WO2009108917A2 - Marqueurs pour la détection améliorée du cancer du sein - Google Patents

Marqueurs pour la détection améliorée du cancer du sein Download PDF

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WO2009108917A2
WO2009108917A2 PCT/US2009/035654 US2009035654W WO2009108917A2 WO 2009108917 A2 WO2009108917 A2 WO 2009108917A2 US 2009035654 W US2009035654 W US 2009035654W WO 2009108917 A2 WO2009108917 A2 WO 2009108917A2
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seq
gene
cancer
methylation
genes
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WO2009108917A3 (fr
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James Herman
Nita Ahuja
Kornel E. Scheubel
Leslie Cope
Valerie Deregowski
Wim Van Criekinge
Stephen Baylin
Leander Van Neste
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MDxHealth SA
Johns Hopkins University
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OncoMethylome Sciences SA
Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates generally to cancer diagnosis and therapeutics.
  • it relates to the identification of a cell proliferative disorder of breast by determining aberrant DNA methylation patterns of particular genes in breast cancer and pre-cancer.
  • Breast cancer is one of the most significant health concerns. Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of cancer death in women. One in eight women (13% of women) will develop breast cancer during her lifetime. Additionally, a small number of men are diagnosed with or die from breast cancer.
  • tumor suppressor function leads to the initiation and progression of human cancer.
  • Inactivation of tumor suppressor genes can result from both genetic mechanisms such as mutation and epigenetic mechanisms such as DNA hypermethylation (Ponder B.A, 2001; Herman et al., 2003).
  • up-regulation of expression of a number of genes e.g. MAGE-Al
  • Identification of these genes provides insight into the biological processes underlying oncogenesis and is useful for developing new therapeutic and diagnostic modalities.
  • Epigenetic silencing is a second mechanism by which abnormal gene inactivation can occur in cancer.
  • a predominant mode of epigenetic alteration in cancer is gene silencing via CpG island promoter hypermethylation (henceforth called hypermethylation).
  • Hypermethylation acts by recruiting methyl-cytosine-binding proteins and histone deacetylases, which in a coordinated fashion, modify nucleosomes to form transcriptionally repressive chromatin (Busslinger et al., 1983; Nan et al., 1998).
  • Repressive histone marks such as methylation of lysine-9 on histone 3 (H3K9) may initiate and help maintain this state of repression (Jenuwein et ah, 2006; Barski et ah, 2007). This results in the activation of many oncogenes and silencing of tumor suppressors to promote proliferation of abnormal cells.
  • hypomethylation of a gene may lead to aberrant expression of certain antigens in a wide variety of tumors. These epigenetic abnormalities (hyper- and hypomethylation) could cooperate with genetic alterations to effect aberrant gene function that results in cancer.
  • DNA methylation markers have been evaluated as potential genetic markers for detection of cancer because they offer certain advantages when compared to mutation markers: DNA hypermethylation appears to be an early event in the etiology of carcinogenesis and appears to precede apparent malignancy in many cases (Esteller et ah, 2001). The methylation profile is in many cases tissue- and tumor-type specific and is also preserved in purified isolated DNA. In addition, methylation markers may serve for predictive purposes as they often reflect the sensitivity to therapy or duration of patient survival. DNA hypermethylation is also prevalent in the highly aggressive HER2/Neu-positive breast cancers.
  • the present invention is based on the finding that several genes are newly identified as being differentially methylated in breast cancers. This information is useful for breast cancer screening, risk-assessment, prognosis, disease identification, disease staging and identification of therapeutic targets. The identification of new genes that are methylated in breast cancer allows accurate and effective early diagnostic assays, methylation profiling using multiple genes and identification of new targets for therapeutic intervention.
  • the invention provides for a method for identifying breast cancer or its precursor, or predisposition to breast cancer.
  • Epigenetic modification is detected in a test sample containing breast cells or nucleic acids from breast cells.
  • the epigenetic modification is of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK
  • the epigenetic modification may be detected by determining the state of methylation of one or more nucleic acids from a subject.
  • a difference in the state of methylation of one or more nucleic acids in a test sample of the subject compared to the state of methylation of one or more nucleic acids from a subject not having a cellular proliferative disorder of breast tissue is indicative of the test sample containing breast cells that are neoplastic, precursors to neoplastic, or predisposed to neoplasia, or as containing nucleic acids from cells that are neoplastic, precursor to neoplastic, or predisposed to neoplasia.
  • the invention also provides for a method for determining the histopathological stage of breast cancer, comprising detecting epigenetic modification of at least one gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2;
  • a method is provided of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer.
  • An epigenetically silenced gene is determined in a cell.
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7
  • a polypeptide encoded by the epigenetic silenced gene in the cell is restored by contacting the cell with one or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor. Unregulated growth of the cell is thereby reduced or inhibited.
  • reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer is accomplished by introduction of a polynucleotide encoding a polypeptide into the cell.
  • the polypeptide is encoded by said gene.
  • the polypeptide is expressed in the cell thereby restoring expression of the polypeptide in the cell.
  • a cancer cell in the patient is determined to have an epigenetic silenced gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKN
  • One or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor is administered to the patient in sufficient amounts to restore expression of the epigenetic silenced gene in the patient's cancer cells.
  • HDAC histone deacetylase
  • a cancer cell in the patient is determined to have an epigenetic silenced gene selected from those shown in HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA
  • a polynucleotide encoding a polypeptide is administered to the patient.
  • the polypeptide is encoded by the epigenetic silenced gene.
  • the polypeptide is expressed in the patient's tumor thereby restoring expression of the polypeptide in the cancer.
  • a method for selecting a therapeutic strategy for treating a cancer patient.
  • a gene is identified whose expression in cancer cells of the patient is reactivated by a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, or a histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A
  • kits for assessing methylation in a test sample comprises at least the following reagents: a reagent that (a) modifies methylated cytosine residues but not non-methylated cytosine residues, or that (b) modifies non- methylated cytosine residues but not methylated cytosine residues; and a pair of oligonucleotide primers that specifically hybridizes under amplification conditions to a region of a gene within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp of said gene's transcription start site, said gene being selected from those shown in HOXDl; SLC2A14; NEFH; HOXA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl;
  • the invention provides oligonucleotide primers and/or probes and their sequences for use in the methods and assays of the invention.
  • FIG. 1A-1B Strategy used to identify common targets of mutation and promoter hypermethylation.
  • FIG. IA Large-scale sequencing of breast and colon cancers identified 189 genes cancer genes (CAN genes) as reported by Sj ⁇ blom et al. Candidate hypermethylated genes were identified via expression microarray analysis. Genes without promoter CpG islands were excluded resulting in 36 target genes.
  • FIG. IB Frequency of promoter methylation in the 36 CAN genes that are subject to hypermethylation. X-axis denotes percent methylation. Methylation status was determined in the 6 colorectal cancer cell lines and 11 breast cancer cell lines as indicated in the example section.
  • FIG. 2 Primers used for Methylation-specific PCR of the 36 target genes. (SEQ ID NO: 1305-1448, respectively)
  • FIG. 3. Promoter methylation frequency in primary colon and breast tumors: 18 out of the 36 common target genes methylated in either breast cancer cell lines, colon cancer cell lines or both, showed cancer-specific methylation. Y-axis denotes percent methylation.
  • FIG. 4 Mutation and methylation plot of the 18 genes showing cancer-specific methylation..
  • FIG. 5 Primary breast tumor grade or stage correlation. Y-axis denotes percent methylation for the different tumor stages/grades.
  • FIG. 6A-6B FIG. 6A. Position of the different primers relative to the TSS.
  • FIG. 6B Sequences of the different primer sets, sense (SEQ ID NO: 1273-1280, respectively) and antisense (SEQ ID NO: 1281-1288, respectively) and amplicons. (SEQ ID NO: 1289-1296, respectively)
  • Fig. 7 Methylation frequency SLC2A14, NEFH and TF. Percentage methylation (Y-axis) is shown for each marker (X-axis)
  • microarray screen identifies hypermethylated genes that are re-expressed following treatment with the DNA methyltransferase (DNMT) inhibitor 5-deoxyazacytidine (DAC) but not following treatment with the HDAC I/II inhibitor trichostatin A (TSA) alone.
