EP1451354A2 - Methoden und nukleinsäuren zur analyse von proliferativen erkrankungen von lymphoidzellen - Google Patents

Methoden und nukleinsäuren zur analyse von proliferativen erkrankungen von lymphoidzellen

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Publication number
EP1451354A2
EP1451354A2 EP02787817A EP02787817A EP1451354A2 EP 1451354 A2 EP1451354 A2 EP 1451354A2 EP 02787817 A EP02787817 A EP 02787817A EP 02787817 A EP02787817 A EP 02787817A EP 1451354 A2 EP1451354 A2 EP 1451354A2
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European Patent Office
Prior art keywords
seq
nucleic acid
oligonucleotides
recited
dna
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EP02787817A
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English (en)
French (fr)
Inventor
Matthias Burger
Charles Caldwell
Bülent GENC
Evelyne Becker
Sabine Maier
Inko Nimmrich
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Epigenomics AG
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Epigenomics AG
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Priority claimed from DE10164501A external-priority patent/DE10164501A1/de
Application filed by Epigenomics AG filed Critical Epigenomics AG
Publication of EP1451354A2 publication Critical patent/EP1451354A2/de
Withdrawn legal-status Critical Current

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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
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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 nucleic acids, oligonucleotides, PNA-oligomers, and to a method for the analysis of lymphoid cell proliferative disorders, the differentiation between subclasses of said disorder or the detection of a predisposition to said disorders, by analysis of the genetic and/or epigenetic parameters of genomic DNA and, in particular, with the cytosine methylation status thereof.
  • Lymphomas e.g. solid tumours of lymphoid cells, fall in two distinct groups: Hodgkin's lymphoma and Non-Hodgkin's lymphoma (NHL).
  • NHL Non-Hodgkin's lymphoma
  • NHL accounts for 5% of new cancers in men and 4 % of new cancers in women each year in the US and is responsible for 5% of deaths. In 1997 NHL was reported to be the leading cause of death from cancer in men between the ages of 20 and 39 (Greenlee et al., Cancer J Clin 2000, 50:7).
  • Non-Hodgkin's lymphoma can be further subclassified into different classes according their origin from different differentiation states of B or T lymphocytes.
  • B-cell NHLs the following classes can be distinguished on a cytological basis: chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL, originating from the mantle zone of a lymphoid follicle) mantle cell lymphoma (MCL, originating in the mantle zone of a lymphoid follicle) follicular lymphoma (FL, originating in the germinal centre of a lymphoid follicle) diffuse large B-cell lymphoma (DLBCL, originating in the germinal centre of a lymphoid follicle) lymphoplasmacytoid lymphoma (LPL, originating in the germinal centre of a lymphoid follicle)
  • CLL/SLL chronic lymphocytic leukemia/small lymphocytic lymphoma
  • MCL mantle cell lymphoma
  • FL originating in the germinal centre of a lymphoid folli
  • Burkitt's lymphoma (BL, originating in the germinal centre of a lymphoid follicle) mucosa-associated lymphoid tissue lymphoma (MALT, originating from the marginal zone of a lymphoid follicle.
  • CLL/SLL which arises from the same zone of the lymphoid follicle as the mantle cell lymphoma and often resembles mantle cell lymphoma regarding morphology, usually progresses rather slowly and is traditionally treated with chlorambucil and cyclophosphamide.
  • MCLs show an overall survival of 3 years with a failure-free survival of 1 year after treatment.
  • MCL is typically treated with regimens containing cyclophosphamide, doxo- rubicin, vincristine and prednisone. Because of the poor outlook young patients with mantle cell lymphoma often undergo autologous or allogeneic bone marrow transplantation (Stewart et al, Ann Oncol 1995, 6: 263). More recently, MCL can be treated with the monoclonal antibody rituximab (Coiffier et al, Haematologica. 1999, 84:14-8.).
  • Each of these neoplasms has a characteristic morphology, which may be sufficient in a given case to permit diagnosis and classification on morphologic grounds alone, if well-prepared sections are available.
  • histologic diagnosis of malignant lymphoma requires much expertise and in cases which present with an atypical morphology classification cannot be achieved on histological grounds alone.
  • additional markers such as immuno- phenotyping and cytogenetics are essential to classify the lymphoma correctly, in most other cases these methods are helpful (Armitage et al., Principles & Practice of Oncology. De Nita NT, Hellmann S, Rosenberg SA. 6 th Edition).
  • Molecular markers offer the advantage that even biopsy samples of very small sizes and samples whose tissue architecture has not been maintained can be analysed quite efficiently.
  • new insights into immunology and genetics of lymphomas will continue to offer new therapeutic approaches, e.g. monoclonal antibodies directed against specific proteins on the surface of malignant lymphoma cells, which have already been widely applied.
