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WO2008066878A9 - Procédés et produits utilisés pour diagnostiquer le cancer - Google Patents

Procédés et produits utilisés pour diagnostiquer le cancer Download PDF

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Publication number
WO2008066878A9
WO2008066878A9 PCT/US2007/024553 US2007024553W WO2008066878A9 WO 2008066878 A9 WO2008066878 A9 WO 2008066878A9 US 2007024553 W US2007024553 W US 2007024553W WO 2008066878 A9 WO2008066878 A9 WO 2008066878A9
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Prior art keywords
methylation
genes
sample
level
cancer
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PCT/US2007/024553
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English (en)
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WO2008066878A3 (fr
WO2008066878A2 (fr
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Marcus Bosenberg
Viswanathan Muthasamy
Sekhar Duraisamy
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Univ Vermont
Marcus Bosenberg
Viswanathan Muthasamy
Sekhar Duraisamy
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Application filed by Univ Vermont, Marcus Bosenberg, Viswanathan Muthasamy, Sekhar Duraisamy filed Critical Univ Vermont
Priority to US12/312,807 priority Critical patent/US20100143899A1/en
Publication of WO2008066878A2 publication Critical patent/WO2008066878A2/fr
Publication of WO2008066878A3 publication Critical patent/WO2008066878A3/fr
Publication of WO2008066878A9 publication Critical patent/WO2008066878A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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 invention pertains to the field of diagnosis of cancer and precancerous conditions.
  • Methylation of bases in DNA serves a number of cellular functions. In bacteria, methylation of cytosine and adenine residues plays a role in the regulation of DNA replication and DNA repair. DNA methylation also constitutes part of an immune mechanism that allows these organisms to distinguish between self and non-self DNA. In mammalian species, DNA methylation occurs only at cytosine residues, and specifically at cytosine residues that lie next to a guanosine residue, i.e., within the sequence CG. Methylation of DNA is carried out by DNA methyltransferases (sometimes called methylases). Generally both DNA strands can accept methyl groups at opposing CG sites.
  • DNA methyltransferases sometimes called methylases
  • Alterations in DNA methylation are one manifestation of the genome instability characteristic of human tumors.
  • a hallmark of human carcinogenesis is the loss of normal constraints on cell growth resulting from genetic alterations in the genes that control cell growth.
  • the consequences of such mutations include the activation of positive growth signals and the inactivation of growth inhibitory signals.
  • Gene function can be lost through mutation or deletion.
  • An alternative mechanism by which gene function can be lost is aberrant DNA methylation. Accordingly, such methylation events can be viewed as key steps in both the initiation and progression of cancer. Summary of the Invention
  • the present invention relates to methods for the detection, diagnosis, and monitoring of cancer or a precancerous condition in a cell or subject.
  • the invention relates to methods for the detection, diagnosis, and monitoring of melanoma in a cell or subject.
  • Methods and kits of the invention are highly sensitive and thus permit the identification of abnormal methylation of one or more genes of the invention in very rare cell events as well as in more common cell events and conditions.
  • Methods and kits of the invention permit quantitative and qualitative evaluation of the methylation state of genes associated with cancer or precancerous conditions, thus permitting assessment of cancer and/or precancerous conditions in cells and subjects.
  • Methods of the invention include determination the methylation state in a cell or sample of one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2, which have now been identified as hypermethylated in cancer and/or precancerous conditions.
  • the methylation state of one or more of these genes may be used to diagnose and monitor cancer or a precancerous condition in a cell or sample.
  • the invention may include assaying the methylation state of one or more of the QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2 genes, and assaying of the methylation state of one or more additional genes, some of which have been previously identified as being abnormally methylated in cancer or precancerous conditions.
  • methods for diagnosing cancer or a precancerous condition in a subject include determining a level of methylation of one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2 in a sample from a subject, comparing the level of methylation of the one or more genes in the sample to a control level of methylation of the one or more genes, wherein a higher level of methylation of the one or more genes in the sample compared to the control level of methylation is diagnostic for cancer or precancerous condition in the subject.
  • the level of two or more of the genes is determined in the sample.
  • the methods also include determining the level of methylation of one or more of the genes CYPlBl, COL1A2, GDFl 5, RARB, TM, 3-OST-2, RASSFlA, ACS/TMS1, BST2, DNAJCl 5, CDKNIc, MGMT, SYK, MiBl, HOXBl 3, PTGS2, DAPK, APC,pl6 INK4A , p27 KipI , PRDX2, PYCARD, CDKN2A, CDKNlB and DALl.
  • the methylation of the one or more genes is located on CpG islands of the one or more genes.
  • the methylation of the one or more genes is located on a nucleotide sequence of SEQ ED No. 51-57.
  • the cancer is melanoma.
  • the cancer is metastatic cancer.
  • the control level of methylation of the one or more genes is the level of methylation of the one or more genes in a non-cancerous cell.
  • the non-cancerous cell is a cultured melanocyte.
  • the sample is a fluid sample, hi some embodiments, the fluid sample is a blood sample, hi certain embodiments, the sample is a tissue sample, hi some embodiments, the tissue sample is a lymph node sample, hi some embodiments, the level of methylation of the one or more genes in the sample is at least 1%, 10%, 20%, 50%, 100%, 200%, 400% or 1000% higher than the control level of methylation of the one or more genes.
  • the level of methylation of the one or more genes in the sample is at least 400% higher than the control level of methylation of the one or more genes, hi certain embodiments, the level of methylation of the one or more genes is determined by methylation-specific PCR, methylation-inhibitor analysis, methylation sensitive restriction analysis, sequencing of bisulfite modified DNA, methylation-sensitive single nucleotide primer extension, MethyLight analysis, pyrosequencing, or combined bisulfite restriction analysis, hi some embodiments, methylation-inhibitor analysis includes adding a DNA methylation inhibitor to the sample and monitoring the change in expression level of the one or more genes upon addition of the DNA methylation inhibitor, wherein if the expression level of the one or more genes increases, the one or more genes have an increased level of methylation.
  • the methylation inhibitor is 5-aza-2'-deoxycytidine.
  • the methods also include isolating a nucleic acid from the sample and analyzing the nucleic acid on a nucleic acid microarray.
  • the methods also include analyzing the sample for one or more mutated genes, hi some embodiments, the one or more mutated genes is pi 6 INK4A ox pl4 ARF .
  • the methods also include analyzing the sample for one or more chromosomal instability loci, hi certain embodiments, the one or more chromosomal instability loci include 6q, 8p, 9p, 10, 13, 2 Iq, 6p, 7, 8q, 1 Iq, q3, 17q and 2Oq. hi some embodiments, the subject is asymptomatic for cancer.
  • methods for determining onset, progression, or regression, of cancer in a subject include determining a level of methylation of one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2 in a sample obtained from the subject, determining the level of methylation of the one or more genes in a second sample obtained from the subject at a later time than the first sample was obtained, comparing the level of methylation of the one or more genes in the first sample with the level of methylation of the one or more genes in the second sample, wherein a lower level of methylation of the one or more genes in the first sample compared with the level of methylation of the one or more genes in the second sample indicates onset or progression of cancer in the subject, and wherein a higher level of methylation of the one or more genes in the first sample compared with the level of methylation of the one or more genes in the second sample indicates regression of cancer in the subject.
  • the level of two or more of the genes is determined in the sample.
  • the methods also include determining the level of methylation of one or more of the genes CYPlBl, COL1A2, GDF15, RARB, TM, 3-OST-2, RASSFlA, ACS/TMS1, BST2, DNAJCl 5, CDKNIc, MGMT, SYK, MiBl, HOXBl 3, PTGS2, DAPK, APC, pl6 INK4A , p27 KipI , PRDX2, PYCARD, CDKN2A, CDKNlB and DALl.
  • the methylation of the one or more genes is located on CpG islands of the one or more genes. In some embodiments, the methylation of the one or more genes is located on a nucleotide sequence of SEQ ID No. 51-57. In some embodiments, the cancer is melanoma. In certain embodiments, the cancer is metastatic cancer. In some embodiments, the control level of methylation of the one or more genes is the level of methylation of the one or more genes in non-cancerous cells. In some embodiments, the non-cancerous cells are cultured melanocytes.
  • the sample is a fluid sample, hi some embodiments, the sample is a blood sample, hi certain embodiments, the sample is a tissue sample, hi some embodiments, the sample is a lymph node sample.
  • the level of methylation of the one or more genes is at least 1%, 10%, 20%, 50%, 100%, 200%, 400%, or 1000% higher in the sample than in the control.
  • the level of methylation of the one or more genes is at least about 400% higher in the sample than in the control, hi some embodiments, the level of methylation of the one or more genes is determined by methylation-specific PCR, methylation-inhibitor analysis, methylation sensitive restriction analysis, sequencing of bisulfite modified DNA, methylation-sensitive single nucleotide primer extension, MethyLight analysis, or combined bisulfite restriction analysis.
  • methylation-inhibitor analysis includes adding a DNA methylation inhibitor to the sample and monitoring the change in expression level of the one or more genes upon addition of the DNA methylation inhibitor, wherein if the expression level of the one or more genes increases, the one or more genes have an increased level of methylation.
  • the methylation inhibitor is 5-aza-2'-deoxycytidine.
  • the method also includes isolating a nucleic acid from the sample and analyzing the nucleic acid on a nucleic acid microarray.
  • the method also includes analyzing the sample for one or more mutated genes.
  • the one or more mutated genes is pi 6 INK4A or pi 4 ARF .
  • the method also includes analyzing the sample for one or more chromosomal instability loci.
  • the one or more chromosomal instability loci include 6q, 8p, 9p, 10, 13, 2 Iq, 6p, 7, 8q, 1 Iq, q3, 17q and 2Oq.
  • the subject is undergoing treatment for cancer.
  • the subject has been diagnosed with cancer.
  • the subject is asymptomatic for cancer.
  • kits for diagnosing cancer or a precancerous condition in a subject include one or more containers, each container containing a nucleic acid for performing methylation-specific PCR to determine the level of methylation of one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8, and LRRC2, and the kit also includes instructions for using the nucleic acids to determine the level of methylation of the one or more genes.
  • the cancer is melanoma.
  • kits for diagnosing cancer or a precancerous condition in a subject are provided.
  • kits include a container containing a DNA methylation-inhibitor and instructions for using the DNA methylation inhibitor to perform methylation-inhibitor analysis to determine the level of methylation of the one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC, and the kit optionally includes a control nucleic acid.
  • the kit also includes an additional one or more containers, each containing a reagent to perform methylation inhibition analysis to determine the level of methylation of the one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2.
  • the kit also includes a nucleic acid microarray, and instructions for using the nucleic acid microarray to determine the level of methylation of the one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2.
  • the cancer is melanoma.
