GASTRIC CANCER BIOMARKER DISCOVERY

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;

FIG. 1 shows a schematic diagram for a systematic method for discovering gastric cancer biomarker. Gene expression level was compared between tumor and paired tumor-adjacent tissue by indirect comparison methods and down regulated genes in tumor cells were obtained from each comparison. Upregulated genes in MKN1, MKN28 and SNU484 cell lines treated with DAC were selected and overlapping common genes were identified as methylation biomarker candidates.

FIG. 2 shows a schematic diagram to conduct methylation assay by enzyme digestion and subsequent gene amplification analysis to determine whether a candidate marker gene is actually methylated.

FIG. 3 shows a flowchart for gastric cancer biomarker discovery.

FIG. 4 shows gene methylation status of 23 identified gastric cancer marker genes. Methylation positive genes in AGS, MKN1, MKN28 and SNU484 cells are depicted. Black pixels: methylated.

FIG. 5 shows gene expression profiles of the 23 identified promoter methylated genes in tumorous and tumor-adjacent non-tumorous gastric tissue. These genes were identified based on the genes that were down regulated in gastric tumor cells.

FIG. 6 shows reactivation of the 23 gastric cancer biomarkers after demethylating agent treatment.

FIGS. 7A and 7B show gene methylation status of 23 identified genes in normal tissue from non-patients, and clinical samples from gastric tumor and paired tumor-adjacent tissue. FIG. 7A shows methylation frequency of 23 identified markers in normal tissue from non-patients (3 samples), tumor tissues (12 samples) and paired tumor-adjacent tissues (10 samples). FIG. 7B shows that these 23 markers are useful for early detection of gastric cancer because they are highly methylated in the paired tumor-adjacent tissues in addition to tumor tissues.

FIG. 8 shows methylation frequency of the 23 identified genes in advanced gastric cancer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present application, “a” and “an” are used to refer to both single and a plurality of objects.

As used herein, “cell conversion” refers to the change in characteristics of a cell from one form to another such as from normal to abnormal, non-tumorous to tumorous, undifferentiated to differentiated, stem cell to non-stem cell. Further, the conversion may be recognized by morphology of the cell, phenotype of the cell, biochemical characteristics and so on. There are many examples, but the present application focuses on the presence of abnormal and cancerous cells in the gastric tissue. Markers for such tissue conversion are within the purview of gastric cancer cell conversion.

As used herein, “demethylating agent” refers to any agent, including but not limited to chemical or enzyme, that either removes a methyl group from the nucleic acid or prevents methylation from occurring. Examples of such demethylating agents include without limitation nucleotide analogs such as 5-azacytidine, 5 aza 2′-deoxycytidine (DAC), arabinofuranosyl-5-azacytosine, 5-fluoro-2′-deoxycytidine, pyrimidone, trifluoromethyldeoxycytidine, pseudoisocytidine, dihydro-5-azacytidine, AdoMet/AdoHcy analogs as competitive inhibitors such as AdoHcy, sinefungin and analogs, 5′deoxy-5′-S-isobutyladenosine (SIBA), 5′-methylthio-5′deoxyadenosine (MTA), drugs influencing the level of AdoMet such as ethionine analogs, methionine, L-cis-AMB, cycloleucine, antifolates, methotrexate, drugs influencing the level of AdoHcy, dc-AdoMet and MTA such as inhibitors of AdoHcy hydrolase, 3-deaza-adenosine, neplanocin A, 3-deazaneplanocin, 4′-thioadenosine, 3-deaza-aristeromycin, inhibitors of ornithine decarboxylase, α-difluoromethylornithine (DFMO), inhibitors of spermine and spermidine synthetase, S-methyl-5′-methylthioadenosine (MTA), L-cis-AMB, AdoDATO, MGBG, inhibitors of methylthioadenosine phosphorylase, difluoromethylthioadenosine (DFMTA), other inhibitors such as methinin, spermine/spermidine, sodium butyrate, procainamide, hydralazine, dimethylsulfoxide, free radical DNA adducts, UV-light, 8-hydroxy guanine, N-methyl-N-nitrosourea, novobiocine, phenobarbital, benzo[a]pyrene, ethylmethansulfonate, ethylnitrosourea, N-ethyl-N′-nitro-N-nitrosoguanidine, 9-aminoacridine, nitrogen mustard, N-methyl-N′-nitro-N-nitrosoguanidine, diethylnitrosamine, chlordane, N-acetoxy-N-2-acetylaminofluorene, aflatoxin B1, nalidixic acid, N-2-fluorenylacetamine, 3-methyl-4′-(dimethylamino)azobenzene, 1,3-bis(2-chlorethyl)-1-nitrosourea, cyclophosphamide, 6-mercaptopurine, 4-nitroquinoline-1-oxide, N-nitrosodiethylamine, hexamethylenebisacetamide, retinoic acid, retinoic acid with cAMP, aromatic hydrocarbon carcinogens, dibutyryl cAMP, or antisense mRNA to the methyltransferase (Zingg et al., Carcinogenesis, 18:5, pp. 869-882, 1997). The contents of this reference is incorporated by reference in its entirety especially with regard to the discussion of methylation of the genome and inhibitors thereof.