  • molecular markers can be used for early detection and monitoring for relapse after or during therapy with a several-fold higher sensitivity compared to standard diagnostic techniques (Corradini et al., Leukemia 1999, 13:1691). Within the last decade numerous genes have been shown to be differentially expressed between different subtypes of ⁇ HLs.
  • CLL/SLL cases show a consistent upregulation of bcl-2 (Schena et al., Blood 1992, 79:2981).
  • Mantle cell lymphomas are typically associated with the t(l l;14)(ql3;q32) translocation, leading to overexpression of bcl-1 (Raffeld et al., Blood 1991, 78;259, Seto et al., Oncogene 1992, 7;1401).
  • follicular lymphomas The most prominent feature of follicular lymphomas is the t(14;18)(q32;q21) translocation, which is associated with a deregulation of bcl-2, a protein with anti-apoptotic properties thought to be responsible for the emergence of long-surviving memory B cells (Hockenberg et al., P ⁇ AS USA 1991, 88:6961). So far, no single marker has been shown to be sufficient for the correct diagnosis of one of the subtypes.
  • Non-Hodgkin lymphoma e.g. TCL1 (Yuille et al., Genes Chromosomes Cancer 2001, 30:336-41), pl5 and AR (Baur et al., Blood. 1999, 94:1773-81, Martinez-Delgado et al., Leukemia. 1998 12:937-41), the androgen receptor (McDonald et al., Genes Chromosomes Cancer. 2000 28:246-57), and the MyoDl gene (Taylor et al., Leukemia. 2001, 15:583-9).
  • TCL1 Yuille et al., Genes Chromosomes Cancer 2001, 30:336-41
  • pl5 and AR Bour et al., Blood. 1999, 94:1773-81, Martinez-Delgado et al., Leukemia. 1998 12:937-41
  • the androgen receptor McDonald et al
  • 5-methylcytosine is the most frequent covalent base modification in the DNA of eukaryotic cells. It plays a role, for example, in the regulation of the transcription, in genetic imprinting, and in tumorigenesis. Therefore, the identification of 5-methylcytosine as a component of genetic information is of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behaviour as cytosine. Moreover, the epigenetic information carried by 5-methylcytosine is completely lost during PCR amplification.
  • a relatively new and currently the most frequently used method for analysing DNA for 5- methylcytosine is based upon the specific reaction of bisulfite with cytosine which, upon subsequent alkaline hydrolysis, is converted to uracil which corresponds to thymidine in its base pairing behaviour.
  • 5-methylcytosine remains unmodified under these conditions. Consequently, the original DNA is converted in such a manner that methylcytosine, which originally could not be distinguished from cytosine by its hybridisation behaviour, can now be detected as the only remaining cytosine using "normal" molecular biological techniques, for example, by amplification and hybridisation or sequencing. All of these techniques are based on base pairing which can now be fully exploited.
  • Fluorescently labelled probes are often used for the scanning of immobilised DNA arrays.
  • the simple attachment of Cy3 and Cy5 dyes to the 5'-OH of the specific probe are particularly suitable for fluorescence labels.
  • the detection of the fluorescence of the hybridised probes may be carried out, for example via a confocal microscope. Cy3 and Cy5 dyes, besides many others, are commercially available.
  • Matrix Assisted Laser Desorption Ionization Mass Spectrometry is a very efficient development for the analysis of biomolecules (Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988 Oct 15;60(20):2299-301).
  • An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by a short laser pulse thus transporting the analyte molecule into the vapor phase in an unfragmented manner.
  • the analyte is ionised by collisions with matrix molecules.
  • An applied voltage accelerates the ions into a field-free flight tube. Due to their different masses, the ions are accelerated at different rates. Smaller ions reach the detector sooner than bigger ones.
  • MALDI-TOF spectrometry is excellently suited to the analysis of peptides and proteins.
  • the analysis of nucleic acids is somewhat more difficult (Gut I G, Beck S. DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Current Innovations and Future Trends. 1995, 1; 147-57).
  • the sensitivity to nucleic acids is approximately 100 times worse than to peptides and decreases disproportionally with increasing fragment size.
  • the ionisation process via the matrix is considerably less efficient.
  • the selection of the matrix plays an eminently important role.
  • Genomic DNA is obtained from DNA of cell, tissue or other test samples using standard methods. This standard methodology is found in references such as Fritsch and Maniatis eds., Molecular Cloning: A Laboratory Manual, 1989.