  • methods for screening for a candidate therapeutic agent for treatment of cancer include contacting an agent with a sample that includes cancer cells, determining the level of methylation of one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2, and comparing the level of methylation of the one or more genes in the sample to the level of methylation of the one or more genes in a control sample, wherein a lower level of methylation of the one or more genes in the sample contacted with the agent compared to the control level, indicates that the agent is a candidate therapeutic agent for treatment of cancer.
  • the cancer is melanoma.
  • the level of methylation of the one or more genes is determined by methylation-specific PCR, methylation-inhibitor analysis, methylation sensitive restriction analysis, sequencing of bisulfite modified DNA, methylation-sensitive single nucleotide primer extension, MethyLight analysis, pyrosequencing, or combined bisulfite restriction analysis.
  • the sample includes cultured cells.
  • the sample is a sample obtained from a subject.
  • methods for monitoring response to a cancer treatment in a subject with cancer include detecting a level of methylation of one or more of the genes QPCT, LXN, PCSKl , MFAP2, WFDCl , CDH8, and LRRC2 in a first sample obtained from the subject, administering the cancer treatment to the subject, detecting the level of methylation of the one or more genes in a second sample, wherein the second sample is obtained from the subject after treatment and at a time later than the first sample, and comparing the level of methylation of the one or more genes in the first sample with the level of methylation of the one or more genes in the second sample, wherein a lower level of methylation of the one or more genes in the second sample than in the first sample indicates that the subject is responsive to the cancer treatment.
  • the cancer is melanoma.
  • the treatment includes chemotherapy, radiation, and/or surgical therapy.
  • the level of methylation of the one or more genes is determined by methylation-specific PCR, methylation-inhibitor analysis, methylation sensitive restriction analysis, sequencing of bisulfite modified DNA, methylation-sensitive single nucleotide primer extension, MethyLight analysis, pyrosequencing, or combined bisulfite restriction analysis.
  • methods for selecting a course of treatment of a subject having or suspected of having cancer include detecting a level of methylation of one or more of the genes QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8I and LRRC2 in a sample obtained from a subject, comparing the level of methylation of the one or more genes to a control level of methylation of the one or more genes, determining the stage and/or type of cancer of the subject based at least in part on the difference in the level of methylation of the one or more genes in the sample compared to the control level of methylation, and selecting a course of treatment for the subject appropriate to the stage and/or type of cancer of the subject.
  • the cancer is melanoma.
  • the level of methylation of the one or more genes is determined by methylation-specific PCR, methylation-inhibitor analysis, methylation sensitive restriction analysis, sequencing of bisulfite modified DNA, methylation-sensitive single nucleotide primer extension, MethyLight analysis, pyrosequencing, or combined bisulfite restriction analysis.
  • the treatment includes chemotherapy, radiation, and/or surgical therapy.
  • Fig. 1 shows a schematic diagram of expression changes induced by 5AzadC treatment in melanoma cell lines.
  • Fig. IA shows changes in expression of selected candidate genes upon 48h 5AzadC treatment validated by quantitative RT-PCR analysis.
  • Fig. IB shows expression of the genes in untreated melanoma cell lines relative to primary melanocytes by quantitative RT-PCR.
  • Fig. 1C provides microarray profile data of genes showing significant expression changes upon 48h 5 AzadC treatment. Genes were ordered by median expression change relative to corresponding untreated cells.
  • Fig. 2 shows panels and a graph indicating methylation status of candidate melanoma associated genes.
  • Fig. 2 A shows the methylation status of candidate genes in nine melanoma cell lines. Black shading box indicates presence of methylation in the promoter region CpG island of the gene. *Indicates additional cell lines used in the bisulfite sequencing analysis originally not included in the microarray screening.
  • Fig. 2B shows the methylation status of the candidate tumor suppressor genes in a panel of twenty melanoma tumor tissues. Black shading box indicates presence of methylation in the promoter region CpG island of the gene.
  • Fig. 2C is a comparison of frequency of methylation of the candidate genes in tumors versus cell lines. Fig.
  • Fig. 3 provides panels indicating methylation status of individual CpG sites of target genes determined by bisulfite sequencing. The methylation status of each gene is indicated in the following panels: Fig. 3 A, LXN; Fig. 3B, BST2; Fig. 3C, GDFl 5; Fig. 3D, WFDCl; Fig. 3E, CDKNlC; Fig. 3F, PTGS2; Fig. 3G, PCSKl; Fig. 3H, LRRC2; Fig. 31, CDH8; Fig. 3 J, QPCT; Fig. 3K, HOXBl 3; Fig. 3L, DNAJCl 5; Fig. 3M, COLl A2; Fig.
  • Fig. 4 provides histograms showing results of analysis of expression changes following
  • Fig. 4A, Fig. 4B, Fig. 4C, Fig. 4D, Fig. 4E, and Fig. 4F show results for HOXBl 3, LXN, GDF15/PLAB, SYK, DNAJCl 5/MCJ, and EPB41L3/DAL1, respectively. Shaded circles on top of the bars indicate presence of methylation in the promoter region of the gene in the corresponding cell line.
  • Fig. 5 shows electropherograms depicting bisulfite sequencing of the QPCT promoter region.
  • Fig. 5A represents the original sequence for comparison (SEQ ID NO:58).
  • Fig. 5B represents the sequence of the QPCT promoter region in primary cultured melanocytes (SEQ ID NO.59);
  • Fig. 5C represents the sequence of the QPCT promoter region in the untreated MeUuSo melanoma cell line (SEQ ID NO:60);
  • Fig. 5D represents the sequence of the QPCT promoter region in the MeIJuSo melanoma cell line after 48 h treatment with 5AzadC (SEQ ID NO:59), demonstrating demethylation of the promoter region.
  • Fig. 6 provides histograms and a digitized image of a Western blot showing SYK expression in melanoma cell lines and melanoma tumor samples.
  • Fig. 6 A shows results of quantitative RT-PCR analysis of SYK mRNA expression in melanoma cell lines compared to primary human melanocytes.
  • Fig. 6B shows a Western blot of SYK expression in melanoma cell lines.
  • Fig. 6C shows results of quantitative RT-PCR analysis of SYK mRNA expression in tumor samples compared to melanocytes.
  • Fig. 7 provides histograms and digitized images of Western blots demonstrating SYK and HOXB 13 expression in Xenografted tumors.
  • Fig. 7 A shows average weights of tumors formed by vector and SYK transfected clone in nude mice.
  • SYK 7 represents grouping of smaller tumors ( ⁇ 80 mg)
  • S YK7* represents grouping of larger tumors (>80 mg) at the endpoint of experiment.
  • Fig. 7B shows results of RT-PCR analysis of SYK expression in tumors formed by the SYK7 clone. Expression of SYK is present in the clonal transfected cell line (SYK7, Fig.
  • Fig. 7D shows results of RT-PCR analysis of HOXB 13 expression in tumors formed by HOXB 13 clones.
  • Fig. 7D shows expression of SYK in stably transfected clonal cell lines compared to primary human melanocytes and the parental MeUuSo cell line.
  • Fig. 7E shows expression of HOXB 13 in stably transfected clonal cell lines compared to primary human melanocytes and the parental MeUuSo cell line.
  • Fig 8 shows graphs, digitized images of expression assays, and digitized images of tumor growth.
  • Fig. 8A-D show tumor suppressor characteristics of HOXBl 3 in-vitro and in-vivo;
  • Fig. 8A shows results of an in-vitro proliferation assay of HOXB 13 transfected MeUuSo clones compared to vector controls (pTRE). The results presented are an average of 3 replicates counted on a flow cytometer (p-value 120 h ⁇ 0.01).
  • Fig. 8B shows representative plates of a colony formation assay comparing vector and HOXBl 3 transfected MeUuSo clones.
  • FIG. 8C shows results of tumor growth of subcutaneous xenografts of vector and HOXBl 3 transfected clones in nude mice. The results presented here represent average of four subcutaneous injections (p-value 10 weeks ⁇ 0.05).
  • Fig. 8D shows images of representative tumors formed by vector controls and HOXB 13 transfected MeUuSo cell line.
  • Figs. 8E-H show tumor suppressor characteristics of SYK in-vitro and in-vivo.
  • Fig. 8E shows results of an in-vitro proliferation assay of SYK transfected MeUuSo clones compared to vector controls. The results represent an average of three replicates counted on a flow cytometer (p-value 12Oh ⁇ 0.01).
  • FIG. 8F shows representative plates of a colony formation assay comparing vector and SYK transfected MeUuSo clones.
  • Fig. 8G shows tumor growth of subcutaneous xenografts of vector and SYK transfected clones in nude mice showing tumor kinetics over a 10 week period. The results presented here represent average of four subcutaneous injections (p-value 10 weeks ⁇ 0.05).
  • Fig. 8H shows average weights of tumors formed by vector control and HOXBl 3, SYK transfected clones at 10 weeks following xenografting. The results represent an average of 8 tumors each of vector, HOXB 13 transfected clones and 4 tumors of SYK transfected clone (p-value ⁇ 0.01).
  • Fig. 9 provides histograms and digitized images of Western blots demonstrating HOXB 13 expression in melanoma cell lines and melanoma tumor samples.
  • Fig. 9A shows results of quantitative RT-PCR analysis of HOXB 13 mRNA expression in melanoma cell lines compared to primary human melanocytes.
  • Fig. 9B shows a Western blot of HOXB 13 expression in melanoma cell lines.
  • Fig. 9C shows results of quantitative RT-PCR analysis of HOXB 13 mRNA expression in tumor samples compared to melanocytes.
  • Cancers may arise from any number of cellular perturbations in a cell. Most of these perturbations take the form of a genetic mutation at the genomic DNA level. Genetic mutations can in turn manifest their effects in a number of ways including alterations in expression levels and/or function of an mRNA or a polypeptide. The end result is an uncontrolled growth of the mutated population of cells as a result of increased proliferative rates, decreased apoptotic rates, and/or failure to respond to normal growth-control signals.
  • Gene loci that are altered in the progression of such disorders are not always the primary or direct target of the initial mutation. Rather, a mutation may exist in a genomic locus that encodes an "upstream" factor. Mutation of the upstream factor may not produce a malignant phenotype to a cell by itself, but the mutation of the upstream factor may impact one or more "downstream” factors, the genomic locus of which remains essentially wild type.
  • One such upstream factor is a factor capable of methylating genomic sequences.
  • Abnormal methylation of genomic loci has been reported to cause altered expression levels from that genomic locus.
  • the mammalian genome is widely methylated except for regions rich in CG dinucleotides (e.g., CpG islands), which in normal cells are undermethylated as compared to the rest of the genome.
  • DNA methylation is one form of epigenetic change and involves the covalent addition of a methyl group to cytosine residues in CpG dinucleotides by DNA methyltransferases.