  • DNMT DNA methyltransferase
  • DAC 5-deoxyazacytidine
  • TSA HDAC I/II inhibitor trichostatin A
  • a further substantial fraction of the cancer gene promoter CpG island DNA hypermethylome is defined through comparison of the hypermethylome genes found by the micro human transcriptome microarray screen and their specificity for certain cell lines such as metastatic cell lines but not for non-metastatic cell lines. Resulting methylation markers are useful in identifying cancers with high metastatic potential and may provide guidance to a preferred method of treatment. For instance, for cancers with high metastatic potential it may be indicated to have radiotherapy treatment following the resection of the cancer.
  • genes include: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34
  • Detection of epigenetic silencing of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of such genes can be used as an indication of cancer or pre-cancer or risk of developing cancer.
  • accession numbers corresponding to the listed genes can be found at the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health website.
  • silencing when used in reference to a gene, means that the gene is not being transcribed, or is being transcribed at a reduced level when compared to the transcription level of the same gene in a corresponding control cell, e.g., a healthy cell, a non-cancerous cell, a non-infected cell.
  • a cell comprising the silenced gene has reduced levels of, or completely lacks, a polypeptide encoded by the gene, i.e. the gene product, such that any function normally attributed to the gene product in the cell is reduced of absent.
  • Assays that recognize only a single marker of the listed markers may be of limited predictive value.
  • a multiplexed analytical approach is particularly suitable for cancer diagnostics, and thus a panel approach may be more consistent with the heterogeneous nature of cancer. Combinations of markers may be applied as desired. Multiplexing in connection to epigenetic silencing of genes has been described in WO05042713 and such methods are applicable in the present invention.
  • the cancer to be identified is preferably a breast cancer.
  • breast cancer There are two major groups of breast cancer: noninvasive carcinoma and invasive carcinoma.
  • the noninvasive carcinomas include lobular carcinoma in situ and ductal carcinoma in situ.
  • breast cancers often grow through the basement membrane and roughly 95% of all breast cancers are infiltrating or invasive carcinomas.
  • the most common type of invasive breast cancer (about 75%) is invasive ductal carcinoma, arising in the milk ducts and spreading through the duct walls.
  • Invasive lobular carcinoma originates in the milk glands and accounts for 10 to 15% of invasive breast cancers.
  • invasive breast cancer Less common types of invasive breast cancer include the following: inflammatory breast cancer, Paget's disease of the nipple, medullary carcinoma, mucinous carcinoma, phyllodes tumor, and tubular carcinoma. Rarely (about 1%), sarcomas (cancer of the connective tissue) develop in the breasts. Individuals may develop one, the other, or a combination of invasive and noninvasive breast cancer.
  • Epigenetic silencing of a gene can be determined by any method known in the art. One method is to determine that a gene which is expressed in normal cells or other control cells is less expressed or not expressed in tumor cells. This method does not, on its own, however, indicate that the silencing is epigenetic, as the mechanism of the silencing could be genetic, for example, by somatic mutation.
  • One method to determine that the silencing is epigenetic is to treat with a reagent, such as DAC (5'-deazacytidine), or with a reagent which changes the histone acetylation status of cellular DNA or any other treatment affecting epigenetic mechanisms present in cells, and observe that the silencing is reversed, i.e., that the expression of the gene is reactivated or restored.
  • a reagent such as DAC (5'-deazacytidine)
  • Another means to detect epigenetic silencing is to determine the presence of methylated CpG dinucleotide motifs in the silenced gene.
  • Methylation of a CpG island at a promoter usually prevents expression of the gene.
  • the islands can surround the 5' region of the coding region of the gene as well as the 3' region of the coding region.
  • CpG islands can be found in multiple regions of a nucleic acid sequence including upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g., exons), downstream of coding regions in, for example, enhancer regions, and in introns. All of these regions can be assessed to determine their methylation status.
  • these reside near the transcription start site and surround the TTS, for example, within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp.
  • RNA can also be assessed using amplification techniques, such as RT-PCR.
  • Sequencing-based methods are an alternative; these methods started with the use of expressed sequence tags (ESTs), and now include methods based on short tags, such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS).
  • SAGE serial analysis of gene expression
  • MPSS massively parallel signature sequencing
  • Differential display techniques provide another means of analyzing gene expression; this family of techniques is based on random amplification of cDNA fragments generated by restriction digestion, and bands that differ between two tissues identify cDNAs of interest.
  • Specific proteins can be assessed using any convenient method including immunoassays and immuno- cytochemistry but are not limited to that. Most such methods will employ antibodies which are specific for the particular protein or protein fragments.
  • the sequences of the mRNA (cDNA) and proteins of the markers of the present invention are known in the art and publicly available.
  • microarray refers broadly to both “DNA microrarray” and “DNA chips”, and encompasses all art-recognized solid supports and methods for affixing nucleic acid molecules thereto or synthesis of nucleic acids thereon.
  • Methylation-sensitive restriction endonucleases can be used to detect methylated CpG dinucleotide motifs. Such endonucleases may either preferentially cleave methylated recognition sites relative to non-methylated recognition sites or preferentially cleave non- methylated relative to methylated recognition sites.
  • Non limiting examples of the former are Aat II, Ace III, Ad I, AcI I, Age I, AIu I, Asc I, Ase 1, AsiS I, Ban I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrV I, BssK 1, BstB I, BstN I, Bs I, CIa I, Eae I, Eag I, Fau I, Fse I, Hha I, mPl I, HinC II, Hpa 11, Npy99 I, HpyCAIV, Kas I, Mbo I, MIu I, MapA 1 1.
  • Msp I Nae I, Nar I, Not 1, PmI I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, SfI I, Sfo I, SgrA I, Sma I SnaB I, Tsc I, Xma I, and Zra I.
  • Non limiting examples of the latter are Ace II, Ava I, BssH II, BstU I, Hpa II, Not I and Mho I.
  • chemical reagents can be used which selectively modify either the methylated or non-methylated form of CpG dinucleotide motifs. Modified products can be detected directly, or after a further reaction which creates products which are easily distinguishable. Means which detect altered size and/or charge can be used to detect modified products, including but not limited to electrophoresis, chromatography, and mass spectrometry. Examples of such chemical reagents for selective modification include hydrazine and bisulfite ions. Hydrazine-modified DNA can be treated with piperidine to cleave it. Bisulfite ion-treated DNA can be treated with alkali. Other means for detection which are reliant on specific sequences can be used, including but not limited to hybridization, amplification, sequencing, and ligase chain reaction. Combinations of such techniques can be used as is desired.
  • the principle behind mass spectrometry is the ionizing of nucleic acids and separating them according to their mass to charge ratio. Similar to electrophoresis, one can use mass spectrometry to detect a specific nucleic acid that was created in an experiment to determine methylation. See Tost, J. et al. Analysis and accurate quantification of CpG methylation by MALDI mass spectrometry.
  • chromatography high performance liquid chromatography
  • DNA is first treated with sodium bisulfite, which converts an unmethylated cytosine to uracil, while methylated cytosine residues remain unaffected.
  • One may amplify the region containing potential methylation sites via PCR and separate the products via denaturing high performance liquid chromatography (DHPLC).
  • DHPLC has the resolution capabilities to distinguish between methylated (containing cytosine) and unmethylated (containing uracil) DNA sequences.
  • Deng, D. et al. describes simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography.
  • Hybridization is a technique for detecting specific nucleic acid sequences that is based on the annealing of two complementary nucleic acid strands to form a double-stranded molecule.
  • One example of the use of hybridization is a microarray assay to determine the methylation status of DNA. After sodium bisulfite treatment of DNA, which converts an unmethylated cytosine to uracil while methylated cytosine residues remain unaffected, oligonucleotides complementary to potential methylation sites can hybridize to the bisulfite-treated DNA.
  • the oligonucleotides are designed to be complimentary to either sequence containing uracil or sequence containing cytosine at the original CpG islands, representing unmethylated and methylated DNA, respectively.
  • Computer-based microarray technology can determine which oligonucleotides hybridize with the DNA sequence and one can deduce the methylation status of the DNA.
  • An additional method of determining the results after sodium bisulfite treatment would be to sequence the DNA to directly observe any bisulfite-modifications.
  • Pyrosequencing technology is a method of sequencing-by-synthesis in real time.
  • PPi pyrophosphate
  • dNTP deoxynucleotide
  • This method presents a DNA template-primer complex with a dNTP in the presence of an exonuclease-deficient Klenow DNA polymerase.
  • the four nucleotides are sequentially added to the reaction mix in a predetermined order. If the nucleotide is complementary to the template base and thus incorporated, PPi is released.
  • the PPi and other reagents are used as a substrate in a luciferase reaction producing visible light that is detected by either a luminometer or a charge-coupled device.