  • the invention provide a method for the analysis of biological samples for features associated with the development of lymphoid cell proliferative disorders , characterised in that the nucleic acid of at least one member of the group comprising MDR1, CSNK2B, EGR4, AR, CDK4, RBI, CDC25A, GPIb beta, MYOD1, CDH3, MYCL1, ELK1, ABL1, APC, BCL2, CDH1, CDKN1A, CDKN1B, CDKN2a, CDKN2B, FOS, GSTP1, HIC-1, MGMT, MLH1, MOS, MYC, PTEN, RBL2, TGFBR2, TP73, CDKN1C, GSK3B, ESR1, APAF1, BAK1, BAX and HOXA5 is/are contacted with a reagent or series of reagents capable of distinguishing between methylated and non methylated CpG dinucleotides within the genomic sequence of interest.
  • the present invention makes available a method for ascertaining genetic and/or epigenetic parameters of genomic DNA.
  • the method is for use in the improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders, more specifically by enabling the improved identification of and differentiation between subclasses of said disorder and the genetic, predisposition to said disorders.
  • the invention presents improvements over the state of the art in that it enables a highly specific classification of lymphomas, thereby allowing for improved informed treatment of patients.
  • the present invention makes available methods and nucleic acids that allow the differentiation between mantle cell lymphoma (hereinafter referred to as MCL), diffuse B-cell lymphoma (hereinafter referred to as DBCL), follicular lymphoma (FL), and chronic lymphocytic leukaemia, also known as small lymphocytic lymphoma (hereinafter referred to as CLL or SLL).
  • MCL mantle cell lymphoma
  • DBCL diffuse B-cell lymphoma
  • FL follicular lymphoma
  • CLL chronic lymphocytic leukaemia
  • the method enables the analysis of cytosine methylations and single nucleotide polymorphisms.
  • the method comprises the following steps:
  • the genomic DNA sample In the first step of the method the genomic DNA sample must be isolated from tissue or cellular sources.
  • tissue or cellular sources may include lymphoid tissue samples, cell lines, histological slides, body fluids, or tissue embedded in paraffin. Extraction may be by means that are standard to one skilled in the art, these include the use of detergent lysates, sonification and vor- texing with glass beads. Once the nucleic acids have been extracted the genomic double stranded DNA is used in the analysis.
  • the DNA may be cleaved prior to the next step of the method, this may be by any means standard in the state of the art, in particular, but not limited to, with restriction endonucleases.
  • the genomic DNA sample is treated in such a manner that cytosine bases which are unmethylated at the 5 '-position are converted to uracil, thymine, or another base which is dissimilar to cytosine in terms of hybridisation behaviour. This will be understood as 'pretreatment' hereinafter.
  • the above described treatment of genomic DNA is preferably carried out with bisulfite (sul- fite, disulfite) and subsequent alkaline hydrolysis which results in a conversion of non- methylated cytosine nucleobases to uracil or to another base which is dissimilar to cytosine in terms of base pairing behaviour.
  • bisulfite solution is used for the reaction, then an addition takes place at the non-methylated cytosine bases.
  • a denaturating reagent or solvent as well as a radical interceptor must be present.
  • a subsequent alkaline hydrolysis then gives rise to the conversion of non-methylated cytosine nucleobases to uracil.
  • the chemically converted DNA is then used for the detection of methylated cytosines.
  • Fragments of the pretreated DNA are amplified, using sets of primer oligonucleotides according to SEQ ID NO: 213 to SEQ ID NO: 290, and a, preferably heat-stable, polymerase. Because of statistical and practical considerations, preferably more than ten different fragments having a length of 100 - 2000 base pairs are amplified.
  • the amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Usually, the amplification is carried out by means of a polymerase chain reaction (PCR).
  • the method may also be enabled by the use of alternative primers, the design of such primers is obvious to one skilled in the art.
  • These should include at least two oligonucleotides whose sequences are each reverse complementary or identical to an at least 18 base-pair long segment of the base sequences specified in the appendix (SEQ ID NO: 61 through SEQ ID NO: 212).
  • Said primer oligonucleotides are preferably characterised in that they do not contain any CpG dinucleotides.
  • the sequence of said primer oligonucleotides are designed so as to selectively anneal to and amplify, only the lymphoid tissue specific DNA of interest, thereby minimising the amplification of background or non relevant DNA.
  • background DNA is taken to mean genomic DNA which does not have a relevant tissue specific methylation pattern, in this case, the relevant tissue being lymphoid, both healthy and diseased.
  • At least one primer oligonucleotide is bound to a solid phase during amplification.
  • the different oligonucleotide and/or PNA- oligomer sequences can be arranged on a plane solid phase in the form of a rectangular or hexagonal lattice, the solid phase surface preferably being composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold, it being possible for other materials such as nitrocellulose or plastics to be used as well.
  • the fragments obtained by means of the amplification can carry a directly or indirectly detectable label.
  • the detection may be carried out and visualised by means of matrix assisted laser desorption/ionisation mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
  • MALDI matrix assisted laser desorption/ionisation mass spectrometry
  • ESI electron spray mass spectrometry
  • the amplificates obtained in the second step of the method are subsequently hybridised to an array or a set of oligonucleotides and/or PNA probes.