  • Abnormal methylation, particularly at CpG islands may be accompanied by gene silencing and may be one mechanism responsible for the inactivation of several tumor suppressor genes in human cancers.
  • the invention described herein is premised, in part, on the identification of genes that are abnormally methylated in cancer.
  • genes that are hypermethylated facilitates analysis of cancer and cancer treatments. For example, it has been discovered that an increased level of methylation of certain genes may lead to gene activation (e.g., epigenic silencing).
  • Epigenic silencing of a gene may prevent normal functioning of the gene, and if the normal function of the gene includes tumor suppression activity, the epigenic silencing resulting from hypermethylation (e.g., in CpG islands of the gene) may result in cancer. Modifying the amount of such hypermethylation of the genes of the invention may be useful to prevent and/or treat cancer.
  • Compounds that decrease hypermethylation of one or more genes of the invention may result in more normal functioning of the genes and a corresponding prevention or treatment of cancer or of a precancerous condition.
  • methods to assess hypermethylation in the genes of the invention may be used to monitor the onset, progression, and/or regression of cancer by monitoring levels of methylation in a cell or subject and determining the effect of a candidate therapeutic compound on the level of methylation of such genes.
  • Such monitoring may also be used to assess the efficacy of treatments administered to an individual subject by monitoring the level of methylation of genes of the invention in a sample or subject before, during, and after administration of a treatment regimen (e.g., a therapeutic agent).
  • the present invention provides methylation assays for assessing a methylation state of a gene in a cell, tissue, and/or subject.
  • methylation assay refers to any assay for determining the methylation state of a CpG dinucleotide within a sequence of DNA.
  • methylation state refers to the presence or absence of 5- methylcytosine ("5-mCyt") at one or a plurality of CpG dinucleotides within a DNA sequence.
  • hypomethylation refers to a methylation state that corresponds to an increased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a normal control DNA sample.
  • hypomethylation refers to the methylation state corresponding to a decreased presence of 5- mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a normal control DNA sample.
  • Methods of the invention may include determining a level of methylation of CpG islands of genes of the invention in a sample and comparing the level to a control level as a measure of whether the amount of methylation is abnormal compared to the control level of methylation (e.g., the level of methylation in a control sample). Such comparisons may be useful for diagnosing cancer in a cell and/or subject and for assays to identify treatments for cancer and for the selection of treatment paradigms for subjects diagnosed with cancer.
  • CpG Island refers to a contiguous region of genomic DNA that satisfies the criteria of (1) having a frequency of CpG dinucleotides corresponding to an "Observed/Expected Ratio”>0.6), and (2) having a "GC Content”>0.5.
  • GC Content refers, within a particular DNA sequence, to the [(number of C bases+number of G bases)/band length for each fragment].
  • observed/expected ratio means the frequency of CpG dinucleotides within a particular DNA sequence, and corresponds to the [number of CpG sites/(number of C bases x number of G bases)] x band length for each fragment.
  • CpG islands are typically, but not always, between about 0.2 to about 1 kb in length. CpG islands are readily identifiable by those of ordinary skill in the art using routine procedures.
  • a CpG island sequence associated with a particular SEQ ID NO sequence of the present invention is that contiguous sequence of genomic DNA that encompasses at least one nucleotide of the particular SEQ ID NO sequence, and satisfies the criteria of having both a frequency of CpG dinucleotides corresponding to an Observed/Expected Ratio>0.6), and a GC Content>0.5.
  • the invention in part, also includes nucleic acid sequences that include methylated CpG islands, compositions comprising nucleic acids that comprise methylated CpG islands that can be used to assess methylation status in a cell or subject.
  • Genes of the invention include the QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8, LRRC2, CYPlBl, COLl A2, GDF15, RARB, TM, 3-OST-2, RASSFlA, ACS/TMS1, BST2, DNAJCl 5, CDKNIc, MGMT, SYK, MiBl, HOXBl 3, PTGS2, DAPK, APC,pl6 INK4A ,p27 Kif " , PRDX2, PYCARD, CDKN2A, CDKNlB and DALl genes.
  • Sequences of exemplary CpG island regions of some genes of the invention including QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8, and LRRC2 are provided in Table 3.
  • the invention relates, in part, to the use of one or more methylation assays for determining the methylation state of CpG islands of at least one or more of the QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2 genes in cells, tissues, and/or subjects to assess the cancer status of the cell, tissue, and/or subject.
  • Methods and kits of the invention are highly sensitive and may permit the identification of abnormal methylation of a gene of the invention in very rare cell events.
  • the methods may be as sensitive as to permit detection of having abnormal methylation of one or more genes of the invention that are as rare as 1 abnormal cell per 10,000 cells, 1 abnormal cell per 100,000 cells, 1 abnormal cell per 1,000,000 cells etc.( including all values below and in between).
  • the methods of the invention permit detection and diagnosis of cancer and/or precancerous conditions based on very rare cellular events (e.g., isolated cancer cells circulating in peripheral blood and/or rare metastatic cells in a lymph node, etc.) These rare events may be clinically significant in the diagnosis and monitoring of cancer and/or precancerous conditions and may be detectable using methods and kits of the invention.
  • cancer status means the presence or absence of cancer, the stage of a cancer, and/or the detection of the presence, absence, or stage of a precancerous condition in a cell, tissue, and/or subject.
  • An elevated level of methylation of a CpG island of one or more of the QPCT, LXN, PCSKl , MFAP2, WFDCl , CDH8 and LRRC2 genes versus a control level of methylation may indicate that the tested cell or subject has cancer or a precancerous condition.
  • a precancerous condition is a condition that would not be clinically diagnosed as cancer but is indicative of an abnormality in the gene function in the cell, tissue, and/or subject that may be a precursor, or may lead to cancer.
  • precancerous conditions although not intended to be limiting include dysplasia, benign neoplasia, hyperplasia, atypical hyperplasia , metaplasia, carcinoma in situ, etc.
  • the presence of hypermethylation of CpG islands of one or more of the QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2 genes may be asymptomatic and yet indicate the subject will develop cancer or more advanced cancer if left untreated.
  • Treatments for precancerous conditions may include surgery, chemotherapy, radiotherapy, etc. and treatments may be selected and their efficacy monitored using methods of the invention.
  • Compounds and strategies for treating pre-cancerous conditions may be identified using assays and screening methods of the invention.
  • treatment of a precancerous condition may prevent or delay its development into cancer.
  • the invention in part, provides methods for analyzing samples for features associated with the development of cancer or precancererous conditions, characterized in that the nucleic acid of at least one of QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8, LRRC2, and optionally at least one of CYPlBl, COLl A2, GDFl 5, RARB, TM, 3-OST-2, RASSFlA, ACSZTMSl 1 BST2, DNAJCl 5, CDKNIc, MGMT, SYK, MiBl, HOXBl 3, PTGS2, DAPK, APC, pl6 lNK4A , p27 Kipl , PRDX2, PYCARD, CDKN2A, CDKNlB and DALl is/are contacted with a reagent or series of reagents capable of distinguishing between methylated and non methylated CpG dinucleotides these genes.
  • the methylation state of only a portion of a gene of the invention that is present in a cell or subject need be assessed in a determination of the methylation state and cancer status of a cell or subject.
  • the entire gene sequence need not be assayed.
  • the term "genomic sequence" is used herein to refer to a region of a gene of the invention that may be assessed in a determination of the cancer status of a cell, tissue, or subject.
  • a genomic sequence may correspond to a region of a gene of the invention that is assayed for methylation state and the genomic sequence may provide information on the methylation state of the gene of the invention that is sufficient to determine the gene's status as abnormally methylated (e.g., hypermethylated or hypomethylated) and the cancer or precancerous condition status of the cell or subject tested.
  • abnormally methylated e.g., hypermethylated or hypomethylated
  • Detection of a methylation state of one or more genes of the invention may be used in combination with assessment of methylation state in one or more other genes of the invention that may also be abnormally methylated (hyper or hypomethylyated) in cancer and/or precancerous conditions.
  • Additional genes whose methylation levels may be assessed in combination with one or more genes of the invention include, but are not limited to: CYPlBl, COLl A2, GDFl 5, RARB, TM, 3-OST-2, RASSFlA, ACS/TMS1, BST2, DNAJCl 5, CDKNIc, MGMT, SYK, MiBl, HOXBl 3, PTGS2, DAPK, APC,pl6 INK4A ,p27 Kipl , PRDX2, PYCARD, CDKN2A, CDKNlB and DALl.
  • CYPlBl CYPlBl
  • COLl A2 GDFl 5
  • RARB RARB
  • TM 3-OST-2
  • RASSFlA ACS/TMS1, BST2
  • DNAJCl 5 CDKNIc
  • MGMT SYK
  • MiBl HOXBl 3
  • PTGS2 DAPK
  • allelic variants of genes and/or sequence provided herein.
  • allelic variation in sequences of the genes assayed for methylation states using methods of invention, including wild-type gene sequences and/or mutant gene sequences.
  • allelic variant means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides with altered amino acid sequences.
  • allelic variations may occur in full- length wild-type and mutant genes and may also occur in CpG island regions of genes.
  • QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2 genes, and other genes disclosed herein as useful in methods of the invention may be allelic variants of wild-type genes or may be mutant gene sequences.
  • determination of a methylation state of one or more genes of the invention may include determination of methylation state of wild-type and/or mutant form of the gene.
  • the invention may include examination of a full CpG island region of a gene of the invention, or may include assessment of a portion of a CpG island region of a gene that is sufficient to provide information on methylation state of the gene. For example, detection of hypermethylation in a portion of a CpG island of a gene of the invention can be used to ascertain the methylation state of the gene as a whole.
  • a portion of a CpG island region that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides shorter (including all integers in between each of the foregoing integers) than the full length CpG island region of a gene may be assessed for its methylation state and the results used in the diagnosis of cancer or a precancerous condition as described herein.
  • Nucleic acid primers and DNA sequences and genes disclosed herein for use in assessing methylation states of genes for diagnose and assessment of cancer and precancerous conditions are meant to be exemplary.
  • Those of ordinary skill in the art will be able to use routine procedures to recognize, design, and/or use alternative primers and/or sequences to assess methylation states of genes of the invention identified herein, including at least the QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2 genes and CYPlBl, COLl A2, GDFl 5, RARB, TM, 3-OST-2, RASSFlA, ACS/TMS1, BST2, DNAJCl 5, CDKNIc, MGMT, SYK, MiBl, HOXBl 3, PTGS2, DAPK, APC, pl6 INK4A ,p27 Kipl , PRDX2, PYCARD, CDKN2A, CDKNlB and DALl genes or other genes useful
  • Methods for assaying methylation states of one or more genes of the invention may be carried out in cells from culture, cells in solution, and/or on samples obtained from subjects.