  • the light produced is proportional to the number of nucleotides added to the DNA primer and results in a peak indicating the number and type of nucleotide present in the form of a pyrogram. Pyrosequencing can exploit the sequence differences that arise following sodium bisulfite-conversion of DNA.
  • present methods use amplification techniques in a reaction for creating distinguishable products.
  • Multiple amplification techniques are known. Some of these techniques employ PCR.
  • Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990), transcription amplification (Kwoh et al. 1989; WO88/10315), selective amplification of target polynucleotide sequences (US Patent No.
  • Sequence variation that reflects the methylation status at CpG dinucleotides in the original genomic DNA offers two approaches to PCR primer design.
  • the primers do not themselves "cover” or hybridize to any potential sites of DNA methylation; sequence variation at sites of differential methylation are located between the two primers.
  • Such primers are used in bisulphite genomic sequencing, COBRA, Ms-SNuPE.
  • the primers are designed to anneal specifically with either the methylated or unmethylated version of the converted sequence.
  • the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations.
  • additional nucleotide residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats.
  • the oligonucleotide primers may or may not be such that they are specific for modified methylated residues.
  • One way to distinguish between modified and unmodified DNA is to hybridize oligonucleotide primers which specifically bind to one form or the other of the DNA. After primer hybridization, an amplification reaction can be performed. The presence of an amplification product indicates that a sample hybridized to the primer. The specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not. For example, bisulfite ions convert non- methylated cytosine bases to uracil bases. Uracil bases hybridize to adenine bases under hybridization conditions.
  • an oligonucleotide primer which comprises adenine bases in place of guanine bases would hybridize to the bisulfite-modified DNA, whereas an oligonucleotide primer containing the guanine bases would hybridize to the non-converted (initial methylated) cytosine residues in the modified DNA.
  • Amplification using a DNA polymerase and a second primer yield amplification products which can be readily observed. This method is known as MSP (Methylation Specific PCR; Patent Nos 5,786,146; 6,017,704; 6,200,756).
  • Preferred primers and primer sets for assessing the methylation status of the concerned gene by way of MSP are provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495-1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288).
  • the amplification products can be optionally hybridized to specific oligonucleotide probes which may also be specific for certain products. Alternatively, oligonucleotide probes can be used which will hybridize to amplification products from both modified and non- modified DNA.
  • present invention provides for a method for detecting breast cancer or its precursor, or predisposition to breast cancer in a test sample containing breast cells or nucleic acids from breast cells comprising:
  • CTAGlA CXCLl; CCND2; CYPlAl; CYP24A1; DACTl; DAPKl; DDX27;
  • NEURL NEURL; NPPB; OGDHL; CDKN2A; PAX3; PCSK6; PDLIM3; PIK3CA;
  • PPPl Rl 4A PROXl; PTEN; PTPRD; PYCARD; RARBeta; RASSFl; REC8L1;
  • TIMP3 TNFRSFI lB; TRPV2; IGSF4; TSPYL6; TWISTl; UCHLl; WIFl;
  • Modified and non-modified DNA can be distinguished with use of oligonucleotide probes which may also be specific for certain products. Such probes can be hybridized directly to modified DNA or to amplification products of modified DNA. Probes for assessing the methylation status of the concerned gene will specifically hybridize to the converted sequence but not to the corresponding non converted sequence. Probes are designed to anneal specifically with the converted sequence representing either the methylated or unmethylated version of the DNA. Preferred converted sequences are provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495- 1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288).
  • Preferred probes anneal specifically with the converted sequence representing the methylated version of the DNA, in particular those sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501- 1503), and Figure 6B (SEQ ID NO: 1289-1304).
  • Oligonucleotide probes can be labeled using detection systems known in the art. These include but are not limited to fluorescent moieties, radioisotope labeled moieties, bioluminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.
  • Real time chemistry allows for the detection of PCR amplification during the early phases of the reactions, and makes quantitation of DNA and RNA easier and more precise.
  • a few variants of real-time PCR are well known. They include Taqman® (Roche Molecular Systems), Molecular Beacons®, Amplifluor® (Chemicon International) and Scorpion® DzyNA®, PlexorTM (Promega) etc.
  • the TaqMan® system and Molecular Beacon® system have separate probes labeled with a fluorophore and a fuorescence quencher.
  • the labeled probe in the form of a hairpin structure is linked to the primer.
  • Quantitation in real time format may be on an absolute basis, or it may be relative to a methylated DNA standard or relative to an unmethylated DNA standard.
  • the absolute copy number of the methylated marker gene can be determined; or the methylation status may be determined by using the ratio between the signal of the marker under investigation and the signal of a reference gene with a known methylation (e.g. ⁇ -actin), or by using the ratio between the methylated marker and the sum of the methylated and the non- methylated marker.
  • identification of methylated CpG dinucleotides may utilize the ability of the MBD domain of the McCP2 protein to selectively bind to methylated DNA sequences (Cross et al, 1994; Shiraishi et al, 1999). Restriction enconuclease digested genomic DNA is loaded onto expressed His-tagged methyl-CpG binding domain that is immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences. Variants of this method have been described and may be used in present methods of the invention.
  • Test samples for diagnostic, prognostic, or personalized medicine uses can be obtained from surgical samples, such as biopsies or fine needle aspirates, from paraffin embedded colon, rectum, small intestinal, gastric, esophageal, bone marrow, breast, ovary, prostate, kidney, lung, brain or other organ tissues, from lymph nodes, or from a body fluid such as blood, serum, lymph, cerebrospinal fluid, saliva, sputum, bronchial -lavage fluid, ductal fluids stool, urine, or semen.
  • a test sample obtainable from such specimens or fluids includes detached tumor cells or free nucleic acids that are released from dead or damaged tumor cells.
  • Nucleic acids include RNA, genomic DNA, mitochondrial DNA, single or double stranded, and protein-associated nucleic acids. Any nucleic acid specimen in purified or non-purified form obtained from such specimen cell can be utilized as the starting nucleic acid or acids. Particular suitable for breast cancer assays are blood derived samples such as serum and plasma. Alternatively, fluid within the mammary glands is used. Such fluid can be obtained from the breast e.g. by nipple aspiration of the milk ducts or by ductal lavage of at least one breast milk duct or from spontaneous nipple discharge. Administration of oxytocin may be used to stimulate expression of mammary fluid from a nipple of the patient.
  • Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA, and leads to significant changes in the distribution of cirDNA between cell-free and cell-surface-bound fractions.
  • Analysis of RARbeta2 and RASSFlA methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women (Skvortsova et ⁇ /. 3 2006).
  • primers and probes useful in MSP carried out on the gene selected from HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH
  • primers specifically hybridize to the sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304).
  • Preferred primers comprise, consist essentially of, or consist of the sequences provided in Table IA (SEQ ID NO: 1-848), Table 3 (SEQ ID NO: 1449-1470), Table 9 (SEQ ID NO: 1495-1500), Figure 2 (SEQ ID NO: 1305-1448) and Figure 6B (SEQ ID NO: 1273-1288).
  • probes specifically hybridize to the sequences provided in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304).
  • Preferred probes (5' - 3') may comprise, consist essentially of or consist of the sequences represented by SEQ ID NO. 1485-1494.
  • Alternative fluorescent donor and acceptor moieties/FRET pairs may be utilized as appropriate.
  • the primers and probes may include modified oligonucleotides and other appending groups and labels provided that the functionality as a primer and/or probe in the methods of the invention is not compromised.
  • an enrichment step prior to the amplification step may be appropriate.
  • the nested MSP approach (US 7,214,485) for instance may provide a solution.
  • the generation and amplification of a DNA library before testing for methylation of any specific gene may be required.
  • Suitable methods on whole genome amplification and libraries generation for such amplification e.g., Methylplex and Enzyplex technology, Rubicon Genomics
  • WO2005/090507 regards library generation/amplification methods that require either bisulphite conversion or non-bisulphite based application.
  • Bisulphite treatment may occur before or after library construction and may require the use of adaptors resistant to bisulphite conversion.
  • Meth- DOP-PCR (Di Vinci et al, 2006), a modified degenerate oligonucleotide-primed PCR amplification (DOP-PCR) that is combined with MSP, provides another suitable method for specific detection of methylation in small amount of DNA. Improved management of patient care may require these existing methods and techniques to supplement the methods of the invention.
  • CA 15-3 CA 27.29, CEA, carcinoembryonic antigen (CEA), estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-I).