  • the hybridisation takes place in the manner described as follows.
  • the set of probes used during the hybridisation is preferably composed of at least 10 oligonucleotides or PNA-oligomers.
  • the amplificates serve as probes which hybridise to oligonucleotides previously bonded to a solid phase.
  • the oligonucleotides are taken from the group comprising SEQ ID NO: 291 to SEQ ID NO: 602.
  • the oligonucleotides are taken from the group comprising SEQ ID NO: 559 to SEQ ID NO: 602. The non-hybridised fragments are subsequently removed.
  • Said oligonucleotides contain at least one base sequence having a length of 10 nucleotides which is reverse complementary or identical to a segment of the base sequences specified in the appendix, the segment containing at least one CpG or TpG dinucleotide.
  • CpG dinucleotide or in the case of TpG, the thiamine, is the 5 tn to 9 tn nucleotide from the 5'- end of the 10-mer.
  • One oligonucleotide exists for each CpG or TpG dinucleotide.
  • the non-hybridised amplificates are removed.
  • the hybridised amplificates are detected.
  • labels attached to the amplificates are identifiable at each position of the solid phase at which an oligonucleotide sequence is located.
  • the labels of the amplificates are fluorescence labels, radionuclides, or detachable molecule fragments having a typical mass which can be detected in a mass spectrometer.
  • the mass spectrometer is preferred for the detection of the amplificates, fragments of the amplificates or of probes which are complementary to the amplificates, it being possible for the detection to be carried out and visualised by means of matrix assisted laser desorption/ionisation mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
  • MALDI matrix assisted laser desorption/ionisation mass spectrometry
  • ESI electron spray mass spectrometry
  • the produced fragments may have a single positive or negative net charge for better detectability in the mass spectrometer.
  • the aforementioned method is preferably used for ascertaining genetic and/or epigenetic parameters of genomic DNA.
  • the invention further provides the modified DNA of genes MDR1, CSNK2B, EGR4, AR, CDK4, RBI, CDC25A, GPIb beta, MYOD1, CDH3, MYCL1, ELK1, ABL1, APC, BCL2, CDH1, CDKN1A, CDKN1B, CDKN2a, CDKN2B, FOS, GSTP1, HIC-1, MGMT, MLH1, MOS, MYC, PTEN, RBL2, TGFBR2, TP73, CDKN1C, GSK3B, ESR1, APAF1, BAK1, BAX and HOXA5 as well as oligonucleotides and/or PNA- oligomers for detecting cytosine methylations within said genes.
  • the present invention is based on the discovery that genetic and epigenetic parameters and, in particular, the cytosine methylation patterns of genomic DNA are particularly suitable for improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders. Furthermore, the invention enables the differentiation between different subclasses of lymphomas or detection of a predisposition to lymphomas.
  • the nucleic acids according to the present invention can be used for the analysis of genetic and/or epigenetic parameters of genomic DNA.
  • nucleic acid containing a sequence of at least 18 bases in length of the pretreated genomic DNA according to one of SEQ ID NO: 61 through SEQ ID NO: 212 and sequences complementary thereto.
  • the modified nucleic acid could heretofore not be connected with the ascertainment of disease relevant genetic and epigenetic parameters.
  • the object of the present invention is further achieved by an oligonucleotide or oligomer for the analysis of pretreated DNA, for detecting the genomic cytosine methylation state, said oligonucleotide containing at least one base sequence having a length of at least 10 nucleo- tides which hybridises to a pretreated genomic DNA according to SEQ ID NO: 61 through SEQ ID NO: 212.
  • the oligomer probes according to the present invention constitute important and effective tools which, for the first time, make it possible to ascertain specific genetic and epigenetic parameters during the analysis of biological samples for features associated with the development of lymphoid cell proliferative disorders.
  • Said oligonucleotides allow the improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders and detection of the predisposition to said disorders. Furthermore, they allow the differentiation of different subclasses of lymphomas.
  • the base sequence of the oligomers preferably contains at least one CpG or TpG dinucleotide.
  • the probes may also exist in the form of a PNA (peptide nucleic acid) which has particularly preferred pairing properties.
  • oligonucleotides according to the present invention in which the cytosine of the CpG dinucleotide is the 5 - 9th nucleotide from the 5 '-end of the 13-mer; in the case of PNA- oligomers, it is preferred for the cytosine of the CpG dinucleotide to be the 4 t ⁇ - 6 ⁇ nucleotide from the 5 '-end of the 9-mer.
  • the oligomers according to the present invention are normally used in so called “sets” which contain at least one oligomer for each of the CpG dinucleotides within SEQ ID NO: 61 through SEQ ID NO: 212.