  • a subject is a human or a non-human animal, including, but not limited to a non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent. In all embodiments, human subjects are preferred.
  • Methods of the invention may be used to detect abnormal levels of methylation or expression products of genes in subjects not yet diagnosed with cancer.
  • methods of the invention may be applied to subjects who have been diagnosed with cancer.
  • a sample may comprise one or more cells.
  • a sample may originate from a subject or culture, may be a lysate of a sample from a subject, and/or may be partially processed prior to use in methods of the invention.
  • a sample from a subject or culture may be processed to obtain DNA for use in assays for methylation as described herein.
  • an initial step in an assay of methylation states that may be used use in methods and/or kits of the invention may include isolation of a genomic DNA sample from a cell, tissue, and/or subject. Extraction of DNA may be by any suitable means, including to routine methods used by those of ordinary skill in the art such as methods that include the use of detergent lysates, sonification, and vortexing with glass beads, etc.
  • genomic double-stranded DNA may be used in further analysis of the methylation state of a sample.
  • Methods of the invention may also include assessment of expression products of one or more genes of the invention in a sample.
  • sample means any animal material containing DNA or RNA, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents.
  • a sample containing nucleic acids can be drawn from any source and can be natural or synthetic.
  • a sample containing nucleic acids may contain of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or copolymers of deoxyribonucleic acids and ribonucleic acids or combinations thereof.
  • a sample may have been subject to purification (e. g. extraction) or other treatment.
  • sample may also refer to a "biological sample.”
  • biological sample may refer to a whole organism or a subset of its tissues, cells or component parts (e. g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, stool, vaginal fluid, and semen, etc.).
  • a “biological sample” may also refer to a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, stool, milk, blood cells, tumors, or organs, etc.
  • a “biological sample” may also refer to medium, such as a nutrient broth or gel in which an organism or cell has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
  • Sample sources may include tissues, including, but not limited to lymph tissues; body fluids (e.g., blood, lymph fluid, etc.), cultured cells; cell lines; histological slides; tissue embedded in paraffin; etc.
  • tissue refers to both localized and disseminated cell populations including, but not limited to: brain, heart, serum, breast, colon, bladder, epidermis, skin, uterus, prostate, stomach, testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid gland, salivary gland, mammary gland, kidney, liver, intestine, spleen, thymus, bone marrow, trachea, and lung.
  • Biological fluids include, but are not limited to, blood, lymph fluid, cerebrospinal fluid, tears, saliva, urine, and feces, etc.
  • a sample comprises a blood or lymph node sample. Invasive and non-invasive techniques can be used to obtain such samples and are well documented in the art.
  • a control cell sample may include a cell, a tissue, or may be a lysate of either.
  • a control sample may be a sample from a cell or subject that is free of cancer and/or free of a precancerous condition.
  • a control sample may be a sample that is from a cell or subject that has cancer or a precancerous condition.
  • a biological sample corresponds to the amount and type of DNA and/or expression products present in a parent cell from which the sample was derived. If the sample is a melanoma tumor tissue sample or a melanoma cell line, cultured melanocytes may be, but need not be, used as a control.
  • the term "cancer” refers to an uncontrolled growth of cells that may interfere with the normal functioning of the bodily organs and systems. Cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • a metastasis is a cancer cell or group of cancer cells, distinct from the primary tumor location resulting from the dissemination of cancer cells from the primary tumor to other parts of the body.
  • the subject may be monitored for the presence of in transit metastases, e.g., cancer cells in the process of dissemination.
  • Methods of the invention may be used to assess the status of primary and/or metastatic cancer.
  • cancer includes, but is not limited to the following types of cancer, breast cancer, biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chromic myelogenous leukemia, multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epi
  • Methods of the invention may be useful to assess characteristics of cancer in a cell or subject (e.g., for assessment of melanoma in a cell or subject). For example, characteristics such as whether a cancer is metastatic or non-metastatic and/or the present status or stage of a cancer may be assessed using methods of the invention, hi general, cancer staging is based on a combination of clinical information obtained by physical examination, radiologic examination, etc. and pathogic information, which may be based on pathological examination of a tumor. Methods of the invention can provide pathological staging information about a cancer in a cell or subject.
  • an amount of methylation of one or more genes in a sample may be useful to assess the pathologic status of a cancer.
  • Genes that form the basis of the invention can be assessed singly or in sets of two or more (e.g, in panels). The use of a single gene assessment and/or use of a gene panel for multiple gene assessment allows for rapid, specific analysis of cancer or a precancerous condition that are associated with methylation states of included genomic sequences.
  • a gene panel may be used with high efficiency for the diagnosis and monitoring of and the analysis of cancer, (e.g., melanoma) and/or precancerous conditions as described herein.
  • a gene panel can also be used in screens for treatments for cancer and/or precancerous conditions.
  • the invention includes assays of methylation states of one or more CpG dinucleotides of more than one gene of the invention and in other embodiments, the invention includes assay of the methylation state of one or more CpG dinucleotides of a single gene of the invention for diagnosis, staging, and analysis of cancer in cells, tissues, and/or subjects.
  • Methods for assaying methylation states of genomic sequences of interest are well- known in the art. Examples of methods of assaying methylation states of genes of interest are provided herein, but are not intended to be limiting. Non-limiting examples of methylation assay methods are disclosed in patent documents such as US Patent No. 5,871,917; US Patent Application No. 20040081976; US Patent No. 6,893,820, US Patent No. 5,871,917; US Patent No. 5,786,146, US Patent No. 4,839,060, US Patent No. 6,200,756, US Patent No. 7,112,404, and US Patent No. 6,331,393, each of which is incorporated by reference herein in its entirety. Those of ordinary skill in the art will recognize that any suitable method that permits the assessment of the level of methylation of a CpG dinucleotide in a gene of the invention can be used in methods and kits of the invention.
  • Assay of Methylation State A level of methylation of one or more genes can be determined using a number of routine techniques and methods described in the art.
  • Useful techniques for assay of methylation state in a sample include, but are not limited to: methylation-specific PCR (MSP) and other PCR based methods, methylation-inhibitor analysis, methylation-sensitive restriction analysis, sequencing of bisulfite-modified DNA, methylation-sensitive single nucleotide primer extension (Ms-SnuPE), MethyLight analysis, and combined bisulfite restriction analysis (COBRA), pyrosequencing, etc.
  • MSP methylation-specific PCR
  • Ms-SnuPE methylation-sensitive single nucleotide primer extension
  • COBRA combined bisulfite restriction analysis
  • a method for determining the level of methylation of one or more genes is methylation specific PCR (i.e., MSP), which is disclosed in US patent 5,786,146 (incorporated by reference herein in its entirety). This method is based on the differential reactivity of cytosine and 5-methylcytosine with sodium bisulfite. In the presence of sodium bisulfite, cytosines are deaminated to uracils and 5-methylcytosines remain as cytosines.
  • MSP methylation specific PCR
  • methylation-specific PCR may provide a quantitative and qualitative assessment of methylation, e.g., may include the use of MethyLight, TaqMan, or Sybr-based systems.
  • two reactions are performed for each sample, one with methylation specific primers, and one with non-methylation-specific primers.
  • Another PCR-based method for determining the level of methylation of one or more genes that can be used in the methods and kits of the invention was reported by McGrew and Rosenthal and involves the use of ligation-mediated PCR (Biotechniques 1993, 15: 722- 729), which is hereby incorporated by reference.
  • Ligation-mediated PCR involves the measurement of conversion of large genomic DNA fragments to shorter DNA fragments as a function of demethylation. The cleavage of large genomic DNA is accomplished using pairs of non-isoschizometic enzymes, one of which is methylation specific.
  • the digestion products are then amplified with ligation-mediated, radiolabeled PCR, and used as a measure of cleavage with the methylation sensitive restriction enzyme. Specifically, the ratio of the two amplified fragments is related to the degree of methylation at the particular restriction site. Internal control of the amplification reaction confers the quantitative aspect of the approach.
  • Methylation-inhibitor analysis comprises differential analysis of the level of methylation of one or more genes in samples treated with a DNA methylation inhibitor compared to untreated samples, and analysis of these samples using any suitable method for analysis of differential expression, including, but not limited to use of a nucleic acid microarray.
  • a sample may include one or more genes that have a high level of methylation. Genes with a high level of methylation will have a low expression level. Treatment of the sample with a DNA methylation inhibitor results in inactivation or repression of the enzyme that methylates DNA, resulting in a lower level of methylation of the one or more genes than would be present in the absence of the inhibitor.
  • a decrease in methylation of the one or ore genes will subsequently result in an increase of expression of the one or more genes.
  • This difference in expression between a DNA methylation-inhibitor-treated sample and a control sample can be analyzed on using any suitable differential expression method, for example, using a nucleic acid microarray.
  • a DNA methylation inhibitor is an agent that directly or indirectly causes a reduction in the level of methylation of a nucleic acid molecule.
  • DNA methylation inhibitors are well known and routinely utilized in the art and include, but are not limited to, inhibitors of methylating enzymes such as methylases and methyltransferases.
  • Non-limiting examples of DNA methylation inhibitors include 5-azacytidine, 5-aza-2'deoxycytidine (also known as Decitabine in Europe), 5, 6-dihydro-5-azacytidine, 5, 6-dihydro-5-aza-2'deoxycytidine, 5- fluorocytidine, 5-fluoro-2'deoxycytidine, and short oligonucleotides containing 5-aza- 2'deoxycytosine, 5, 6-dihydro-5-aza-2'deoxycytosine, and 5-fluoro-2'deoxycytosine, and procainamide, Zebularine, and (-)-egallocatechin-3-gallate.
  • Methylation-sensitive restriction analysis is derived from the existence in nature of restriction enzymes that are methylation sensitive (i.e., these enzymes do not recognize restriction sites that contain methylated residues). For example, the restriction enzyme Notl recognizes the sequence containing 2 'CG' dinucleotides. If either of the CG sites is methylated, the enzyme will not digest DNA. This fact has been utilized in the analysis of DNA methylation in genomic DNA. In this approach, DNA is digested with a methylation-sensitive restriction enzyme and then electrophoresed on an agarose gel that separates DNA based on its size.
  • the DNA is then transferred to a membrane and hybridized to a detectably labeled probe (e.g., a radiolabeled or fluorescently labeled probe), as is routinely done in a Southern analysis. Based on the sizes of bands that hybridize to the probe, the digested, and therefore unmethylated, DNA can be distinguished from the undigested, and therefore methylated, DNA.
  • a detectably labeled probe e.g., a radiolabeled or fluorescently labeled probe
  • Other methylation-sensitive enzymes include, but are not limited to: SacII, Eagl, Smal, Thai, Hpall, all of which are commercially available.