As used herein, “direct comparison” refers to a competitive binding to a probe among differentially labeled nucleic acids from more than one source in order to determine the relative abundance of one type of differentially labeled nucleic acid over the other.

As used herein, “early detection” of cancer refers to the discovery of a potential for cancer prior to metastasis, and preferably before morphological change in the subject tissue or cells is observed. Further, “early detection” of cell conversion refers to the high probability of a cell to undergo transformation in its early stages before the cell is morphologically designated as being transformed.

As used herein, “hypermethylation” refers to the methylation of a CpG island.

As used herein, “indirect comparison” refers to assessing the level of nucleic acid from a first source with the level of the same allelelic nucleic acid from a second source by utilizing a reference probe to which is separately hybridized the nucleic acid from the first and second sources and the results are compared to determine the relative amounts of the nucleic acids present in the sample without direct competitive binding to the reference probe.

As used herein, “sample” or “bodily sample” is referred to in its broadest sense, and includes any biological sample obtained from an individual, body fluid, cell line, tissue culture, depending on the type of assay that is to be performed. As indicated, biological samples include body fluids, such as semen, lymph, sera, plasma, and so on. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. A tissue biopsy of the gastric is a preferred source.

As used herein, “tumor-adjacent tissue” or “paired tumor-adjacent tissues” refers to clinically and morphologically designated normal appearing tissue adjacent to the cancerous tissue region.

Screening for Methylation Regulated Biomarkers

The present invention is directed to a method of determining biomarker genes that are methylated when the cell or tissue is converted or changed from one type of cell to another. As used herein, “converted” cell refers to the change in characteristics of a cell or tissue from one form to another such as from normal to abnormal, non-tumorous to tumorous, undifferentiated to differentiated and so on. See FIG. 1.

Thus, the present invention is directed to a systematic approach to identifying methylation regulated marker genes in gastric cancer cell conversion. In one aspect of the invention, (1) the genomic expression content between a converted gastric cancer and unconverted cell or cell line is compared and a profile of the more abundantly expressed genes in the unconverted cell or cell line is categorized; (2) a converted gastric cancer cell or cell line is treated with a methylation inhibitor, and genomic expression content between the methylation inhibitor treated converted gastric cancer cell or cell line and untreated converted gastric cancer cell or cell line is compared and a profile of the more abundantly expressed genes in the methylation inhibitor treated converted gastric cancer cell or cell line is categorized; (3) profiles of genes from those obtained in (1) and (2) above are compared and overlapping genes are considered to be methylation regulated marker genes in converting a cell from the unconverted state to the converted gastric cancer cell form.

In addition to the above, in order to further fine-tune the list of candidate biomarkers and also to determine whether the candidate biomarkers so obtained above are indeed methylated under conversion conditions, a nucleic acid methylation detecting assay is carried out. Any number of numerous ways of detecting methylation on a DNA fragment may be used. By way of example only and without limitation, one such way is as follows. Genomic DNA is treated with a methylation sensitive restriction enzyme, and probed with marker specific gene sequence directed to the methylation region. Detection of an uncleaved probed region indicates that methylation has occurred at the probed site.

One way to practice the invention is by utilizing microarray technology as follows:

(1) Converted cell expression library and non-converted cell expression library are differentially labeled with preferably fluorescent labels, Cy3 which produces green color, and Cy5 which emanates red color. They are competitively bound to a microarray immobilized with a set of known gene probes. The genes that are differentially more expressed in the unconverted cells are identified. Alternatively, an indirect comparison method may be used.