  • CEA carcinoembryonic antigen
  • ER estrogen receptor
  • PgR progesterone receptor
  • HER2 human epidermal growth factor receptor 2
  • uPA urokinase plasminogen activator
  • PAI-I plasminogen activator inhibitor 1
  • BRACAnafysis® assesses a woman's risk of developing breast or ovarian cancer based on detection of mutations in the BRCAl and BRCA2 genes.
  • HER2/neu gene Approximately 25-30 percent of breast cancers have an amplification of the HER2/neu gene or overexpression of its protein product. Overexpression of this receptor is associated with highly aggressive breast cancer that requires special treatment, increased disease recurrence and worse prognosis. DNA hypermethylation is prevalent in these highly aggressive HER2/Neu-positive breast cancers. To aid prognostication, it may be necessary to combine the methods of the invention with this established marker for breast cancer prognosis.
  • stage of a cancer is a descriptor (usually numbers I to IV) of how much the cancer has spread.
  • the stage takes into account the size of a tumor, how deep it has penetrated, whether it has invaded adjacent organs, if and how many lymph nodes it has metastasized to, and whether it has spread to distant organs. Staging of cancer is important because the stage at diagnosis is the biggest predictor of survival, and treatments are often changed based on the stage.
  • the "grade” of a cancer refers to cell appearance (differentiation) and DNA make up.
  • the invention also provides for a method for determining the stage of cancer comprising determining epigenetic silencing of a gene in cancers, cancer precursors, and pre-cancers.
  • the genes include: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK
  • SYNEl and COL7A1 are preferentially methylated in advanced tumors.
  • PTPRD, SYNEl, and EVL are preferentially hypermethylated in high grade tumors.
  • These markers are preferred markers for determining the stage and/or grade of the breast cancer. Since grade is a strong predictor of local recurrence and metastasis, such markers are useful in predicting aggressive clinical behavior of the cancer and prognosis.
  • Testing can be performed diagnostically or in conjunction with a therapeutic regimen. Testing can be used to monitor efficacy of a therapeutic regimen, whether a chemotherapeutic agent or a biological agent, such as a polynucleotide. Testing can also be used to determine what therapeutic or preventive regimen to employ on a patient. Moreover, testing can be used to stratify patients into groups for testing agents and determining their efficacy on various groups of patients. [69] According to another aspect of the invention a method is provided of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer. An epigentically silenced gene is determined in a cell.
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7A
  • a polypeptide encoded by the epigenetic silenced gene in the cell is restored by contacting the cell with one or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor. Unregulated growth of the cell is thereby reduced or inhibited.
  • a CpG dinucleotide demethylating agent a DNA methyltransferase inhibitor
  • HDAC histone deacetylase
  • Another aspect of the invention is a method of reducing or inhibiting neoplastic growth of a cell which exhibits epigenetic silenced transcription of at least one gene associated with a cancer.
  • An epigenetic silenced gene is determined in the cell.
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK
  • Yet another aspect of the invention is a method of treating a cancer patient.
  • a cancer cell in the patient is determined to have an epigenetic silenced gene selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDK
  • One or more agents selected from the group consisting of a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, and a histone deacetylase (HDAC) inhibitor is administered to the patient in sufficient amounts to restore expression of the epigenetic silenced gene in the patient's cancer cells.
  • HDAC histone deacetylase
  • Yet another aspect of the invention is a method of treating a cancer patient.
  • a cancer cell in the patient is determined to have an epigenetic silenced gene selected from those shown in HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA
  • a polynucleotide encoding a polypeptide is administered to the patient.
  • the polypeptide is encoded by the epigenetic silenced gene.
  • the polypeptide is expressed in the patient's tumor thereby restoring expression of the polypeptide in the cancer.
  • a method for selecting a therapeutic strategy for treating a cancer patient.
  • a gene is identified whose expression in cancer cells of the patient is reactivated by a CpG dinucleotide demethylating agent, a DNA methyltransferase inhibitor, or a histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • the gene is selected from the group consisting of HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS 15; ADAMTS 18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl; CNIH3; CNNl; CNN3; C0L7
  • Demethylating agents can be contacted with cells in vitro or in vivo for the purpose of restoring normal gene expression to the cell.
  • Suitable demethylating agents include, but are not limited to 5-aza-2'-deoxycytidine, 5-aza-cytidine, Zebularine, procaine, and L- ethionine. This reaction may be used for diagnosis, for determining predisposition, and for determining suitable therapeutic regimes.
  • expression or methylation can be tested of a gene selected from HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; F0XL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl; AXIN2; BACHl; BEXl; BHMT2; BIK; BRCAl; C10orfl3; CALCA; CCK; CCND2; CD34; CDKNlA; CDKN2B; CFTR; CHLl
  • kits according to the present invention are assemblages of reagents for testing methylation. They are typically in a package which contains all elements, optionally including instructions. The package may be divided so that components are not mixed until desired. Components may be in different physical states. For example, some components may be lyophilized and some in aqueous solution. Some may be frozen. Individual components may be separately packaged within the kit.
  • the kit may contain reagents, as described above for differentially modifying methylated and non-methylated cytosine residues.
  • the kit will contain oligonucleotide primers which specifically hybridize to regions within about 10 kbp, within about 5 kbp, within about 3 kbp, within about 1 kbp, within about 750 bp, within about 500 bp, within 200 bp or within 100 bp kb of the transcription start sites of the genes/markers: HOXDl; SLC2A14; NEFH; H0XA4; GDA; CKM; TF; DSC3; NPTX2; CDOl; TACl; FOXL2; GPNMB; GREMl; H0XA9; RARRESl; TCERGlL; NDRG2; SALL4; LTB4R; RARRES2; ABHD3; ACP5; ACTN2; ADAM23; ADAMTS15; ADAMTS18; ADRA2A; APC; APC2; AQP5; ARMCX2; ATP2A2; ATXNl
  • the kit will contain both a forward and a reverse primer for a single gene or marker. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Exemplary of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats.
  • the oligonucleotide primers may or may not be such that they are specific for modified methylated residues.
  • the kit may optionally contain oligonucleotide probes.
  • the probes may be specific for sequences containing modified methylated residues or for sequences containing non-methylated residues.
  • the kit may optionally contain reagents for modifying methylated cytosine residues.
  • the kit may also contain components for performing amplification, such as a DNA polymerase and deoxyribonucleotides. Means of detection may also be provided in the kit, including detectable labels on primers or probes.
  • Kits may also contain reagents for detecting gene expression for one of the markers of the present invention. Such reagents may include probes, primers, or antibodies, for example. In the case of enzymes or ligands, substrates or binding partners may be sued to assess the presence of the marker.
  • the invention provides for oligonucleotide primers and/or probes and their sequences for use in the methods and assays of the invention.
  • Preferred primers and their sequences bind to at least one of the polynucleotide sequences listed in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304) or to the complement sequence thereof.
  • Preferred probes and their sequences bind to at least one of the polynucleotide sequences listed in Table IB (SEQ ID NO: 849-1272), Table 4 (SEQ ID NO: 1471-1481), Table 10 (SEQ ID NO: 1501-1503), and Figure 6B (SEQ ID NO: 1289-1304) or to the complement sequence thereof.
  • Preferred probes (5' - 3') may comprise, consist essentially of or consist of the sequences represented by SEQ ID NO. 1485-1494, or the complement sequence thereof.
  • the invention also provides for an isolated polynucleotide which consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-1503.