  • Genomic sequencing of bisulfite modified DNA is another method for determining the level of methylation of one more genes. Like other methylation assay methods described herein, this method is based on the differential reactivity of cytosine and 5-methylcytosine with sodium bisulfite. In this approach however, primers are designed to avoid potential methylation sites (e.g., CG dinucleotides) and a non-specific PCR is performed in order to amplify all alleles equally. Following amplification, the PCR product is sequenced directly, or can be subcloned into a plasmid and individual subclones sequenced. The sequence of amplified product is compared to that of non-bisulfite treated DNA.
  • primers are designed to avoid potential methylation sites (e.g., CG dinucleotides) and a non-specific PCR is performed in order to amplify all alleles equally.
  • the PCR product is sequenced directly, or can be subcloned into a
  • CG dinucleotides present in the non-bisulfite-treated sample that read as TG as a result of bisulfite treatment were unmethylated in the original sample, and those that continue to read as CG even after bisulfite treatment were originally methylated.
  • This approach is quantitative to the extent that it provides an absolute number of methylated residues in a particular nucleic acid sequence.
  • Cytosine methylation can also be measured using an approach that combines automated genomic DNA sequencing and GENESCAN analysis. This approach has been reported by Paul et al. (Biotechniques 1996, 21:126-133), which is hereby incorporated by reference. This technique also requires bisulfite treatment and PCR amplification of DNA. Cloning and sequencing of the modified and amplified products is then performed to determine the methylation of individual DNA molecules. The sequencing of the entire population of amplified products provides the average methylation status over the population, and thus may not be appropriate if the methylation status of individual molecules is desired. By employing fluorescence-based automated genomic sequencing, Paul et al. were able to directly quantitate methylation status of any cytosine residue in a DNA molecule. The technique involves sequencing only cytosine and thymine residues of modified and amplified DNA and using fluorescent dyes to identify and visualize signals from these residues.
  • GENESCAN analysis is then performed to estimate methylation at every cytosine in a rapid and accurate manner.
  • the approach permits a rapid overview of DNA methylation profiles for a number of DNA molecules.
  • Ms-SnuPE Methylation-sensitive single nucleotide primer extension
  • genomic DNA is first reacted with sodium bisulfite to convert unmethylated cytosine to uracil without modification of 5-methylcytosine, as in other approaches described herein.
  • the bisulfite-treated DNA is then amplified using PCR primers specific for bisulfite- converted DNA.
  • the amplified product is then used as a template for methylation analysis at the CpG site(s) of interest.
  • the method is amenable to the analysis of small amounts of DNA.
  • Ms-SNuPE can be used in the analysis of microdissected pathology sections and other samples.
  • MethyLight analysis is another PCR-based technique that can be used in methods and kits of the invention.
  • MethyLight analysis includes analysis of bisulfite-treated DNA. Bisulfite treatment of DNA results in conversion of unmethylated cytosines to uracil but leaves methylated cytosines unaffected.
  • Detectable-label-based PCR e.g., fluorescence-label based PCR
  • primers that either overlap CpG methylation sites or primers that do not overlap any CpG sequences.
  • Quantitative sequence discrimination can occur either at the level of the PCR amplification process or at the probe hybridization process or both. The technique is described in detail in Eads et al. (Nucleic Acid Res. 2000, 28: e32), which is hereby incorporated by reference.
  • COBRA bisulfite restriction analysis
  • nucleic acids used in methods of the invention may be detectably labeled.
  • detectable label means a molecule preferably selected from, but not limited to, fluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent, and bioluminescent molecules.
  • detectable labels are available for use in methods of the invention and may include labels that provide direct detection (e.g., fluorescence, colorimetric, or optical, etc.) or indirect detection (e.g., enzyme- generated luminescence, epitope tag such as the FLAG epitope, enzyme tag such as horseradish peroxidase, labeled antibody, etc.).
  • a non-limiting example of use of a detectable label in a method of the invention is the incorporation of a fluorescent or radioactive label in an amplification reaction (e.g., in PCR).
  • Other methods that may use detectable labels include, but are not limited to methylation assays such as the MethyLight assay, etc.
  • a variety of methods may be used to detect a detectable label depending on the nature of the label and other assay components. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for using and detecting labels are well known to those of ordinary skill in the art.
  • Methods of the invention relate, in part, to assessment of abnormal methylation amounts and patterns of genes of the invention.
  • abnormal methylation amounts and/or patterns in genes may result in differential expression of the abnormally methylated gene.
  • Methods of the invention include, in addition to methods of assessing DNA to determine methylation levels, may also include additional methods of assaying for differential gene expression of one or more genes of the invention. Differentially expressed genes may be indicated by expression products (i.e., mRNA and/or proteins/polypeptides) that are differentially expressed in a sample as compared to a control. Abnormal methylation patterns in the DNA of a cell may result in differential expression of that DNA.
  • a gene that is differentially expressed or has a differential level of methylation in a sample compared to a control may be referred to herein as a cancer-associated gene.
  • a level of methylation of one or more cancer-associated genes of the invention can be at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%,. 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 10,000%, or more higher (including all percentages in between the listed percentages) than a control level of methylation of the one or more genes of the invention.
  • Differentially expressed genes can be identified in a number of ways. If the expression product is a nucleic acid (i.e., an mRNA), then the differentially expressed gene may be identified using techniques such as subtractive hybridization, differential display, representational difference analysis, or reverse transcriptase-quantitative PCR (RT-qPCR). An approach aimed at identifying differentially expressed transcripts may include conversion of RNA to at least a first strand cDNA.
  • RT-qPCR reverse transcriptase-quantitative PCR
  • Another technique that is useful for identifying differentially expressed transcripts involves DNA chip technology and cDNA microarray hybridization. Standard and custom- made DNA chips are commercially available from manufacturers such as Affymetrix and InCyte. High-throughput screening for difference expressed genes and sequences is readily accomplished (Von Stein, et al., Nucleic Acids Res, 1997, 25:2598-602; Carulli, et al., J Cell Biochem Suppl 1998, 30-31: 286-96). These and other methods of assaying for differential gene expression may be in methods of the invention to assess methylation of one or more genes of the invention.
  • the invention may also include methods of detecting expression products of genes of the invention. Such methods may be used to identify expression products of genes of the invention, e.g., proteins/polypeptides encoded by a gene of the invention. Such methods may be used for a number of purposes, including, but not limited to confirming the identity of one or more genes (e.g., as tumor suppressor genes), confirmation of a diagnosis, etc. of cancer and/or precancerous condition in cells and subjects, etc. Methods of the invention for detecting expression products of genes of the invention may include, but are not limited to ELISA, immunohistochemistry, immunofluorescence, Western blotting, etc. Those of skill in the art will be able to use such methods and/or alternative methods for the detection and assessment of expression products of one or more genes of the invention.
  • a level of methylation of one or more genes of the invention can be determined in a number of ways when carrying out the various methods of the invention.
  • a level of methylation of one or more genomic sequences is measured in relation to a level of unmethylated genomic sequences.
  • the measurement may be a relative measure, which can be expressed, for example, as a percentage of total positions for methylation in one or more genes of the invention, or as a percentage of the overall methylation level in set or panel of one or more genes of the invention.
  • relative amounts of methylated and unmethylated genomic sequences may be determined by measuring either the relative amount of methylated genomic sequence or the relative amount of unmethylated genomic sequence.
  • measuring the level of methylated genomic sequence may be carried out using a method that measures the relative amount of unmethylated genomic sequence.
  • Another measurement of the level of methylation of one or more genes of the invention may be a measurement of absolute level methylation of one or more genes of the invention. This could be expressed, for example, in methylation levels per unit of cells or tissue, or as fraction of methylated target sites/total target sites.
  • target site means a CpG dinucleotide.
  • Another measurement of the level of methylation of one or more genes of the invention may be a measurement of the change in the level of methylation of the one or more genes of the invention over time. This may be expressed in an absolute amount or may be expressed in terms of a percentage increase or decrease over time.
  • Methods of the invention for assessing levels of methylation of genomic sequences of the invention are useful to characterize methylation levels by monitoring changes in the absolute or relative amounts of methylation levels in a subject or sample (e.g., a cell culture) over time. For example, it is expected that an increase in methylation of one or more genes of the invention in a cell or tissue correlates with the presence and/or severity of cancer in the cell or tissue. Accordingly one can monitor levels of methylation of one or more genes of the invention over time to determine if the status of a subject's cancer or cancer cells are changing. Changes (e.g., an increase) in relative or absolute methylation levels of one or more genes of the invention that are greater than 0.1% may indicate an increasing abnormality.
  • the change in a level of methylation of the one or more genes of the invention that indicates an abnormality is greater than 0.2%, greater than 0.5%, greater than 1.0%, 2.0%, 3.0% , 4.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more.
  • Increases in amounts of methylation of one or more genes of the invention over time may indicate a change in cancer or precancerous condition status in a sample or subject.
  • levels of methylation of one or more genes of the invention can be determined using methods of the invention and are advantageously compared to controls according to the invention.
  • the control may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as in groups having normal amounts of methylation of the genomic sequence of the invention and groups having abnormal amounts of methylation of one or more genes of the invention.
  • Another example of comparative groups may be groups having cancer or a precancerous condition, groups that have symptoms of cancer or a precancerous condition, groups without cancer or a precancerous condition, and groups without symptoms of cancer or of a precancerous condition.
  • Another comparative group may be a group with a family history of cancer or a precancerous condition and a group without such a family history.
  • a predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest risk and lowest amounts of methylation of a genomic sequence of the invention and the highest quadrant or quintile being individuals with the highest risk and highest amounts of methylation of a genomic sequence of the invention.
  • the predetermined value will depend upon the particular population selected. For example, an apparently healthy population will have a different 'normal' range than will a population that is known to have a cancer or a precancerous condition. Accordingly, the predetermined value selected may take into account the category in which an individual or cell falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
  • "abnormal" means not normal as compared to a control. By abnormally high it is meant high relative to a selected control. Typically the control will be based on apparently healthy normal individuals in an appropriate age bracket or apparently healthy cells.
  • controls according to the invention may be, in addition to predetermined values, samples of materials tested in parallel with the experimental materials. Examples include samples from control populations or control samples generated through manufacture to be tested in parallel with the experimental samples.
  • One aspect of the present invention relates to the use of the methods of the invention for detecting levels of methylated genomic sequences in an in vitro (e.g., histological or cytological specimens, biopsies, fluid samples, and the like), and, in particular, to distinguish the level of methylation of one or more genes of the invention in the sample from the level of methylation of the one or more genes of the invention in a control sample or a subject.
  • This method involves utilizing a suitable method to assay the methylation state of one or more genes of the invention in a cell and or tissue sample.