(2) Converted cell line is treated with a demethylating agent and the expression library is labeled with a fluorescent label. A differentially labeled expression library from a converted cell line that has not been treated with the demethylating agent is also obtained. The two libraries are competitively bound on a microarray substrate immobilized with a set of known gene probes. The genes that are differentially more expressed in the converted cells treated with the demethylating agent are identified. These genes are presumably reactivated under demethylating conditions. Alternatively, an indirect comparison method may be used.

(3) The identified genes from the two sets of experiments above are compared and genes common to both lists are chosen.

Again, it is understood that such comparison in gene expression between the converted and unconverted cells and between cells treated with demethylating agent and not treated with demethylating agent may be carried out by direct competitive binding to a set of probes. Alternatively, the comparison may be indirect. For instance, the expressed genes may be bound to a set of known reference gene probes each separately. Thus, the relative abundance of expressed genes from the various cells can be compared indirectly. The set of reference gene probes are generally optimized so that they contain as complete a set of expressed genes as possible. See FIG. 1.

(4) The nucleic acid sequence of the promoter regions of the genes are examined to determine whether there are CpG islands within them. Genes with promoters that do not possess CpG islands are discarded. The remaining genes are assayed for their level of methylation. This can be accomplished using a variety of means. In one embodiment, the genome from converted cells is digested with methylation sensitive restriction endonuclease. Nucleic acid amplification is carried out using various primers wherein the methylation site is located within the region to be amplified. When the nucleic acid amplification step is carried out, successful amplification indicates that methylation has occurred because the gene was not cleaved by the methylation sensitive restriction endonuclease. The absence of an amplified product indicates that methylation did not occur because the gene was digested by the methylation sensitive restriction endonuclease.

Gastric Cancer Biomarkers

Biomarkers for gastric cancer detection are provided in the present application.

Gastric Cancer Biomarker—Using Cancer Tumor Cells for Comparison with Normal Cells

In practicing the invention, it is understood that “normal” cells are those that do not show any abnormal morphological or cytological changes. “Tumor” cells are cancer cells. “Non-tumor” cells are those cells that were part of the diseased tissue but were not considered to be the tumor portion.

Gastric tumor cell gene expression content was indirectly compared between non-tumor cell and tumor cell gene expression content in a microarray competitive hybridization format. A common reference was competed with non-tumor tissue, such as tumor-adjacent tissue, gene content; and common reference was also competed with tumor cell gene content. Genes that were repressed in tumor cells as compared with non-tumor cells were found and noted as the tumor suppressed genes.

Alternatively, the gene expression content from tumor may be directly competed with non-tumor and/or normal cells in a microarray hybridization format to obtain the tumor suppressed genes. Also, both direct and indirect methods may be used to obtain the tumor suppressed genes.

Separately, gastric cancer cell lines MKN1, MKN28, and SNU484 were treated with a demethylating agent DAC and assayed for reactivation of genes that are normally repressed in tumor cells. Overlapping genes between the tumor suppressed gene set and the demethylation reactivated gene set were considered to be candidate genes for gastric cancer biomarkers. Sixty one (61) such overlapping genes were found (FIG. 3). These genes were then analyzed in silico to determine whether they contained the requisite CpG island motif. A few genes (21 genes) did not contain them and were removed. Further biochemical testing of the remaining 40 genes was needed to determine whether the candidate genes were actually methylated when isolated from tumor cells. Methylation sensitive enzyme/nucleic acid sequence based amplification analysis such as Hpa II/MspT enzyme digestion/PCR (or enzyme digestion post-PCR) further removed a few other genes (17 genes) that were not methylated in any of the gastric cancer cell lines. To further confirm biochemically that the candidate genes were indeed methylated in tumor cells, bisulfite sequencing assays were conducted and methylation of the final 23 genes was verified.

Gene expression profiles of the 23 genes were created. The expression level of the 23 genes was measured in tumor and tumor-adjacent non-tumor tissue (FIG. 5). Methylation status of the genes was also measured using methylation sensitive enzyme/nucleic acid sequence based amplification analysis such as Hpa II/MspI enzyme digestion/PCR (or enzyme digestion post-PCR) method on clinical samples and the results for the 23 genes is shown in FIG. 5. The identified genes are not methylated in normal cells. However, they are methylated in tumor cells as well as in tumor-adjacent non-tumor cells. FIGS. 7 and 8 further show that the frequency of methylation in tumor cells is higher than in tumor-adjacent tissue.