  • ACP5_55536 54 ACP5 TTTTGTATTTGCGGTCGGT AAAAACTCCTAAATATCCGCGA
  • ACP5_55540 54 ACP5 CGGTTTATTTTATAGATGCGGAG AAAAACGAACGCCTCGACC
  • ACTN2_55535 88 ACTN2 GTGTTTTGTTTGTTTTCGGAC TAACCGCGTAACCTAACGACT
  • ADRA2A_57683 150 ADRA2A GGTAGAGTTCGCGTTTTAGTTTC TCGAAAATCCTATATCTATCGCC
  • ADRA2A_57684 150 ADRA2A GTTTTTGTTTTAATTCGCGTTG ATTCTCCTAAACGTCCGCCT
  • ARMCX2_13030 9823 ARMCX2 TTTTACGTGATTAGGAGTTGACG ATTAAACGCACGACAACGAA
  • ATXN1_56027 6310 ATXN 1 TACGGGGATAGATTTGGGAGC ATAAACCGACAAAAACCTCGATA
  • BIK_22360 638 BIK TTTTTGGAGTTTCGGTTTTTAC CACGAATAACCTCCGTTCG
  • CCND2_1 894 CCN D2 GAGTTTCGGGGTTGTTTTATTC CCAACTTACGTCACCGCTT
  • CCND2_25209 894 CCN D2 GAAGGTAGCGTTTTTCGATG AAATAAACCCGATCCGCAA
  • CDKN2A_9696 1029 CDKN2A TTTAAGTTTTTAGGGCGTCGTT AAATAACGCTTCGATTCTCCG
  • CFTR_55912 1080 CFTR TCGAGAGATTATGTAGAGGTCGTT CCTCCTCTTTCGTAAACACGTA
  • CFTR_55913 1080 CFTR AGTTTTTCGGGGAGTCGGT GCAAATAAACGACAATCGC
  • CNIH3_55879 1491 11 CNIH3 TTATTAAATCGTTGAGGAGAGACGA GCTAAACACTATCCGTTTACGCC
  • CNN3 1266 CNN3 GCCTAAACCTTCGAAAATTAACCG
  • COL7A1_55842 1294 COL7A1 CGGTTATTAGAAGTCGTAGCGT GAACCTACCAAAACCACGAAA
  • COL7A1_55846 1294 COL7A1 GGGTGGTACGGTGTAGTGTTTC TAACAAAAACGCCCCGAAT
  • COL9A3_161 15 1299 COL9A3 TAGGTTTACGGGGGTATTTGC TCCTAACCGAACTTCCCGAAC
  • COL9A3_161 18 1299 COL9A3 TCGCGTAGTGTGTTTTTCGTT ATAAATCCTCGCTAACCCGAAT
  • CTAG2 246100 CTAG 1 A GTGAGAATCGGTTACGTGTTTC GAAATCCTCAAAACGCCTACG
  • CYP1A1_23831 1543 CYP1A1 TATTCGTGGTGGTGTTGAGC AAAATATAAAAATCGAACCGACC
  • ESR2_55653 2100 ESR2 TTTTTAGTTTTGGGGACGC CTATAAAATCCAACGCCCG
  • FAM20B_55630 9917 FAM20B ATATAGGGATACGCGGTTCGAG CCGTCCCCGAATAACTACG
  • FAS_18139 355 FAS GGAGTTGTTTCGTTTGTTTAGC AACCCGAATACTCAAAACGC
  • FBLN1_54935 2192 FBLN 1 ATTTCGAGTTTTCGTGGTTTC CGACCTTACGCTACTAACGACC
  • GALE_new2 2582 GALE GTTATTGGGATTTGGCGTC GCTATAACGAAAACTACGCAACG
  • GJB2_22306 2706 GJB2 TGCGGAGTATAGAGGATAACGA AACGAAAACGACTAAAAATCGAAA
  • GPNMB_52603 10457 GPNMB TTAATTTTTAGTTTTTCGATTGCG GTACTACGCGCTAACACCGA
  • GPNMB_52605 10457 GPNMB GAAGCGGTTAAAGGCGTAG TAAAAATTAAAACGCGACCG
  • GREM1_29777 26585 GREM1 GAATTTGGTACGATTTTACGGAG ATCTAAACTTTCCCTATCGACCG
  • HIST1 H3G_57040 8355 HIST1 H3G CGATTTTTCGTATTAGGCGTTG AAAACCTCATCGCTACCGTC
  • HIST1 H3G_57042 8355 HIST1 H3G CGGGTGAAAGTAGGCGGTT AAACATTCAACTCGCTCGC
  • HOXA10_57002 3206 HOXA10 TTTATTCGGTAAGATCGGGG AATCTAAATCCCGAAACGCA
  • HOXA5_1 3202 HOXA5 GTAGTTCGGGTTATTTGGATAGC AAAAATACTATAAACGCACAAACGA
  • ICAM1 3383 ICAM1 TAAAGACGTTTTCGCGGTTAAGGTC ACCACGTCCGAAAAAATCGACG
  • ID4_56952 3400 ID4 GCGAGTAGGGTTTAGGCGTT AAAACTACGAAAATATACGACCGA
  • INHBB_56906 3625 INHBB GTATTTCGTGTCGCGGTTC CCGAATACAATACCCTCGCT
  • ITIH5_56887 80760 ITIH5 CGTTGTAAAGCGTGTTTCGT CTACGCCTCTTCCTACGACC
  • KCNG3_56878 170850 KCNG3 GAGGGTTGCGTATATCGAGG ACGAAAAACTATTCCGCCC
  • KRTCAP3_56842 200634 KRTCAP3 GTTGATGCGTGTGGGTTTC AAAATTAACCACGTACCGCAA
  • LZTFL1_56661 54585 LZTFL1 GTCGGTTATTTATTATGGCGGT ACGATCCCAAACTTAACGAAA
  • MAL 1 4118 MAL TTCGGGTTTTTTTGTTTTTAATTC GAAAACCATAACGACGTACTAACG 254 MAL_56656 4118 MAL GTGGCGGTGGTTTAGTTTC TTCATTTTTCCGCTAAATACGTT
  • NAGS_57828 162417 NAGS GGGTGTCGAGTTTTAGGTTTTC TAAATATCCCGCTACACCAACGA
  • NDP_1 4693 NDP TTTCGTGGAAGGGAGTCGAG CGCACTAACAAATAAAAATACCACG
  • NEF3_13215 4741 NEF3 TGAGTTATACGTTGGATTCGTTG AAAAACCGCTAACGCGACT
  • NPPB_13212 4879 NPPB GGAGTACGGGGTGATTTATAGC CGACGAACAAATACTACGCTACG
  • NPTX2_57773 4885 NPTX2 TTTTAGTTTGTGACGTTCGCGTT TAAAACTCTCGAAAACCTCGACT
  • NPTX2_57779 4885 NPTX2 GCGTCGTTTTGTATGGGTATC CCCGATAACCGCTTCGTAT
  • OGDHL_19631 55753 OGDHL GTTTTAGTTTCGTTTTGCGGTT GCTCCTAACGCTATCCACG
  • PIK3CA_66163 5290 PIK3CA AAAAATAGGGGCGACGGAG GACACCGAAACTACCGCTT
  • PIK3CA_66165 5290 PIK3CA ATAGAGTTATCGCGGTCGGG CTCTAACTACCGCCTCGCTC
  • RARRES1_57581 5918 RARRES 1 GCGAAATCGTAGGGGAAAC ATAAAAACGCCTCCCCGAAA
  • RASSF1_8476 11 186 RASSF1 TCGTTTTTAGGAATGATTTTATCG CACTCTTATACGCTTACCCGAAC
  • RASSF1_8480 11 186 RASSF1 GTTTGCGAGTTAGCGAGGTTC TAACCTAAAACAACACGACGA
  • RPRC1_56336 55700 RPRC1 TTTTTATTTAGTTTTTAGGCGTTGC ACCGAACGAACTCCACGAC
  • SESN1_29753 27244 SESN 1 TTTTAGATAGGGCGGGTTTTC AAAAACGACTCACGATACGCAA
  • SESN1_29754 27244 SESN 1 ATTAGGACGAGGTATTTGGGC AAAAACAACGTAACCTCCGTAT
  • SFN 2810 SFN TGGTAGTTTTTATGAAAGGCGTC CCTCTAACCGCCCACCACG
  • TIMP3 7078 TIMP3 GCGTCGGAGGTTAAGGTTGTT CTCTCCAAAATTACCGTACGCG
  • TNFRSF11 B_30721 4982 TNFRSF11 B TATCGGGTTGAGGAATAAGGC AAAAACTAACCGCTAACGAAA
  • TNFRSF11 B_30722 4982 TNFRSF11 B TGGTTTAGGGATTTATTACGAGC AACTACGAAAACGCACCGAAA