  • Methods of the invention may be used for a variety of diagnostic and experimental methods, including, but not limited to (1) diagnosis of cancer and precancerous conditions; (2) determining onset, progression, and/or regression of cancer or a cancerous condition in a subject; (3) characterizing the impact of the level of methylated gene sequences on cancer or a precancerous condition in a subject; (4) evaluating a treatment for altering the level of methylation of a gene of the invention in a subject; (5) evaluating treatments for cancer or a precancerous condition; (6) selecting a treatment for cancer or a precancerous condition in a subject; and (7) monitoring efficacy of a treatment for cancer or for a precancerous condition.
  • subjects can be characterized, treatment regimens can be monitored, treatments can be selected and disease status can be better understood using the assays of the present invention.
  • Methods of the invention are useful in one aspect in methods for measuring the level of methylation of one or more genes of the invention as a diagnosis of cancer or a precancerous condition.
  • the impact of the level of methylation of one or more genes of the invention on a cell or subject thus can be measured due to the positive correlation between the level of methylation of the sequence and the stage of cancer or a precancerous condition in the cell or subject.
  • the level of methylation in a gene of the invention thus may correlate with the status of cancer or a precancerous condition in a subject.
  • a relatively high level of methylation of a one or more genes of the invention may reflect a more severe type of cancer and/or a more advanced stage of cancer or a precancerous condition in a subject and a lower level of methylation (although still above normal levels, e.g., levels in a cancer-free subject) may reflect a less severe type of cancer and/or a less advanced stage of cancer or a precancerous condition in a subject.
  • diagnosis means the initial recognition of cancer or a precancerous condition in a cell, tissue, and/or subject and also may mean determination of the status or stage of cancer or a precancerous condition in the cell, tissue, and/or subject.
  • a diagnosis of cancer or a precancerous condition in a subject using a methods of the invention may include the determination of the stage of cancer, and/or pathogenic features of cancer in the subject.
  • the level of expression and/or the level of methylation of one or more genes of the invention can be used to determine the stage or status of cancer in a subject.
  • High levels of methylation of one or more genes of the invention in a sample from a subject may be correlated with advanced stage cancer, with concomitant advanced pathologic features in the cells and tissues of the subject.
  • a lower level of methylation of one or more genes of the invention in a sample from a subject may be correlated with a less advanced stage cancer, (e.g.
  • the relative levels and changes in the level of methylation and/or expression of one or more genes of the invention provide diagnostic information about the stage and status of cancer in a cell, tissue, and/or subject.
  • the level of methylation of some cancer-associated genes may be higher in cancer and some may be lower in cancer when compared to a control.
  • determination of the level of methylation of a set of cancer-associated genes in a sample may include some cancer-associated genes with higher methylation levels and some with lower methylation levels than that found in a control sample.
  • Some cancer-associated genes may be methylated at an early presymptomatic or precancerous stage at a level that is higher than a normal methylation level (e.g., a control level) and the methylation level may decrease (or increase) at a later presymptomatic and/or symptomatic state.
  • Some cancer-associated genes may be methylated or their expression repressed when the subject is asymptotic for the cancer. It will be understood that the level of methylation of some cancer-associated genes may be higher at more progressive stages of cancer, including metastasis. Due to methylation levels, expression of some cancer- associated genes of the invention may increase and some may decrease at different stages of cancer, (e.g., high methylation may result in low levels of expression and low or no methylation levels may result in higher levels of expression). Thus, in some embodiments, determination of a methylation pattern of more than one cancer-associated gene may be used as an indication of the stage or status of cancer in a cell, tissue, or subject.
  • the diagnosis of cancer is not limited to analysis of the methylation pattern of one or more genes and may be combined with diagnosis methods routine in the art.
  • diagnostic assays include but are not limited to histopathology, immunohistochemistry, flow cytometry, cytology, pathophysiological assays, including MRI and tomography, and biochemical assays.
  • Biochemical assays include but are not limited to mutation analysis, chromosomal analysis, ELISA analysis of specific proteins, platelet count etc.
  • Methods and/or kits of the invention can be used to screen patients for diseases associated with the presence of abnormal levels methylation (increased or decreased levels versus a control level) of one or more genes of the invention in which an abnormal level of methylation is associated with cancer or a precancerous condition.
  • the term "increased” means higher, for example higher versus a control level.
  • Methods of the invention may be used to diagnose the status and/or stage of cancer or a precancerous condition by assessing the level of methylation in one or more genes of the invention in a sample from a subject or culture of cells that have cancer or a precancerous condition.
  • the invention in some aspects, includes various assays to determine levels of methylation of one or more genes of the invention for which abnormal methylation (e.g., hypermethylation) is associated with cancer.
  • Methods and assays of the invention e.g., methylation assays, examples of which are provided herein
  • methods of the invention may be used to monitor changes in methylation state of genes in a cell sample and or a subject over time.
  • methods of the invention may be used to examine changes in specific methylation patterns of one or more genomic sequences (e.g., genes) of the invention, in a subject or cell sample (e.g., cell culture) over time.
  • Methods of the invention also permit monitoring of a cell or subject for residual disease (e.g., minimal residual cancer or a precancerous condition) and permit monitoring of a cell or subject's response to treatment of cancer or treatment of a precancerous condition.
  • methods of the invention may be used to diagnose or assess cancer or a precancerous condition in a subject.
  • Methods and/or kits of the invention can be used to obtain useful prognostic information by providing an early indicator of disease onset, progression, and/or regression.
  • the invention includes methods to monitor the onset, progression, or regression of cancer in a subject by, for example, obtaining samples at sequential times from a subject and assaying such samples for the level of methylation of one or more genes.
  • a subject may be suspected of having cancer or may be believed not to have cancer and in the latter case, the sample may serve as a normal baseline level for comparison with subsequent samples.
  • Onset of a condition is the initiation of the changes associated with the condition in a subject. Such changes may be evidenced by physiological symptoms, or may be clinically asymptomatic.
  • the onset of cancer may be followed by a period during which there may be cancer-associated pathogenic changes in the subject, even though clinical symptoms may not be evident at that time.
  • the progression of a condition follows onset and is the advancement of the pathogenic (e.g. physiological) elements of the condition, which may or may not be marked by an increase in clinical symptoms.
  • Onset of a cancer condition may be indicated by a change in the level of methylation of one or more genes of the invention in samples obtained form the subject. For example, if the level of methylation of one or more genes of the invention is lower in a first sample from a subject, than in a second or subsequent sample from the subject, it may indicate the onset or progression of cancer.
  • the regression of a condition may include a decrease in physiological characteristics of the condition, perhaps with a parallel reduction in symptoms, and may result from a treatment or may be a natural reversal in the condition.
  • Progression and regression of cancer may be generally indicated by the increase or decrease, respectively, of the level of methylation of one or more genes of the invention in a subject's samples over time. For example, if the level of methylation of one or more genes of the invention is low in a first sample from a subject and increased levels of methylation are determined to be present in a second or subsequent sample from the subject, it may indicate the progression of cancer and/or additional cancer pathogenesis. Regression of cancer and/or a reduction in pathogenesis may be indicated by finding that the level of methylation of one or more genes of the invention in a sample from a subject are decreased in a second or subsequent sample from the subject relative to the level in a first sample obtained from a subject at an earlier time. Methods of the invention can also be used to detect the presence of minimal residual disease. Assays for Efficacy of Treatment
  • Methods of the invention may also be used to assess the efficacy of a therapeutic treatment of cancer or a precancerous condition and for assessing the level of methylation of one or more genes of the invention in a subject at various time points.
  • a level of a subject's methylation of one or more genes of the invention can be obtained prior to the start of a therapeutic regimen (either prophylactic or as a treatment of cancer or a precancerous condition), during the treatment regimen, and/or after a treatment regimen, thus providing information on the effectiveness of the regimen in the subject.
  • Methods of the invention may be used to compare levels of methylation of one or more genes of the invention in two or more samples obtained from a subject at different times.
  • a sample is obtained from a subject, the subject is administered a treatment for cancer or a precancerous condition and a subsequence sample is obtained from the subject.
  • a comparison of a subject's levels of methylation of one or more genes of the invention measured in samples obtained at different times and/or on different days provides a measure of the status of the subject's cancer or precancerous condition and can be used to determine the effectiveness of any treatment for cancer or a precancerous condition in a subject.
  • treatment encompasses both prophylactic and therapeutic treatment, and it embraces the prevention of cancer and precancerous conditions, and the inhibition and/or amelioration of pre-existing cancers and precancerous conditions.
  • a subject receive treatment because the subject has been determined to be at risk of developing cancer or a precancerous condition, or alternatively, the subject may have such a disorder. Thus, a treatment may reduce or eliminate cancer altogether or prevent it from becoming worse.
  • evaluation of treatment means the comparison of a subject's levels of methylation of one or more genes of the invention is measured in samples obtained from the subject at different sample times, preferably at least one day apart.
  • the time to obtain the second sample from the subject is at least 5, 10, 20, 30, 40, 50, minutes after obtaining the first sample from the subject. In certain embodiments, the time to obtain the second sample from the subject is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, 96, 120 or more hours after obtaining the first sample from the subject.
  • a treatment may be a surgical treatment, a chemotherapy treatment, a radiation treatment, etc.
  • Cancer treatment methods are known in the art and the invention embraces all cancer treatment methods.
  • Non-limiting examples of cancer treatment are chemotherapy, radiation and surgical therapy.
  • Non-limiting examples of chemotherapy are monoclonal antibody therapy, the administration of DNA topoisomerase inhibitors, DNA synthesis inhibitors (like cisplatin), or inhibitors of cell surface receptors (like Gleevec® (immatinib mesylate)) and cytokine therapy.
  • a cancer treatment may include administration of an agent or compound that reduces methylation of one or more genomic sequences of the invention.
  • a treatment strategy may include administration of a methylation inhibitor to a cell or subject to reduce methylation of one or more genes of the invention that are hypermethylated in cancer or in a precancerous condition.
  • DNA methylation inhibitors include 5-azacytidine, 5-aza-2'deoxycytidine (also known as Decitabine in Europe), 5, 6- dihydro-5-azacytidine, 5, 6-dihydro-5-aza-2'deoxycytidine, 5-fluorocytidine, 5-fluoro- 2'deoxycytidine, and short oligonucleotides containing 5-aza-2'deoxycytosine, 5, 6-dihydro- 5-aza-2'deoxycytosine, and 5-fluoro-2'deoxycytosine.
  • assessments of candidate therapeutics can be tested in vitro by assessing any change in methylation levels of one or more genomic sequences of the invention that occur in response to contact of the cell with a candidate agent for treatment of cancer or a precancerous condition.
  • methods of the invention may be used to help select a treatment for a subject with cancer or a precancerous condition.