Thus, one aspect of the invention is in part based upon the discovery of the relationship between gastric cancer and the above 23 exemplified promoter hypermethylation of the following genes: MTCBP-1 (NT_—022270)—Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1; MTPN (NT_—007933)—Myotrophin; MTSS1 (NT_—008046)—Metastasis suppressor 1; PEL12 (NT_—026437)—Pellino homolog 2 (Drosophila); PLEKHF2 (NT_—008046)—Pleckstrin homology domain containing, family F (with FYVE domain) member 2; RERG (NT_—009714)—RAS-like, estrogen-regulated, growth inhibitor; THBD (NT_—011387)—Thrombomodulin; TP531NP1 (NT_—008046)—Tumor protein p53 inducible nuclear protein 1; MGC11324 (NT_—016354)—Hypothetical protein MGC11324; ZFHX1B (NT_—005058)—Zinc finger homeobox 1b; ADRB2 (NT_—029289)—Adrenergic, beta-2-, receptor, surface; AR (NT_—011669)—Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease); BLVRB (NT_—011109)—Biliverdin reductase B (flavin reductase (NADPH); CALCR (NT_—007933)—Calcitonin receptor; CDH2 (NT_—010966)—Cadherin 2, type 1, N-cadherin (neuronal); CKAP4 (NT_—019546)—Cytoskeleton-associated protein 4; CYBRD1 (NT_—005403)—Cytochrome b reductase 1; GFPT1 (NT_—022184)—Glutamine-fructose-6-phosphate transaminase 1; GOLPH2 (NT_—023935)—Golgi phosphoprotein 2; HHEX (NT_—030059)—Hematopoietically expressed homeobox; LAMA2 (NT_—025741)—laminin, alpha 2 (merosin, congenital muscular dystrophy); CPEB3 (NT_—030059)—cytoplasmic polyadenylation element binding protein 3; HTR1B (NT_—007299)-5-hyroxytryptamine (serotonin) receptor 1B gene.

In another aspect, the invention provides early detection of a cellular proliferative disorder of gastric tissue in a subject comprising determining the state of methylation of one or more nucleic acids isolated from the subject, wherein the state of methylation of one or more nucleic acids as compared with the state of methylation of one or more nucleic acids from a subject not having the cellular proliferative disorder of gastric tissue is indicative of a cellular proliferative disorder of gastric tissue in the subject. A preferred nucleic acid is a CpG-containing nucleic acid, such as a CpG island.

Another embodiment of the invention provides a method of determining a predisposition to a cellular proliferative disorder of gastric tissue in a subject comprising determining the state of methylation of one or more nucleic acids isolated from the subject, wherein the nucleic acid may encode MTCBP-1 (NT_—022270)—Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1; MTPN (NT_—007933)—Myotrophin; MTSS1 (NT_—008046)—Metastasis suppressor 1; PEL12 (NT_—026437)—Pellino homolog 2 (Drosophila); PLEKHF2 (NT_—008046)—Pleckstrin homology domain containing, family F (with FYVE domain) member 2; RERG (NT_—009714)—RAS-like, estrogen-regulated, growth inhibitor; THBD (NT_—011387)—Thrombomodulin; TP531NP1 (NT_—008046)—Tumor protein p53 inducible nuclear protein 1; MGC11324 (NT_—016354)—Hypothetical protein MGC11324; ZFHX1B (NT_—005058)—Zinc finger homeobox 1b; ADRB2 (NT_—029289)—Adrenergic, beta-2-, receptor, surface; AR (NT_—011669)—Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease); BLVRB (NT_—011109)—Biliverdin reductase B (flavin reductase (NADPH); CALCR (NT_—007933)—Calcitonin receptor; CDH2 (NT_—010966)—Cadherin 2, type 1, N-cadherin (neuronal); CKAP4 (NT_—019546)—Cytoskeleton-associated protein 4; CYBRD1 (NT_—005403)—Cytochrome b reductase 1; GFPT1 (NT_—022184)—Glutamine-fructose-6-phosphate transaminase 1; GOLPH2 (NT_—023935)—Golgi phosphoprotein 2; HHEX (NT_—030059)—Hematopoietically expressed homeobox; LAMA2 (NT_—025741)—laminin, alpha 2 (merosin, congenital muscular dystrophy); CPEB3 (NT_—030059)—cytoplasmic polyadenylation element binding protein 3; HTR1B (NT_—007299)-5-hyroxytryptamine (serotonin) receptor 1B gene, and combinations thereof, and wherein the state of methylation of one or more nucleic acids as compared with the state of methylation of said nucleic acid from a subject not having a predisposition to the cellular proliferative disorder of gastric tissue is indicative of a cell proliferative disorder of gastric tissue in the subject.