  • ACTN2_55534 ACTN2 GCGGCGCGGTTATTAAGTCGCGCGGTAGTTGTTCGTAGTCGGAGTTGGT GTTTCGTTCGAGATTTAGCGTTTAGGCGTGTCGT
  • ACTN2_55535 ACTN2 GTGTTTTGTTTGTTTTCGGACGTGTTTTTTTTTTTAAGGGGTTTTCGGGGT
  • ADAM23_2 8745 ADAM23 GAAGGACGAGAAGTAGGCGGTAGGGCGGCGTGCGGGTCGGGGCGTTG
  • ADAM23_A 8745 ADAM23 GAGGTTTTAAGTTGGCGGAGCGGCGAGGATTTTTGGATTTTTTTGCGTTT
  • ADAMTS15_56144 170689 ADAMTS 15 TAGTAGAAGTATGGCGTCGGGTAGCGTATCGTCGCGTTGTGGGAAGGG
  • ADRA2A_57683 150 ADRA2A GGTAGAGTTCGCGTTTTAGTTTCGGGTCGGGTCGGGTTAGAATCGTAGC
  • ADRA2A_57684 150 ADRA2A GTTTTTGTTTTAATTCGCGTTGTCGTCGGATTTCGGTTTATTTAGTAGCGT
  • APC(2) 324 APC TTATATGTCGGTTACGTGCGTTTATATTTAGTTAATCGGCGGGTTTTCGAC GGGAATGGGGAGCGTTTTGGTTC
  • APC2 10297 APC2 GTCGTTTGTTTAGGTTCGGATCGGGTTTTGTTCGTTTCGGAGTTTTTGTT CGCGTCGCGGAGATTTCGGAGTTCGCGCGTTTCGAGGTTATTTCGGGTC
  • APC2_56103 10297 APC2 TCGGATGGTGAAGTTCGTGAGTGGGTGTGTGCGTAGGATCGGTTGTAGA AACGTTGACGTTTAGTTTATCGGGATTTAGTTT
  • AQP5_56090 362 AQP5 TCGGGATCGAGTTTCGTTTTTTAGGGAGTTCGGGGCGTACGGTATCGAG GAGAGCGCGGGAGTTAATTTGGGCGTATTATGCGTAGGG
  • ARMCX2_13030 9823 ARMCX2 TTTTACGTGATTAGGAGTTGACGTGGGTAAAGGTATTTAAAGTTTTGATC
  • ARMCX2_13032 9823 ARMCX2 TGGCGATGTAGTTTTTACGTGATTAGGAGTTGACGTGGGTAAAGGTATTT
  • TCGTAAGAGGAGG ATP2A2_23355 488 ATP2A GAGGGTTCGAGAAGCGAAGAGGTTTAGGGAAGGCGAGGCGAGGATCGT
  • ATXN1_56027 6310 ATXN 1 TACGGGGAT AGATTTGGGAGCGTTGGGCGGGGAGTAGTTTAGTTTTGTG
  • ATXN1_56032 6310 ATXN 1 GGAGTAGTTGGTTGTCGTCGTCGTAGTTTAAGGATTCGATTTTATGGGGG TGGGGGGTATTTTTTGGCGGGCGGTTCGGCGAGTCGTATAGAGTACGG
  • AXIN2_56020 8313 AXIN2 GAGAGATAGAGAGATTACGTCGATTGTTGAGAGGAATTGGAAGAAGAAA
  • BACH1_56015 571 BACH1 TTTTGTAATTTTTCGCGTGGGTTTTCGGTCGCGGCGATTTTTGTTTCGCG
  • BACH1 56017 571 BACH1 TTTTGTGGGGTTAGCGTTCGTTTTTTTTTTTTTGTTGTTCGCGGGTATTCGG GTACGCGGCGATTCGGTTGAATTAGGGCGTTT
  • BEX1 55859 BEX1 GAGTATTAGTTAATTGGTCGTCGGTTCGTGGGGGTTGGTGAGAAGGAGG GTGAGTTTGGCGGTGACGTACGGTTTTTACGTGATCG
  • BEX1_12842 55859 BEX1 TCGGGGTTTTTATTTGGTTCGTTTTTTTTCGGGTCGGATGTTAGTTCGTC GAGCGTAGGGTAGCGGGGAGTTGGTAGCGAGATACGAGTGACGATT
  • BEX1_12850 55859 BEX1 TTAGTT AATTGGTCGTCGGTTCGTGGGGGTTGGTGAGAAGGAGGGTGAG TTTGGCGGTGACGTACGGTTTTTACGTGATCGGGAGTTGTAGAGT
  • BHMT2_55998 23743 BHMT2 TTCGTTTAAGTGTTTTGTCGAGGTTTTAGGGTTTGGTTAGGGATGGACGT CGGGTGTGAACGGAGTCGTTCGTAGTTTAGCGGTT
  • BIK_17885 638 BIK TTTAGAGTTCGGAGTCGGGTGTTCGGAAGTCGTATTGGAGGATTGTGCG
  • BIK_22360 638 BIK TTTTTGGAGTTTCGGTTTTTACGTGGGTAAAATGATCGTGAAAAAAAGTAT CGAGGAGTATTTTGAGTTCGAACGGAGGTTATTCGTG
  • BRCA1_JH 672 BRCA1 TCGTGGTAACGGAAAAGCGCGGGAATTATAGATAAATTAAAATTGCGATT GCGCGCGGCGTGAGTTCGTTGAGATTT
  • CALCA_2 796 CALCA CGTTTTTATAGGGTTTTGGTTGGACGTCGTCGTCGTCGTTGTTATCGTTT
  • CCK_13382 885 CCK ATTGCGAGGGTTTTTAATGCGGTTGAGAAGAAAGTGAAGATTTCGATTTT TTTTTTTTTTCGAAAGAGTCGATAT AGGTATGAAGATTAGCG
  • CCND2_1 894 CCND2 GAGTTTCGGGGTTGTTTTATTCGTATCGGTTTTTTTTTTAAAATTGGTTTC GTTTTTTTTTGTTCGTTTTTTTTCGTTTTGAAGCGGTGACGTAAGTTGG
  • CCND2_25209 894 CCN D2 GAAGGTAGCGTTTTTCGATGGTGAGTAGGTTTTGTAGGACGCGGTCGTT TCGGAGTAGGTTGCGGTTTCGTACGGTTTTGCGGATCGGGTTTATTT
  • CD34_55940 947 CD34 GCGGTATTTTGGGTTTTGCGCGCGCGTTTTTGCGGATTAGTATTTTTTTC GCGCGGTTTAGAGAGACGTATCGAGTGGAAGATATTA
  • CDKN1A_24717 1026 CDKN1A GGGAAATGTGTTTAGCGTATTAACGTAGGCGAGGGATTGGGGGAGGAG GGAAGTGTTTTTTTGTAGTACGCGAGGTTTCGGGATCGGTTGGTTT
  • CDKN2A_8472 1029 CDKN2A GTTTTGGCGAGGGTTGTTTTCGGTTGGTGTTTTCGGGGGAGATTT AATTT GGGGCGATTTTAGGGGTGTTATATTCGTTAAGTGTTCGGAGTTAATAGT
  • CDKN2A_9696 1029 CDKN2A TTTAAGTTTTTAGGGCGTCGTTAGGAGGAGGTTTGTGATTATAAATTTTTT
  • CDO1_55929 1036 CD01 GTTTACGCGATTTTTGGGACGTCGGAGATAACGGGGTTTTTGGGAAGGC
  • CFTR_55912 1080 CFTR TCGAGAGATTATGTAGAGGTCGTTTTTGGAAAAGGTTAGCGTTGTTTTTA
  • CFTR_55913 1080 CFTR AGTTTTTCGGGGAGTCGGTTTTTTCGTCGGTGGTTTTTTTTGTTTTTTAGC
  • CHL1_55888 10752 CHL1 GAATCGAGTGAAATTATCGGGGAGGGGGTGGGGGGCGTTTTTTTTAAAT
  • CNIH3_55879 1491 11 CNIH3 TTATTAAATCGTTGAGGAGAGACGAGCGTTTCGTTTCGGATTTTTTCGCG
  • CNN3 1266 CNN3 GTTTTCGCGGTTTTTTAATTGGTCGTATCGTTTTTCGGCGTAGAGGCGGG
  • COL7A1_55842 1294 COL7A1 CGGTTATTAGAAGTCGTAGCGTTATTTTAGGTAGTAAAAGTCGTTAGTTA
  • COL7A1_55846 1294 COL7A1 GGGTGGTACGGTGTAGTGTTTCGGGTCGGGTTTTTTTTTGCGGTGTTTAT TTTTTTTTTTGTGTTGGGTTCGTATTCGGGGCGTTTTTGTTA
  • COL9A3_161 15 1299 COL9A3 TAGGTTTACGGGGGTATTTGCGTTTTTTAATGAGTTTTTTTCGTTTTAGAG
  • COL9A3_161 18 1299 COL9A3 TCGCGTAGTGTGTTTTTCGTTTTTTTTCGGCGGCGGGAATAAAGGTTTTT
  • CRIP1J 1396 CRIP1 GTCGTTTTAGGGATTTAGCGTTTTCGGTTTTTTTGAGCGGTTTTTAGTTTC
  • CSPG2_23363 1462 CSPG2 TATTGTAGCGTTGCGCGATTGGGTTCGGCGTTGTTTAGGCGGGTTATATA