  • Selection of a treatment for a cancer or precancerous condition may be based upon selecting subjects who have abnormally high levels of methylation of one or more genomic sequences of the invention or may be based on a pattern of hypermethylation of one ore more genes of the invention.
  • Methods of selecting a treatment may be useful to assess and/or adjust treatment of subjects already receiving a drug or therapy (e.g., radiation treatment or surgery) for treating cancer or a precancerous condition. Based on the determination of the methylation state of one or more genes of the invention, it may be appropriate to alter a therapeutic regimen for a subject.
  • detection of an increase in methylation of one or more genes of the invention in a subject who has received or is receiving a cancer or precancerous-condition treatment may indicate that the treatment regimen should be adjusted (e.g., the dose or frequency of dosing, increased, new treatment initiated, etc.).
  • a subject may be free of any present treatment for cancer and monitoring of methylation levels according to methods of the invention may identify the subject as a candidate for a treatment for cancer or a precancerous condition, (e.g., treatment to decrease the methylation level of one or more genes of the invention).
  • subjects may be selected and treated with elevated levels of the same drugs or with different therapies as a result of assays of methylation states of genes of the invention.
  • some subjects may be free of symptoms otherwise calling for treatment with a particular therapy, and determining the level of methylation of one or more genes of the invention may identify the subject as needing treatment.
  • determining the level of methylation of one or more genes of the invention may identify the subject as needing treatment.
  • the subject would not according to convention as of the date of the filing of the present application have symptoms calling for treatment with a particular cancer therapy or a therapy for a precancerous condition.
  • the subject become a candidate for treatment with the therapy.
  • Screening for candidate therapeutic agents The invention also embraces methods for screening for candidate therapeutic agents or candidate treatments to prevent and/or treat cancer and/or precancerous conditions. Assessment of efficacy of candidate therapeutic agents and strategies may be done using assays of the invention in subjects (e.g., non-human animals) and in cells from culture.
  • a candidate therapeutic agent is defined as a compound or molecule that can change the methylation level of one or more of the genomic sequences of the invention.
  • a candidate treatment may be a surgical treatment, radiation treatment, etc.).
  • administration to the subject of an agent, or exposing a sample to the candidate agent or candidate treatment will result in an increase of the level of methylation of one or more genomic sequences of the invention.
  • administration to the subject of a candidate agent or treatment, or exposing a sample to the candidate agent or treatment will result in a decrease of the level of methylation of one or more genomic sequences of the invention.
  • a sample comprising cancer cells is exposed to a candidate therapeutic agent or treatment.
  • a sample comprising similar cancer cells, or cancer cells of the same lineage and passage, as the cancer cells exposed to the candidate, that have not been exposed to the agent or treatment will function as a control.
  • the level of methylation of one or more cancer-associated genes is monitored upon administration of the candidate therapeutic or treatment, and the change in level of methylation is compared to the change in level of methylation of the one or more genes in the control.
  • the agent is a candidate therapeutic or candidate treatment.
  • the level of methylation of one or more genes of the invention in the sample exposed to the agent or treatment is lower than the level of methylation of one or more genes of the invention in the control.
  • Screening methods may include mixing the candidate agent with cells or tissues or in a subject or exposing cells or tissues or a subject to the candidate treatment and using methods of assaying methylation state of one or more gene of the invention to determine the level of methylation before and after contact with the candidate agent or treatment.
  • a decrease in the amount of methylation of a gene of the invention compared to a control is indicative that the candidate agent or treatment is capable of treating cancer or a precancerous condition in a cell, tissue, and/or subject.
  • an assay mixture for testing a candidate agent comprises a candidate agent.
  • a candidate agent may be an antibody, a small organic compound, or a polypeptide, and accordingly can be selected from combinatorial antibody libraries, combinatorial protein libraries, or small organic molecule libraries.
  • pluralities of reaction mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection.
  • Any molecule or compound can be a candidate therapeutic.
  • candidate therapeutics are small molecules, RNA including siRNAs, DNA including aptamers, and proteins including antibodies and antibody fragments.
  • the invention also embraces candidate therapeutic with different modes of action.
  • modes of action of candidate therapeutics are methylation inhibitors, DNA modifying agents and agents that bind and hybridize to DNA.
  • Candidate agents encompass numerous chemical classes, although typically they are organic compounds, proteins or antibodies (and fragments thereof that bind antigen).
  • the candidate agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500.
  • Candidate agents comprise functional chemical groups necessary for structural interactions with polypeptides and/or nucleic acids, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of the functional chemical groups and more preferably at least three of the functional chemical groups.
  • the candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups.
  • Candidate agents also can be biomolecules such as polypeptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random or non-random polypeptides, combinatorial libraries of proteins or antibodies, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc.
  • reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc., which may be used to facilitate optimal protein-protein and/or protein-agent binding. Such a reagent may also reduce non-specific or background interactions of the reaction components.
  • reagents that improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.
  • incubation temperature typically are between 4°C and 40°C.
  • Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.
  • the presence or absence of and/or the level of methylation of one or more genomic sequences of the invention may be detected by any suitable method available to the user. Examples of suitable methods are provided herein and it will be understood by those of ordinary skill in the art that the invention may also encompass the use of additional methods of assaying methyation levels of one or more genomic sequences of the invention.
  • kits are provided.
  • Kits of the invention may contain a nucleic acid or other molecule of the invention for use in vitro diagnosis, prognosis, monitoring of cancer or a precancerous condition, and/or testing of candidate cancer or precancerous condition treatments by the methylation assay methods described above.
  • kits of the kits can be packaged either in aqueous medium or in lyophilized form.
  • Reagents for use in methylation assays may also be include in kits of the invention as can detectable labeling agents in the form of intermediates or as separate moieties to be conjugated as part of procedures to assay methylation levels, hi some embodiments of a kit of the invention, the kit may include instructions for determining methylation levels and may also include control values (e.g., reference numbers) that can be used for interpreting results of methylation methods used in the invention.
  • Kits Also within the scope of the invention are kits comprising the compositions of the invention and instructions for use. Kits of the invention may be useful for diagnosing cancer or a precancerous condition. Kits of the invention may include a component for determining the level of DNA methylation of one or more cancer-associated genes (e.g., genomic sequences). An example of such a kit may include methods for determining the level of methylation of one, two, or more of the genes of the invention.
  • a kit of the invention may include nucleic acid or protein microarrays nucleic acids of one or more genes of the invention or the polypeptides they encode. Kits may include materials for use in standard techniques of microarray technology to assess expression of one or more genes of the invention.
  • kits of the invention may include PCR components, e.g. primers, solutions, polymerase, etc for amplifying mRNA from a sample or subject, hi some embodiments the kit includes components for methylation-specific PCR.
  • a kit may include one or more antibodies to one or more polypeptides encoded by the cancer- associated genes along with components useful for use of the antibodies to determine expression of one or more cancer-associated genes in a cell, tissue or subject.
  • kits may include components for methylation-inhibitor analysis. These components may include the DNA methylation-inhibitor and means for analyzing differential methylation inhibition, such as a nucleic acid microarray.
  • a kit may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like.
  • a first of said container means or series of container means may contain primers for amplifying one or more genomic sequence of the invention or a methylated or unmethylated control sequence.
  • a second container means or series of container means may contain a label or linker-label intermediate for use in a assay of methylation state in a cell, tissue, or subject.
  • a kit of the invention may also include instructions. Instructions typically will be in written form and will provide guidance for carrying-out the assay embodied by the kit and for making a determination based upon that assay.
  • Epigenetic silencing can be reversed by methylation inhibitors. This property of epigenetic silencing has been exploited to perform a microarray based assay to screen for genes that are re-expressed following treatment of melanoma cells with 5AzadC (5-Aza-2'- deoxycytidine). Both DNA methylation and silencing of seventeen genes in melanoma cell lines and in uncultured melanoma tumor samples has been observed.
  • the seventeen genes are PCSKl, BST2, CYPlBl, LXN, SYK, COLl A2, DNAJCl 5, MFAP2, QPCT, CDH8, LRRC2, CDKNlC, GDFl 5, H0XB13, DALl, PTGS2, and WFDCl.
  • DNA methylation has not been previously demonstrated in QPCT, LXN, PCSKl, MFAP2, WFDCl, CDH8 and LRRC2 in any form of cancer.
  • QPCT was methylated in all melanomas and encodes a glutaminyl cyclase that converts precursor glutaminyl peptides to their bioactive pyro-glutaminyl peptide forms (25, 26).
  • LXN was methylated in 95% of melanomas and encodes a global inhibitor of mammalian carboxypeptidases and may function to limit prostate tumor aggressiveness by inhibiting carboxypeptidase 4 (CP A4) (27, 28).
  • COLl A2 which was found to be methylated in 80% of melanomas, has been found to be frequently hypermethylated in several human malignancies including breast cancer, hepatocellular carcinomas, and colorectal cancer (29).
  • DNAJC15 was methylated in 50% of melanomas.
  • CYPlBl is a member of the cytochrome P450 family of mono-oxygenases and has a wide range of substrates, including estrogen, androgens and chemotherapeutic drugs (30). Methylation of CYPlBl is associated with a poor prognosis in breast cancer (31).
  • DNAJCl 5 also known as DNAJDl and MCJ
  • Loss of DNAJCl 5 confers resistance to various chemotherapeutic agents used in the treatment of ovarian cancers (32).
  • methylation specific PCR assays for CYPlBl, QPCT, and LXN on blood or serum of melanoma patients could be used as possible staging markers as has recently been described using the lower frequency markers RARB (70%) and RASSFlA (55%)(46).
  • any gene that is methylated in uncultured metastatic melanoma can be used as a marker for metastatic melanoma.
  • the SYK cytoplasmic tyrosine kinase plays a role in coupling activated immune receptors to downstream signaling effectors (35). It is expressed in normal breast epithelial tissue and has tumor suppressor properties in breast cancer cells and may affect mitotic progression (36, 37).
  • HOXB 13 is a member of the highly conserved HOX transcription factors that regulate differentiation and pattern formation during embryogenesis (40). HOXB 13 has been found to negatively regulate wound healing, possibly by downregulating hyaluronic acid (41, 42).
  • HOXB 13 was found to be downregulated in prostate and colorectal cancer cells, where it has an antiproliferative role (43, 44). More recently HOXB 13 was found to be epigenetically inactivated in a subset of renal cell carcinomas and had growth inhibitory effects in vitro (45). The data presented in this study show that HOXB 13 has tumor suppressive properties in melanoma and is frequently inactivated by promoter region hypermethylation.
  • Stable MeUuSo cell lines expressing HOXB 13 and SYK were generated by RT-PCR amplification of full-length coding sequence from primary melanocytes and subcloning into the pTRE expression vector (Clontech; Mountain View, CA). The inserts were sequenced to ensure an absence of introduced mutations.