  • a set which contains at least one oligomer for each of the CpG dinucleotides, from SEQ ID NO: 291 through SEQ ID NO: 602 .
  • a set comprising SEQ ID NO: 559 to SEQ ID NO: 602.
  • oligonucleotide is bound to a solid phase. It is further preferred that all the oligonucleotides of one set are bound to a solid phase.
  • the present invention moreover relates to a set of at least 10 n (Oligonucleotides and/or PNA- oligomers) used for detecting the cytosine methylation state of genomic DNA using treated versions of said genomic DNA (according to SEQ ID NO: 61 through SEQ ID NO: 212 and sequences complementary thereto).
  • These probes enable improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders progression. In particular they enable the differentiation between different sub classes of lymphoid cell proliferative disorders and the detection of a predisposition to said disorders.
  • the set comprises SEQ ID NO: 39 to SEQ ID NO: 602.
  • the set of oligomers may also be used for detecting single nucleotide polymorphisms (SNPs) using pretreated genomic DNA according to one of SEQ ID NO: 61 through SEQ ID NO: 212.
  • SNPs single nucleotide polymorphisms
  • an arrangement of different oligonucleotides and/or PNA-oligomers made available by the present invention is present in a manner that it is likewise bound to a solid phase.
  • This array of different oligonucleotide- and/or PNA-oligomer sequences can be characterised in that it is arranged on the solid phase in the form of a rectangular or hexagonal lattice.
  • the solid phase surface is preferably composed of silicon, glass, polystyrene, aluminium, steel, iron, copper, nickel, silver, or gold.
  • nitrocellulose as well as plastics such as nylon which can exist in the form of pellets or also as resin matrices are suitable alternatives.
  • a further subject matter of the present invention is a method for manufacturing an array fixed to a carrier material for the improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders, the differentiation between different subclasses of lymphomas and/or detection of the predisposition to lymphoid cell proliferative disorders.
  • at least one oligomer according to the present invention is coupled to a solid phase.
  • Methods for manufacturing such arrays are known, for example, from US Patent 5,744,305 by means of solid-phase chemistry and photolabile protecting groups.
  • a further subject matter of the present invention relates to a DNA chip for the improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders. Furthermore the DNA chip enables detection of the predisposition to lymphoid cell proliferative disorders and the differentiation between different subclasses of lymphomas.
  • the DNA chip contains at least one nucleic acid according to the present invention. DNA chips are known, for example, in US Patent 5,837,832.
  • kits which may be composed, for example, of a bisulfite-containing reagent, a set of primer oligonucleotides containing at least two oligonucleotides whose sequences in each case correspond or are complementary to a 18 base long segment of the base sequences specified in the appendix (SEQ ID NO: 61 through SEQ ID NO: 212), oligonucleotides and/or PNA-oligomers as well as instructions for carrying out and evaluating the described method.
  • a kit along the lines of the present invention can also contain only part of the aforementioned components.
  • the oligomers according to the present invention or arrays thereof as well as a kit according to the present invention are intended to be used for the improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders. Furthermore the use of said inventions extends to the differentiation between different subclasses of lymphomas and detection of the predisposition to lymphoid cell proliferative disorders.
  • the method is preferably used for the analysis of important genetic and/or epigenetic parameters within genomic DNA, in particular for use in improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders, detection of the predisposition to said disorders and the differentiation between subclasses of said disorders.
  • the methods according to the present invention are used, for example, for improved diagnosis, treatment and monitoring of lymphoid cell proliferative disorders progression, detection of the predisposition to said disorders and the differentiation between subclasses of said disorders.
  • a further embodiment of the invention is a method for the analysis of the methylation status of genomic DNA without the need for pre-treatment.
  • the genomic DNA sample In the first step of the method the genomic DNA sample must be isolated from tissue or cellular sources. Such sources may include cell lines, histological slides, body fluids, or tissue embedded in paraffin. Extraction may be by means that are standard to one skilled in the art, these include the use of detergent lysates, sonification and vortexing with glass beads. Once the nucleic acids have been extracted the genomic double stranded DNA is used in the analysis.
  • the DNA may be cleaved prior to the treatment, this may be any means standard in the state of the art, in particular with restriction endonucleases.
  • the DNA is then digested with one or more methylation sensitive restriction enzymes. The digestion is carried out such that hydrolysis of the DNA at the restriction site is informative of the methylation status of a specific CpG dinucleotide.
  • the restriction fragments are amplified. In a preferred embodiment this is carried out using a polymerase chain reaction.
  • the amplificates are detected.
  • the detection may be by any means standard in the art, for example, but not limited to, gel electrophoresis analysis, hybridisation analysis, incorporation of detectable tags within the PCR products, DNA array analysis, MALDI or ESI analysis.
