomal region / ' and marker j.
  • each single ratio value is considered and classified according to these criteria:
  • Trisomy if the allele ratio allele ratio greater than 1 .8 (to achieve this, our algorithm always divides the larger peak area by the smaller peak area);
  • the alleles of each region are evaluated together.
  • the majority of classifications of individual regional markers should be consistent.
  • 5 markers are used. Therefore, at least three markers indicating the same copy number status are required for making a classification.
  • One marker i.e. D18S499
  • another marker i.e. D18S391
  • this marker had to be designated as "lost”.
  • the three remaining markers have to yield consistent results for an unambiguous classification.
  • countAmp, countUI, countDel, countNI are counters for alleles with amplified, uninformative, deleted and normal status, respectively.
  • countAmp, countUI, countDel, countNI are counters for alleles with amplified, uninformative, deleted and normal status, respectively.
  • one of these counters is equal or greater than 3 than the region will be assigned the respective status.
  • Figure 5 illustrates such a simulation.
  • the peak areas of alleles 1 but not of alleles 2 were duplicated assuming that a trisomy will be reflected by an exact duplication of the peak areas of one of the two alleles.
  • Figure 5 shows the normalized values. Apparently, for the majority of markers with two alleles there is a clear difference between the two peak areas.
  • FIG. 1 The three different microsatellite marker sets were adjusted and the testing was performed for chromosomal regions 8p (5 markers), 8q (5 markers), 13q (4 markers), and 17p (5 markers).
  • marker generation is easy and straightforward so that a person skilled in the art can easily switch to other chromosomal regions.
  • FIG. 1 figures 6 to 8 each show graphs for the source data for un-amplified genomic DNA (top panel) and for two single cells (center and bottom panel). Each row depicts for the individual markers the number of alleles (here: always one or two), the size of the marker and the peak areas.
  • a table is added, which summarizes the exact locations of the microsatellite markers.
  • Figure 6 shows experimental results of an analysis for a first mix of seven different markers.
  • the first mix consists of three markers for chromosome 8p, one marker for chromosome 8q, one marker for chromosome 13q, and two markers for chromosome 17p.
  • the first marker represents a region on chromosome 8p and has two alleles, one at 1 14 bp and the second, which has a very small peak area, at 128 bp. These two alleles were identified also in the two single cells. The same is true for the other markers in this probe set.
  • Figure 7 shows experimental results of an analysis for a second mix of seven different markers.
  • the second mix consists of one marker for chromosome 8p, four markers for chromosome 8q, and two markers for chromosome 17p.
  • Figure 7 illustrates marker analysis with the same DNA and single cells, respectively and demonstrates how an evaluation algorithm according to an exemplary embodiment handles variability, which may occur in single cell analysis due to bias during the whole genome amplification process or other events, e.g. during the cell selection.
  • the first marker for chromosome 8q has two alleles at 120 bp and 124 bp. In the single cell amplification products the second allele at 124 bp shows each a markedly reduced peak area.
  • the algorithm would not classify this microsatellite marker as lost because both alleles are still present and thus there is no LOH. Although the peak area of the allele at 120 bp is much larger as compared to the 124 bp allele in the two single cells, the algorithm would vice versa not call this region amplified, because the peak area at 120 bp in the single cell amplification products has not increased compared to the area with the genomic DNA.
  • marker 17p2 which consists of two alleles at 218 bp and 224 bp.
  • the first single cell has clearly a LOH as the allele at 218 bp cannot be detected. Still, the region would not be called lost by the algorithm as marker 17p-5 ( Figure 6) clearly shows the presence of two alleles.
  • Figure 8 shows experimental results of an analysis for a third mix of five different markers.
  • the third mix consists of one marker for chromosome 8p, three markers for chromosome 13q, and one marker for chromosome 17p,
  • Figure 8 summarizes the other markers, all of them were correctly identified in the single cells.
  • the above described results illustrate an example, which may demonstrate the reliability which could be achieved with the fine tuning of the QF-PCR.
  • the algorithm turned-out to process the data sets very accurately and generates thus reliable data on both the quality of the amplification product and the copy number status of the tested regions.
  • the resolution (sensitivity) that can be achieved with the method depends on the number of markers used for each region. An increase of the number of markers along the length of each chromosomal region increases the likelihood of detecting unbalanced chromosome rearrangements. On the other hand, each additional marker increases the complexity of the multiplex-PCR and makes it more expensive.
  • markers are located in regions, which show frequently chromosomal gains and losses in tumors to increase the chance to identify cells with copy number changes.
  • Tables 1 and 2 show such regions in breast and colon cancer.
  • the number of markers needed to identify a loss is probably lower than for identification of a gain.
  • the identification of a loss is relatively straightforward as it depends on the presence of two alleles in the non-amplified DNA and loss of one allele in the cells or amplification product, respectively. To avoid wrong interpretation due to amplification artifacts (allelic drop out) interpretation depends not on a single, but on several markers. For the unequivocal identification of a loss three markers may be sufficient.
  • Further resolution-determining factors include the number of markers showing a consistent result.
  • the copy number status of a region depends on the classification of the majority of markers, i.e. if at least 3 of the 5 markers indicate a certain copy number status the entire region is set to this value.
  • 4/5 markers or even 5/5 markers should show consistent results, which would make the entire procedure more stringent but, on the other hand, also increase the likelihood to find more unclassifiable cells.
  • the selection of thresholds and stringency criteria may depend on the specific application and on the preferences of individual investigators.
  • clonality may take advantage of the clonality in tumor cell populations. For example, if a gain of a particular chromosome has a 2:1 ratio for a given allele 1 over allele 2, and if this clone expands, multiple progenitor cells will be generated having the same ratio shift between alleles 1 and 2. Thus, this strategy provides also information about clonality, which should again facilitate identification of gained regions.
  • cells of interest e.g. based on certain patterns of gains and losses
  • further analyses e.g. array-CGH.
  • array-CGH it is conceivable to pool cells with identical patterns of gains and losses as such cells may likely be from the same clone.
  • Such an "intelligent pooling" or “smart pooling" of cells may also be important, if the amplification products are subjected to subsequent sequencing and furthermore it will also increase resolution of array-CGH.