  • Stable transfectants were produced by transfection with Lipofectamine (Invitrogen) and selected by growth in media containing G418 (Invitrogen)(800 micrograms/milliliter). Colonies were ring cloned, expanded, and analyzed for transgene expression using RT- QPCR. 5AzadC treatment and microarray analysis.
  • RNA and DNA were isolated from a batch of 5AzadC treated cells every 24 hours starting with 0 hour controls. RNA was isolated following 0 hr and 48 hr 5AzadC treatment of six melanoma cell lines (MeUuSo, UACC 903, c8161, Neo-6 c8161, WM1205 and WM35) and used for the re-expression microarray analysis.
  • Baseline analyses were performed using 0 hr treatment array signals as a baseline for the respective 48 hr treatment.
  • the data was further processed using customized programs written for conditional formatting analyses to select for genes whose expression upon 5 AzadC treatment was significantly altered in more than one cell line. These genes were examined for presence of CpG islands in their promoter regions using the NCBI mapviewer and the EMBL CpGPlot program (www.ebi.ac.uk/emboss/cpgplot/).
  • Criteria used to identify prospective methylated genes included: 1) upregulation of expression (>4 fold) upon 5 AzadC treatment in at least one melanoma cell line; 2) significant expression (MAS 5.0 score of present) in cultured melanocytes, but no significant changes in expression ( ⁇ 2 fold) of the gene upon 5 AzadC treatment (in melanocytes); 3) downregulation of expression of the gene in untreated melanoma cell lines compared to primary melanocytes (>4 fold downregulation in at least 2 melanoma cell lines); and 4) presence of a CpG island in the promoter region.
  • Bisulfite sequencing and quantitative PCR DNA was isolated from cells and tissues using standard phenol-chloroform extraction. Bisulfite modification was performed as previously described (24) on DNA from melanocytes, melanoma cell lines prior to and after 48 hr treatment with 5 AzadC and DNA from melanoma tissues. PCR reactions were carried out using 50 ng of bisulfite modified DNA in a 30 ⁇ l volume with a 0.2 ⁇ M primer concentration (primer sequences are provided in the Table 2 with the amplified regions of selected genes provided in Table 3). PCR products were purified using the Qiaquick Gel Extraction kit (Qiagen) and directly sequenced on the ABI 3100-Avant automated DNA sequencer.
  • Qiaquick Gel Extraction kit Qiagen
  • Western Blotting Western Blotting experiments were performed by separation of 15 ⁇ g of cell lysate per sample on SDS-PAGE, transfer to Immobilon-P membranes (Millipore; Billerica, MA), blocking with 0.1 M phosphate buffered saline containing 0.2% Tween 20 and 5% non fat milk, and incubation with antisera to HOXB13 (F-9: sc28333), SYK (4D10: sc- 1240) (both from Santa Cruz Biotechnology; Santa Cruz, CA) or actin (AC-40)(Sigma).
  • In-vitro proliferation assays and tumor formation in nude mice were performed by plating three replicates each of 1000, 500 or 250 cells of the vector and HOXB 13 or SYK transfected cells in 6 well cell culture plates. Colonies were allowed to form for 2 weeks, stained with 0.005% crystal violet.
  • MFAP2 SEQ ID NO:54 ggccaaggggggactgggaatcctggagggccaggtctgggggagagttaggaggtcgtgaatttggacttggatgcttgttgaag ctgatgcatcttgaggacatttgtgggacacataggctgggtcagggctgaaagaggtgctggttatggccgggggcagggactcat gcctgtaatcccaacagcccaggaggatgagacaggaggaatgcttgaggccaaaatttcgaggccggaagttccagacgagcctg ggcaagaccctgtctctagaaaaaggaaagaagaaccgctggttgtggaagccagccatggcccagagctcagtgtagg
  • WFDCl SEQ ID NO:55 gtgctggacgcggacacatgatccgagggaccctgctgggtggaactaagaaagtccagcagactgtgcacgctcctgtccccactc acaggcccacgcagcgaggggggcccctcttgtgtgcgtctggaaggtcgctgcccagggaggaaatgcctttaaccggcgtgg ggccgggcagctgcaggaggcagatcatccgggctctgtgtgctcttgctacttctcctccacgccggctctgccaagaatatctggaa acgggcattgcctgcgaggctggccgagaaatccccgtgtaagtgcctggggggggaggg
  • a subset of 25 candidate genes was selected for further validation by bisulfite sequencing using the additional criterion of > 4 fold downregulation of expression of the gene in at least 2 untreated melanoma cell lines compared to primary melanocytes. Around two-thirds, 68% (17/25), of these genes were densely methylated in the promoter region in at least one of 9 melanoma cell lines or 20 melanoma tumor tissue samples (Fig. 2A and B). No DNA methylation was detected in primary human melanocytes at any of the loci. DNA methylation of specific genes occurred at similar frequencies in cell lines and tumor tissues, and was present both in melanoma cell lines and melanoma tissue samples for all genes tested (Fig. 2C).
  • SYK mRNA expression was markedly reduced in all 9 melanoma cell lines compared to primary cultured human melanocytes (Fig. 6A) and 5AzadC treatment resulted in >2 fold upregulation of SYK in a subset (4/8) of cell lines with SYK methylation (Fig. 4B). Immunoblotting showed complete absence of protein expression in all but one cell line (Fig. 6B). SYK mRNA expression was reduced >4 fold in 77% of the primary melanoma tissue samples (10/13) (Fig. 6C). Promoter region methylation of SYK was detected in 89% of the melanoma cell lines and 30% of primary tumors (Fig.
  • HOXB 13 mRNA expression was markedly reduced (>16 fold) in all the melanoma cell lines (Fig. 9A). However, one of the cell lines had retained protein expression (Fig. 9B), which may indicate increased protein stability in that cell line.
  • Fig. 9B In the melanoma tumor samples greater than four fold reduction of HOXB 13 mRNA was observed, compared to melanocytes in more than 60% (8/13) of the samples (Fig. 9C).
  • Promoter region methylation was detected in 33% of the melanoma cell lines (Fig. 2A) and 20% of the tumor samples (Fig. 2B) tested. Re-expression of HOXB 13 occurred upon treatment with 5AzadC in 2 out of 3 cell lines with promoter region methylation (Fig. 4A).
  • HOXB 13 was stably expressed in a HOXB13-deficient cell line (MeUuSo) at similar levels to that seen in primary melanocytes (Fig. 7) in order to characterize the possible tumor suppressor function of HOXB 13 in melanoma cells.
  • Cell proliferation and colony formation were reduced in HOXBl 3 transfected clones compared to vector controls (Fig. 8A).
  • HOXB 13 transfected MeUuSo lines exhibited greater than 4 fold reduction in tumor size relative to vector controls in xenografting experiments in immunodeficient mice (Fig. 8A and C).
  • DNA was prepared from ten samples of blood from individuals using standard methods. The DNA was assessed using bisulfite modification as described above herein. The results indicated that in normal blood samples, e.g. those without known disease (e.g., cancer) lacked detectable LXN methylation as detected by bisulfite modification and DNA sequencing. The results indicated that there is a very low level of background of methylation detected in normal blood. The detection of methylation levels in normal blood samples provides a baseline level of methylation, (e.g., a control) that may be used for testing blood from subjects suspected of having cancer. The results indicated the suitability of the method and assay for use with blood samples for the determination of methylation levels of candidate genes, and for the diagnosis of cancer and/or precancerous conditions.
  • a baseline level of methylation e.g., a control
  • Blood samples from melanoma patients are examined for the level of LXN methylation as described for DNA assessment herein.
  • the level of LXN methylation is compared between melanoma patient blood samples (e.g., subject sample) and control blood samples (e.g., non-cancer containing samples or a prior sample from a subject undergoing treatment).
  • control blood samples e.g., non-cancer containing samples or a prior sample from a subject undergoing treatment.
  • methylation status of other candidate genes presented in Figure 3 is examined and compared between melanoma patient blood samples (subject sample) and non-cancer control blood samples.
  • a difference in methylation levels of genes such as LXN and control levels of methylation in a control blood sample, indicates that the presence of cancer in the sample.
  • analysis of methylation of genes such as LXN in blood samples contributes to the diagnosis of cancer. Additional genes that are evaluated include QPCT, PCSKl, MFAP2, WFDCl, CDH8, and LRRC2.
  • Determination of levels of methylation of one or more of the following gene of one or more of the genes, CYPlBl, COLl A2, GDFl 5, RARB, TM, 3-OST-2, RASSFlA, ACS/TMS1, BST2, DNAJCl 5, CDKNIc, MGMT, SYK, MiBl, HOXBl 3, PTGS2, DAPK, APC,pl6 INK4 ⁇ ,p27 Kipl , PRDX2, PYCARD, CDKN2A, CDKNlB, and DALl is also performed in blood samples from a subject and from control blood samples.
  • the levels of methylation of candidate genes is compared in the subject sample and control sample to determine whether or not the subject has cancer or a precancerous condition and/or to assess efficacy of a cancer treatment.
  • Assessment of methylation of candidate genes in a blood sample from a subject permits detection and diagnosis of cancer and/or precancerous conditions in the subject.

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Abstract

Cette invention concerne des procédés et des kits utilisés pour détecter et diagnostiquer le cancer ou des états précancéreux. La méthylation de gènes spécifiques s'avère un élément indicateur du cancer, et les procédés de l'invention concernent, en partie, la détection des taux de méthylation dans les cellules utilisée comme élément de détermination du cancer ou d'un état précancéreux de la cellule.
PCT/US2007/024553 2005-05-17 2007-11-29 Procédés et produits utilisés pour diagnostiquer le cancer WO2008066878A2 (fr)

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US20110076283A1 (en) * 2008-01-24 2011-03-31 Universite De Lausanne Method for predicting and diagnosing brain tumor
WO2012081017A1 (fr) 2010-12-15 2012-06-21 Yeda Research And Development Co. Ltd. Compositions et procédés pour le traitement du cancer et de maladies neurodégénératives
EP3037545A1 (fr) * 2014-12-23 2016-06-29 The Provost, Fellows, Foundation Scholars, & the other members of Board, of the College of the Holy & Undiv. Trinity of Queen Elizabeth near Dublin Test de méthylation d'ADN pour le cancer de la prostate
US20210324479A1 (en) * 2018-07-26 2021-10-21 Beijing Exellon Medical Technology Co., Ltd Method for identifying breast cancer status and kit
CN114410792B (zh) * 2022-02-10 2023-07-25 博尔诚（北京）科技有限公司 用于肾癌筛查的标志物、探针组合物及其应用
CN118441051B (zh) * 2024-05-28 2025-05-02 南通大学 一种与肺腺癌相关的meQTL-SNPs生物标志物及其应用

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