  • the present invention moreover relates to the diagnosis and/or prognosis of events which are disadvantageous or relevant to patients or individuals in which important genetic and/or epigenetic parameters within genomic DNA, said parameters obtained by means of the present invention may be compared to another set of genetic and/or epigenetic parameters, the differences serving as the basis for the diagnosis and/or prognosis of events which are disadvantageous or relevant to patients or individuals.
  • genes that form the basis of the present invention can be used to form a "gene panel", i. e. a collection that comprises the particular genes of the present invention and/or their respective informative methylation sites.
  • a gene panel i. e. a collection that comprises the particular genes of the present invention and/or their respective informative methylation sites.
  • the formation of gene panels enables a fast and specific analysis of the disorders related therewith.
  • the gene panels as used and described in the present invention can be employed for the diagnosis, treatment and monitoring and the analysis of a predisposition for the disorders described herein with surprisingly high efficiencies.
  • the use of a plurality of CpG-sites from a divers array of genes that specifically regulate cell proliferative disorders allows for a relatively high degree of sensitivity and specificity in comparison with tools that comprise singular gene-diagnosis- and gene- determination-instruments.
  • the panel as described herein in contrast to other methods available, can be designed to fit to a more specific use in the analyses of multiple disorders that are all specifically caused by cell proliferative disorders.
  • hybridisation is to be understood as a bond of an oligonucleotide to a completely complementary sequence along the lines of the Watson- Crick base pairings in the sample DNA, forming a duplex structure.
  • mutations are mutations and polymorphisms of genomic DNA and sequences further required for their regulation.
  • mutations are, in particular, insertions, deletions, point mutations, inversions and polymorphisms and, particularly preferred, SNPs (single nucleotide polymorphisms).
  • epigenetic parameters are, in particular, cytosine methylations and further modifications of DNA bases of genomic DNA and sequences further required for their regulation.
  • Further epigenetic parameters include, for example, the acetyla- tion of histones which, cannot be directly analysed using the described method but which, in turn, correlates with the DNA methylation.
  • Figure 1 shows the source of all samples that were used in the analyses described in examples 1 and 2.
  • Columnough1 indicates the sample number, which is applicable also for figures 2 to 7.
  • Columnough2 shows the diagnosis of the disorder, wherein MCL is mantle cell-lymphoma, FL means follicular lymphoma and CLL/SLL means chronic lymphocytic leukaemia/small lymphocytic lymphoma, and DLBCL means diffuse large B-cell-lymphoma.
  • Column Legend3 indicates the age of the patient, from which the sample was obtained.
  • Column Legend4" indicates the gender age of the patient, from which the sample was obtained.
  • Figure 2 shows the differentiation of MCL, DLBCL and CLL/SLL from FL I and II, according to example 2.
  • the markers on the left side of the plot are gene- and CpG-identifiers, these correspond to the ones of table 3.
  • the markers on the right side indicate the significance (p- value, T-test) of the difference between the means of the two groups.
  • Each row correspond to a single CpG and each column to the methylation grade of a sample.
  • the CpGs are sorted according to their contribution for the distinctiveness of the differential diagnosis of the two lymphoma, with increasing contribution from top to bottom. Black indicates a complete methylation of a particular CpG-position, white indicates no methylation at the particular position, with graduations of methylation which are indicated in grey, from light (small portion of methylation) to dark (large portion of methylation).
  • Figure 3 shows the differentiation of MCL, DLBCL and CLL/SLL from FI I and II (only male samples), according to example 2.
  • the markers on the left side of the plot are gene- and CpG- identifiers, these correspond to the ones of table 4.
  • the markers on the right side indicate the significance (p-value, T-test) of the difference between the means of the two groups.
  • Each row correspond to a single CpG and each column to the methylation grade of a sample.
  • the CpGs are sorted according to their contribution for the distinctiveness of the differential diagnosis of the two lymphoma, with increasing contribution from top to bottom.
  • Black indicates a complete methylation of a particular CpG-position
  • white indicates no methylation at the particular position, with graduations of methylation which are indicated in grey, from light (small portion of methylation) to dark (large portion of methylation).
  • Figure 4 shows the differentiation of MCL from follicular lymphoma, according to example 2.
  • the markers on the left side of the plot are gene- and CpG-identifiers, these correspond to the ones of table 5.
  • the markers on the right side indicate the significance (p-value, T-test) of the difference between the means of the two groups.
  • Each row correspond to a single CpG and each column to the methylation grade of a sample.
  • the CpGs are sorted according to their contribution for the distinctiveness of the differential diagnosis of the two lymphoma, with increasing contribution from top to bottom. Black indicates a complete methylation of a particular CpG-position, white indicates no methylation at the particular position, with graduations of methylation which are indicated in grey, from light (small portion of methylation) to dark (large portion of methylation).