  • CST6_17991 1474 CST6 TTAGTTTTAGGTCGCGGGGGCGTATCGCGGGCGTCGGGCGGGGCGGTT
  • CTAG2 246100 CTAG1A GTGAGAATCGGTTACGTGTTTCGGGGTTTATTCGGGGTTTTTTAGGGTCG
  • CYP1A1_23831 1543 CYP 1A1 TATTCGTGGTGGTGTTGAGCGGTTTGGATATTATTCGGTAGGTTTTGGTG CGGTAGGGCGATGATTTTAAGGGTCGGTTCGATTTTTATATTTT
  • CYP24A1_55769 1591 CYP24A1 AATTACGGTCGTCGTTGTCGGTTTTTGTTCGTCGGGGGAGGGCGGGGA GGCGCGTTCGAAGTATATTCGGTGAATTTCGGGTTTCGTATGATTTTT
  • DNAJA4_8894 55466 DNAJA4 TTTTTAGTTTTATTTTTCGGCGTAGGGTTTCGGTTAATATAGTTTTTTAGGT
  • EPB41L3_19072 23136 EPB41L3 GCGTGGGTTTTCGTCGTAGTTTCGCGGAGTTTCGGTGTTTTTTGTAATAG
  • EREG_18104 2069 EREG TTTTCGGGTTTTAACGGGGTGAGGTTAAGAGTGTTTAGAGTTTTATAATT GGTTCGAGGGAGGAGCGGTAGGAGGATCGTTA
  • ESR2_55651 2100 ESR2 GATTGTAATTTGAGCGCGGTTTTTTTAGTAGAGTAAGTATATTTATCGGAT
  • ESR2_55653 2100 ESR2 TTTTTAGTTTTGGGGACGCGGTGTAGAAGTGTGAGGGCGTTCGGTTTTTA
  • ESR2_55656 2100 ESR2 TGAGTTGTAGGAGGTGCGTTCGTTTTTTTTAATAGGTGGCGGCGGGGCG
  • ESR2_55657 2100 ESR2 TTTTCGTTAGGAGGTAGTTGTAAGCGCGGAGGTTGCGAGAAATAATTGTT
  • FAM20B_55630 9917 FAM20B ATATAGGGATACGCGGTTCGAGCGGGTTTTTTAGGAAGTTTCGGGTTTTC GTTCGGGATTCGTTCGTAGTTATTCGGGGACGG
  • FAM84A_55594 151354 FAM84A CGTAGATTTTCGTTTTTGGTTTTCGTTTCGTTATTTTATTGATGGGTAATTA ATTGGATCGTATTATTTATTTTAATTATAGCGAGTTGTTTATAGGGGATTC
  • FAM84A_55600 151354 FAM84A GTCGGTTAGTTTACGTGTTTTTATCGGAGTTATTTTTTTTAGTCGAGTTTTT CGTGTTTTTTTAGGGGTACGAGGGGCGGAGAAGTCGTTATAG
  • FAM84A_55624 151354 FAM84A CGGAGTAGTCGGTTTTAGGGTTCGTTTTAGGACGCGTTTATTGGTTGGG GCGGGTGTTCGAGTTGTGTTAATTTTCGTCG
  • FBLN1_54935 2192 FBLN 1 ATTTCGAGTTTTCGTGGTTTCGAAATTCGTAGGGAGCGCGAGGTCGAGG
  • FBLN2_13328 2199 FBLN2 TAGAGCGGAGGAAGTTGCGGATTTGGGGTGGGGGAATTCGTTCGCGGA TTTTTGGTTTTTATTTCGCGTCGGTTTTTGTGTTCGTATTTG
  • GADD45G_57135 10912 GADD45G TATATTAGAAAGCGGGTGTCGGTTAATAGGCGCGTAGTTTCGCGTTAGTT
  • GALE_new2 2582 GALE GTTATTGGGATTTGGCGTCGGCGGCGTTTTTCGCGGGGTGGTATCGTTT
  • GDA_57108 9615 GDA GTATCGGTAATCGTTCGGGTAAGCGGGGGTAGGATAAGGTCGGAGTTTG TGTTCGTTCGGTAGTCGTTCGTAGTTGTAGAGAGTTTCGTTG
  • GDF10_57718 2662 GDF10 CGGGGATATGAGTTATGGCGTGGTGAGGGCGGTAAAGGGTCGAAGTTT AGGAGGAGGAAGGCGAGCGTTGGCGTATCGGAGGTTGCGGATTG
  • GDF10_57726 2662 GDF10 GTTATAGTCGTTCGGAGTAGCGTAGAGTCGAGTCGAGTTCGAGTCGGCG CGTTGTTTTGGCGGATTCGCGTCGCGAAAGTTTGTAGTTTATTGCG
  • GDF10_57728 2662 GDF10 TAGTTGGGGTTCGGGTTTCGGGTTGGTTCGAGCGGGGATATGAGTTATG GCGTGGTGAGGGCGGTAAAGGGTCGAAGTTTAG
  • GDF10_57734 2662 GDF10 TTTTTATATTCGCGGGCGTATATTTCGGCGCGCGTACGTTGTTATATACG GGCGTACGTATACGGTAGTCGGGTTAGGGACGATTTT
  • GJB2_22306 2706 GJ B2 TGCGGAGTATAGAGGATAACGATTATAGTTATTTTTGAATTTCGTTTACGG
  • GPNMB_52603 10457 GPNMB TTAATTTTTAGTTTTTCGATTGCGATTGCGATTCGGGAAAACGTTTACGTT
  • GPNMB_52605 10457 GPNMB GAAGCGGTTAAAGGCGTAGCGGTTTTTGGTTAGAGTCGTAGAGGTTTGA
  • GREM1_29777 26585 GREM1 GAATTTGGTACGATTTTACGGAGATTTCGTTTTTTTTAGCGTAGTTTTCGT
  • Gst-Pi New1 2950 GSTP1 TCGTTATTAGTGAGTACGCGCGGTTCGCGTTTTCGGGGATGGGGTTTAG AGTTTTTAGTATGGGGTTAATTCGTAGT ATTAGGTTCGGGTTTTCGGTAG GGTTTTTCGTTTATT 165 Gst-P ⁇ _New3 2950 GSTP1 ATTTAGTATTGGGGCGGAGCGGGGCGGGATTATTTTTATAAGGTTCGGA
  • HIST1 H3G_57040 8355 HIST1H3G CGATTTTTCGTATTAGGCGTTGGAAAGGTAATTTGCGGATTAGTAGTTTA
  • HIST1 H3G_57042 8355 HIST1H3G CGGGTGAAAGTAGGCGGTTTTTAAAAGAGTTTTTTTAAGTTGGATAGAAT
  • HMG20B_57034 10362 HMG20B GGTGAAAATAGTCGCGGAATTTCGGGTTTTGAGAGGGGGCGGGGGTGT
  • HOXA10_57000 3206 HOXA10 GTTGTTTTATTGCGTTTGTCGTTTAGCGTGGGGAAGAGTTCGTAGTTTTG TAGTTCGTAGGGTAGGTCGGCGGCGGGCGGTAGGTAGATTTCG
  • HOXA10_57002 3206 HOXA10 TTTATTCGGTAAGATCGGGGCGCGTTTAGTTATAGGTTTATGGGCGAGG GTTCGTAGTCGTGCGTTTCGGGATTTAGATT
  • HOXA5_1 3202 HOXA5 GTAGTTCGGGTTATTTGGAT AGCGATCGTAAAATGAGTTTATAAAAT AAG
  • HUS1B_12677 135458 HUS1B GTTGGGCGGTAGGTAGTTTCGTTATATTTTTTTGGGAAGTATTCGTACGG
  • ICAM1 3383 ICAM1 TAAAGACGTTTTCGCGGTTAAGGTCGAAAGGGGAAGCGAGGAGGTCGTC
  • GAATTAGAGAGT 205 ING1_24685 3621 ING1 TCGAAATAGAATTGGTAATCGTAGTAGTTAATTTATTTGTTAATATTATTTT

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Abstract

Plusieurs gènes sont identifiés comme présentant une méthylation différentielle dans le cancer du sein. Cette information est utile pour le dépistage, l’évaluation de risque, le pronostic du cancer du sein, l’identification de la maladie, la stadification de la maladie, et l’identification de cibles thérapeutiques. L’identification de gènes qui sont méthylés dans le cancer du sein permet des dosages pour le diagnostic précoce précis et efficaces, l’établissement de profil de méthylation au moyen d’une pluralité de gènes et l’identification de nouvelles cibles pour une intervention thérapeutique.