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Abstract

La présente invention concerne un procédé de criblage d'une ou plusieurs cellule(s) issue(s) d'un patient pour déterminer la présence d'un génotype particulier, consistant à : identifier dans la ou les cellules(s) une ou plusieurs région(s) chromosomique(s), chaque région chromosomique comprenant au moins deux marqueurs génétiques candidats; soumettre la ou les cellule(s) à une amplification des acides nucléiques; déterminer dans les produits d'amplification des acides nucléique obtenus dans l'étape précédente pour chacun des marqueurs génétiques candidats dans chaque région chromosomique au moins un paramètre indicateur du statut allélique du marqueur; sélectionner les régions chromosomiques dans lesquelles la majorité des deux marqueurs génétiques ou plus compris dans une région chromosomique présente des résultats cohérents dans l'étape précédente; déterminer pour chacun des marqueurs génétiques candidats dans chaque région chromosomique sélectionnée dans l'étape précédente au moins un paramètre indicateur du nombre de copies alléliques du marqueur génétique candidat; et sélectionner les régions chromosomiques dans lesquelles la majorité des deux marqueurs génétiques ou plus compris dans une région chromosomique présente des résultats cohérents dans l'étape précédente, l'une quelconque de la ou des régions chromosomique(s) sélectionnée(s) dans l'étape précédente étant indicatrice de la présence d'un génotype particulier. La présente invention concerne en outre un procédé correspondant de diagnostic et/ou de surveillance dans un échantillon de la présence d'une tumeur ou d'une prédisposition à développer une tumeur, consistant à effectuer ledit procédé de criblage, ainsi que l'utilisation dudit procédé de criblage pour diagnostiquer et/ou surveiller la présence d'une tumeur, la présence d'un type de tumeur particulier, la présence d'une étape tumorale particulière, la prédisposition à développer une tumeur, et la réponse à un traitement contre la tumeur.
PCT/EP2011/063499 2010-08-05 2011-08-05 Procédés de criblage de cellules tumorales Ceased WO2012017062A2 (fr)

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Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
ALLARD, W.J. ET AL., CLIN. CANCER RES., vol. 10, 2004, pages 6897 - 6904
AUSUBEL, F.M. ET AL.: "Current Protocols in Molecular Biology", 2001, WILEY & SONS
DEAN, F.B. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 99, 2002, pages 5261 - 5266
LUDECKE, H.J. ET AL., NATURE, vol. 338, 1989, pages 348 - 350
MANN, K. ET AL., LANCET, vol. 358, 2001, pages 1057 - 1061
NAGRATH, S. ET AL., NATURE, vol. 450, 2007, pages 1235 - 1239
PANTEL, K. ET AL., NAT. REV. CANCER, vol. 8, 2008, pages 329 - 340
PERTL, B. ET AL., MOL. HUM. REPROD., vol. 5, 1999, pages 1176 - 1179
SAMBROOK, J., RUSSEL, D.W.: "Molecular cloning: A laboratory manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SCHOUTEN, B. ET AL., NUCL. ACIDS RES., vol. 30, 2002, pages E57
TELENIUS, H. ET AL., GENOMICS, vol. 13, 1992, pages 718 - 725
ZHANG, L. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 5847 - 5851

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