  • Figure 5 shows the differentiation of MCL from from follicular lymphoma (only male samples), according to example 2.
  • the markers on the left side of the plot are gene- and CpG- identifiers, these correspond to the ones of table 6.
  • the markers on the right side indicate the significance (p-value, T-test) of the difference between the means of the two groups.
  • Each row correspond to a single CpG and each column to the methylation grade of a sample.
  • the CpGs are sorted according to their contribution for the distinctiveness of the differential diagnosis of the two lymphoma, with increasing contribution from top to bottom. Black indicates a complete methylation of a particular CpG-position, white indicates no methylation at the particular position, with graduations of methylation which are indicated in grey, from light (small portion of methylation) to dark (large portion of methylation).
  • Figure 6 shows the differentiation of FL from CLL/SLL, according to example 2-.
  • the markers on the left side of the plot are gene- and CpG-identifiers, these correspond to the ones of table 7.
  • the markers on the right side indicate the significance (p-value, T-test) of the difference between the means of the two groups.
  • Each row correspond to a single CpG and each column to the methylation grade of a sample.
  • the CpGs are sorted according to their contribution for the distinctiveness of the differential diagnosis of the two lymphoma, with increasing contribution from top to bottom.
  • Black indicates a complete methylation of a particular CpG-position
  • white indicates no methylation at the particular position, with graduations of methylation which are indicated in grey, from light (small portion of methylation) to dark (large portion of methylation).
  • Figure 7 shows the differentiation of FL from CLL/SLL (only male samples), according to example 2.
  • the markers on the left side of the plot are gene- and CpG-identifiers, these correspond to the ones of table 8.
  • the markers on the right side indicate the significance (p-value, T-test) of the difference between the means of the two groups.
  • Each row correspond to a single CpG and each column to the methylation grade of a sample.
  • the CpGs are sorted according to their contribution for the distinctiveness of the differential diagnosis of the two lymphoma, with increasing contribution from top to bottom. Black indicates a complete methylation of a particular CpG-position, white indicates no methylation at the particular position, with graduations of methylation which are indicated in grey, from light (small portion of methylation) to dark (large portion of methylation).
  • SEQ ID NO: 1 to SEQ ID NO: 38 represent 5' and/or regulatory regions of the genomic DNA of genes MDR1, CSNK2B, EGR4, AR, CDK4, RBI, CDC25A, GPIb beta, MYOD1, CDH3, MYCL1, ELK1, ABL1, APC, BCL2, CDH1, CDKN1A, CDKN1B, CDKN2a, CDKN2B, FOS, GSTP1, HIC-1, MGMT, MLH1, MOS, MYC, PTEN, RBL2, TGFBR2, TP73, CDKN1C, GSK3B, ESR1, APAF1, BAK1, BAX and HOXA5.
  • sequences are derived from Genbank and will be taken to include all minor variations of the sequence material which are currently unforeseen, for example, but not limited to, minor deletions and SNPs.
  • SEQ ID NO: 61 to SEQ ID NO: 212 exhibit the pretreated sequence of DNA derived from genes MDR1, CSNK2B, EGR4, AR, CDK4, RBI, CDC25A, GPIb beta, MYOD1, CDH3, MYCL1, ELK1, ABL1, APC, BCL2, CDH1, CDKN1A, CDKN1B, CDKN2a, CDKN2B, FOS, GSTP1, HIC-1, MGMT, MLH1, MOS, MYC, PTEN, RBL2, TGFBR2, TP73, CDKN1C, GSK3B, ESR1, APAF1, BAK1, BAX and HOXA5.
  • sequences will be taken to include all minor variations of the sequence material which are currently unforeseen, for example, but not limited to, minor deletions and SNPs.
  • SEQ ID NO: 213 to SEQ ID NO:290 exhibit the sequence of primer oligonucleotides for the amplification of pretreated DNA according to Sequence IDs 61 to 212.
  • SEQ ID NO: 291 to SEQ ID NO: 602 exhibit the sequence of oligomers which are useful for the analysis of CpG positions within genomic DNA according to SEQ ID NO: 1 to SEQ ID NO: 38.
  • SEQ ID NO: 559 to SEQ ID NO: 602 exhibit the sequence of oligomers which are useful for the analysis of CpG positions within genomic DNA according to SEQ ID NO: 1 to SEQ ID NO: 38.
  • the genomic DNA was isolated from the cell samples using the Wizzard kit from (Promega).
  • the isolated genomic DNA from the samples are treated using a bisulfite solution (hydrogen sulfite, disulfite).
  • the treatment is such that all non methylated cytosines within the sample are converted to thiamine, conversely 5-methylated cytosines within the sample remain unmodified.
  • the treated nucleic acids were then amplified using multiplex PCRs, amplifying 8 fragments per reaction with Cy5 fluorescently labelled primers.