PCT/US2009/035654 2008-02-29 2009-03-02 Marqueurs pour la détection améliorée du cancer du sein Ceased WO2009108917A2 (fr)

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WO2010105815A3 (fr) * 2009-03-17 2010-11-25 Oncomethylome Sciences S.A. Détection améliorée de l'expression de gènes
WO2013033333A1 (fr) * 2011-08-30 2013-03-07 Dcb-Usa Llc Biomarqueurs génétiques pour la prédiction de la sensibilité à des néoplasmes ovariens et/ou le pronostic ou la malignité de cancers de l'ovaire
WO2013186639A2 (fr) 2012-06-13 2013-12-19 King Abdullah University Of Science And Technology Biomarqueurs de méthylation pour le cancer du sein
US9090944B2 (en) * 2011-12-16 2015-07-28 The Chinese University Of Hong Kong Biomarker DACT1 for gastric cancer
US9365851B2 (en) 2011-09-20 2016-06-14 National University Of Singapore Spalt-like transcription factor 4 (SALL4) and uses thereof
EP3054299A3 (fr) * 2010-08-13 2016-11-30 Arizona Board of Regents, a body corporate acting on behalf of Arizona State University Biomarqueurs pour la détection précoce du cancer du sein
US10378060B2 (en) 2011-10-14 2019-08-13 Dana-Farber Cancer Institute, Inc. ZNF365/ZFP365 biomarker predictive of anti-cancer response
WO2019199696A1 (fr) * 2018-04-12 2019-10-17 Singlera Genomics, Inc. Compositions et méthodes d'évaluation et de traitement d'un cancer ou d'une néoplasie
CN111020034A (zh) * 2019-12-31 2020-04-17 上海奕谱生物科技有限公司 新型的诊断肿瘤的标志物及其应用
CN111899789A (zh) * 2020-08-03 2020-11-06 北京市肿瘤防治研究所 二代测序鉴定brca1/2大片段重排的方法及系统
WO2021014187A1 (fr) * 2019-07-19 2021-01-28 Vilnius University Caractérisation du cancer de la prostate à l'aide d'un système de dosage de méthylation de l'adn
EP3828290A4 (fr) * 2018-07-26 2022-03-23 Excellen Medical Technology Co., Ltd. Méthode pour identifier l'état d'un cancer du sein et kit
WO2022125417A1 (fr) * 2020-12-07 2022-06-16 Institute For Cancer Research D/B/A The Research Institute Of Fox Chase Cancer Center Traitement de femelles ayant des mutations brca1/2 avec de la gonadotrophine chorionique humaine pour réduire le risque de développer un cancer du sein
CN114959038A (zh) * 2022-06-13 2022-08-30 湖南宏雅基因技术有限公司 一种hoxa9基因甲基化检测试剂及其应用
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WO2023082141A1 (fr) * 2021-11-11 2023-05-19 华大数极生物科技(深圳)有限公司 Marqueur de méthylation hist1h3g pour la détection du cancer hépatique
US11697674B2 (en) 2017-10-16 2023-07-11 Institute For Cancer Research Human chorionic gonadotropin variant peptides and treatment of breast cancer
WO2023165035A1 (fr) * 2022-03-03 2023-09-07 北京起源聚禾生物科技有限公司 Combinaison de marqueurs de méthylation de l'adn, utilisation, sonde d'amorce pour la détection précoce du cancer ovarien et kit
WO2024006577A1 (fr) * 2022-07-01 2024-01-04 Ohio State Innovation Foundation Procédés et compositions pour identifier des signatures de gène hox afin d'affecter des thérapies spécifiques et efficaces dans la leucémie myéloïde aiguë et d'autres cancers

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WO2010105815A3 (fr) * 2009-03-17 2010-11-25 Oncomethylome Sciences S.A. Détection améliorée de l'expression de gènes
US10802026B2 (en) 2010-08-13 2020-10-13 Arizona Board of Regents, a body corporate acting for and on behalf of Arizona State University Biomarkers for the early detection of breast cancer
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EP3054299A3 (fr) * 2010-08-13 2016-11-30 Arizona Board of Regents, a body corporate acting on behalf of Arizona State University Biomarqueurs pour la détection précoce du cancer du sein
US9857374B2 (en) 2010-08-13 2018-01-02 Arizona Board of Regents, a body corporate acting for and on behalf of Arizona State University Biomarkers for the early detection of breast cancer
WO2013033333A1 (fr) * 2011-08-30 2013-03-07 Dcb-Usa Llc Biomarqueurs génétiques pour la prédiction de la sensibilité à des néoplasmes ovariens et/ou le pronostic ou la malignité de cancers de l'ovaire
US9365851B2 (en) 2011-09-20 2016-06-14 National University Of Singapore Spalt-like transcription factor 4 (SALL4) and uses thereof
US10378060B2 (en) 2011-10-14 2019-08-13 Dana-Farber Cancer Institute, Inc. ZNF365/ZFP365 biomarker predictive of anti-cancer response
US9090944B2 (en) * 2011-12-16 2015-07-28 The Chinese University Of Hong Kong Biomarker DACT1 for gastric cancer
EP2861766A4 (fr) * 2012-06-13 2016-03-02 Univ King Abdullah Sci & Tech Biomarqueurs de méthylation pour le cancer du sein
US9982307B2 (en) 2012-06-13 2018-05-29 King Abdullah University Of Science And Technology Methylation biomarkers for breast cancer
WO2013186639A2 (fr) 2012-06-13 2013-12-19 King Abdullah University Of Science And Technology Biomarqueurs de méthylation pour le cancer du sein
US11697674B2 (en) 2017-10-16 2023-07-11 Institute For Cancer Research Human chorionic gonadotropin variant peptides and treatment of breast cancer
WO2019199696A1 (fr) * 2018-04-12 2019-10-17 Singlera Genomics, Inc. Compositions et méthodes d'évaluation et de traitement d'un cancer ou d'une néoplasie
EP3828290A4 (fr) * 2018-07-26 2022-03-23 Excellen Medical Technology Co., Ltd. Méthode pour identifier l'état d'un cancer du sein et kit
WO2021014187A1 (fr) * 2019-07-19 2021-01-28 Vilnius University Caractérisation du cancer de la prostate à l'aide d'un système de dosage de méthylation de l'adn
CN111020034A (zh) * 2019-12-31 2020-04-17 上海奕谱生物科技有限公司 新型的诊断肿瘤的标志物及其应用
CN111020034B (zh) * 2019-12-31 2023-09-19 上海奕谱生物科技有限公司 新型的诊断肿瘤的标志物及其应用
CN111899789A (zh) * 2020-08-03 2020-11-06 北京市肿瘤防治研究所 二代测序鉴定brca1/2大片段重排的方法及系统
WO2022125417A1 (fr) * 2020-12-07 2022-06-16 Institute For Cancer Research D/B/A The Research Institute Of Fox Chase Cancer Center Traitement de femelles ayant des mutations brca1/2 avec de la gonadotrophine chorionique humaine pour réduire le risque de développer un cancer du sein
WO2023082141A1 (fr) * 2021-11-11 2023-05-19 华大数极生物科技(深圳)有限公司 Marqueur de méthylation hist1h3g pour la détection du cancer hépatique
CN118043481A (zh) * 2021-11-11 2024-05-14 深圳华大基因股份有限公司 用于检测肝癌的hist1h3g甲基化标志物
WO2023165035A1 (fr) * 2022-03-03 2023-09-07 北京起源聚禾生物科技有限公司 Combinaison de marqueurs de méthylation de l'adn, utilisation, sonde d'amorce pour la détection précoce du cancer ovarien et kit
CN114959038A (zh) * 2022-06-13 2022-08-30 湖南宏雅基因技术有限公司 一种hoxa9基因甲基化检测试剂及其应用
WO2024006577A1 (fr) * 2022-07-01 2024-01-04 Ohio State Innovation Foundation Procédés et compositions pour identifier des signatures de gène hox afin d'affecter des thérapies spécifiques et efficaces dans la leucémie myéloïde aiguë et d'autres cancers
CN116064771A (zh) * 2022-09-27 2023-05-05 中山大学 一种促进细胞铁死亡的基因的功能确定及应用

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