  • PCR primers used are described in Table 1. PCR conditions were as follows.
  • each multiplex PCR product was diluted in 10 x Ssarc buffer (10 x Ssarc:230 ml 20 x SSC, 180 ml sodium lauryl sarcosinate solution 20% , dilute to 1000 ml with dH2O).
  • the reaction mixture was then hybridised to the detection oligonucleotides as follows. De- naturation at 95°C, cooling down to 10 °C, hybridisation at 42°C overnight followed by washing with 10 x Ssarc and dH 2 O at 42°C.
  • Fluorescent signals from each hybridised oligonucleotide were detected using genepix® scanner and software. Ratios for the two signals (from the CG oligonucleotide and the TG oligonucleotide used to analyse each CpG position) were calculated based on comparison of intensity of the fluorescent signals.
  • the information is then sorted into a ranked matrix (as shown in Figures 2 to 7) according to CpG methylation differences between the two classes of tissues, using an algorithim.
  • the most significant CpG positions are at the bottom of the matrix with significance decreasing towards the top.
  • Black indicates total methylation at a given CpG position, white represents no methylation at the particular position, with degrees of methylation represented in gray, from light (low proportion of methylation) to dark (high proportion of methylation).
  • Each row represents one specific CpG position within a gene and each column shows the methylation profile for the different CpGs for one sample.
  • the SNM (as discussed by F. Model,P. Adorjan,A. Olek,C. Piepenbrock, Feature selection for D ⁇ A methylation based cancer classification. Bioinformatics. 2001 Jun; 17 Suppl 1 :S 157-64) constructs an optimal discriminant between two classes of given training samples. In this case each sample is described by the methylation patterns (CG/TG ratios) at the investigated CpG sites.
  • the SVM was trained on a subset of samples of each class, which were presented with the diagnosis attached. Independent test samples, which were not shown to the SNM before were then presented to evaluate, if the diagnosis can be predicted correctly based on the predictor created in the training round.
  • the first group is composed of 42 samples of MCL, DLBCL & CLL/SLL from both sexes, which are compared to 38 samples of FL I and II.
  • the p-value shows a clear discrimination between the two groups, 9 CpG positions from 7 distinct genes allow discrimination between the 2 groups (p ⁇ 0.05).
  • the crossvalidation accuracy performed by a SNM is calculated as 75.8% with a standard deviation of 2.8%.
  • the significant genes and detection oligonucleotides are shown below in Table 3.
  • the analysis may be refined by performing the comparison between members of the same sex.
  • the comparison of male samples only (Figure 3) increased the accuracy to 80.4% with a standard deviation of 2.9%.
  • the significant genes and detection oligonucleotides are shown in Table 4.
  • a fragment of the gene CDKNIC (Seq ID NO: 32) was PCR amplified using primers CATTTGGGGAGGCAGATA (Seq ID NO: xx) and TGTCCTTGAGAGGTGCGA (Seq ID NO: yy).
  • the resultant fragment (262 bp in length) contained an informative CpG at position 63.
  • the amplificate DNA was digested with the restriction endonuclease Eael, recogniton site YGGCCR. Hydrolysis by said endonuclease is blocked by methylation of the CpG at position 106 of the amplificate. The digest was used as a control.
  • gene fragments were amplified by PCR performing a first denaturation step for 14 min at 96 °C, followed by 30 - 45 cycles (step 2: 60 sec at 96°C, step 3: 45 sec at 52 °C , step 4: 75 sec at 72 °C) and a subsequent final elongation of 10 min at 72 °C.
  • step 2 60 sec at 96°C
  • step 3 45 sec at 52 °C
  • step 4 75 sec at 72 °C
  • the presence of PCR products was analysed by agarose gel electrophore- sis.
  • PCR products were detectable with Eael hydrolysed DNA isolated from upmethylated tissue, when step 2 to step 4 of the cycle program were repeated 34, 37, 39, 42 and 45 fold. In con- trast significant levels of PCR products were only detectable with Eael hydrolysed DNA isolated from downmethylated (and the control sample) when step 2 to step 4 of the cycle program were repeated 42 and 45 fold.

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WO2007137597A1 (en) * 2006-05-26 2007-12-06 Cnr Consiglio Nazionale Delle Ricerche Tests for detection of hot spot mutations and methylation of retinoblastoma-like 2 gene (rbl2) as diagnostic and prognostic markers of tumors
US20150259747A1 (en) 2007-03-29 2015-09-17 Vanda Pharmaceuticals Inc. Method of predicting a predisposition to qt prolongation
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EP2481810A1 (de) 2005-04-15 2012-08-01 Epigenomics AG Verfahren zur Bereitstellung von DNA-Fragmenten, die von einer dezentralen Probe abgeleitet sind

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