As used herein, “predisposition” refers to an increased likelihood that an individual will have a disorder. Although a subject with a predisposition does not yet have the disorder, there exists an increased propensity to the disease.

Another embodiment of the invention provides a method for diagnosing a cellular proliferative disorder of gastric tissue in a subject comprising contacting a nucleic acid-containing specimen from the subject with an agent that provides a determination of the methylation state of nucleic acids in the specimen, and identifying the methylation state of at least one region of at least one nucleic acid, wherein the methylation state of at least one region of at least one nucleic acid that is different from the methylation state of the same region of the same nucleic acid in a subject not having the cellular proliferative disorder is indicative of a cellular proliferative disorder of gastric tissue in the subject.

The inventive method includes determining the state of methylation of one or more nucleic acids isolated from the subject. The phrases “nucleic acid” or “nucleic acid sequence” as used herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. As will be understood by those of skill in the art, when the nucleic acid is RNA, the deoxynucleotides A, G, C, and T are replaced by ribonucleotides A, G, C, and U, respectively.

The nucleic acid of interest can be any nucleic acid where it is desirable to detect the presence of a differentially methylated CpG island. The CpG island is a CpG rich region of a nucleic acid sequence. The nucleic acids includes, for example, a sequence encoding the following genes (GenBank Accession Numbers are shown):

1. MTCBP-1 (NT_—022270); Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1

Amplicon size: 231 bp

(SEQ ID NO: 1)

cagg acagggtctg ggcttgaatg cctttgcttc cgaaaacagc

ggagccgtgg ggcctccccc gcgccggccc tgcctccaac

cagccgccca atcccggctc ccatgcgcgt ccacgcctcc

ctataagaca aagcgcggcc gacgggctcc gagcgcggcc

cctgggttcg aacacggcac ccgcactgcg cgtcatggtg

caggcctggt atatggacga cgccccg

(SEQ ID NO: 2)

MTCBP-1-F:

5′-caggacagggtctgggcttg-3′

(SEQ ID NO: 3)

MTCBP-1-R:

5′-cggggcgtcgtccatatacc-3′

2. MTPN (NT_—007933); Myotrophin

Amplicon size: 249 bp

(SEQ ID NO: 4)

tggtggtgca gaagcgtcct aatcccatcg aaagtactct

cactcttcct ccattcgctg tcttgacctc ccccgggcct

ccttcattct gcctcccagt cccgcccact tgtcgccggc

tcctaccctc cactctagcc ttcccggcag cctgtacatt

gcgcatgcgc actagtggcg ttcgcgccgc ggacccagag

agaggcgttc cgcggaggag aagaggaaga ggaagttggg

ggagggtcc

(SEQ ID NO: 5)

MTPN-F:

5′-tggtggtgcagaagcgtcct-3′

(SEQ ID NO: 6)

MTPN-R:

5′-ggaccctcccccaacttcct-3′

3. MTSS1 (T_—008046); Metastasis suppressor 1

Amplicon size: 236 bp

(SEQ ID NO: 7)

ta caagcgggct ctgggctagg cgcgccaccc gtgcaagtcc

ccggggagcg gcggtgcacc ctccgtcccg cgcgctcgca

gccattgtag gggtgggcgc tcgccaggca gggtgccgac

acgccctctc cgcgctgcga cgggcggccg ggggaggaga

gggtgcgctg tgcgcaccgg agggagaggc tccggcccag

cgccgcctgc ccgccagcag accagcaggc tcct

(SEQ ID NO: 8)

MTSS1-F:

5′-tacaagcgggctctgggcta-3′

(SEQ ID NO: 9)

MTSS1-R:

5′-aggagcctgctggtctgctg-3′

4. PELI2 (NT_—026437); Pellino homolog 2 (Drosophila)

Amplicon size: 216 bp

(SEQ ID NO: 10)

gcgccttcg aaacgtcctc tacgccagca ccaactggca

aaaccttcta attttctaga cgcctttctg cttggttttg

gaaggggagg cacccaagtg ggtgtgtgcg acacctctag

ttgtaagccg ggacacagtg acgtcgagag agcgctattc

tactcggaga ggaagttaat cccatcgaac tccagccagg

aaaacgtggg cttggga

(SEQ ID NO: 11)

PELI2-F:

5′-gcgccttcgaaacgtcctct-3′

(SEQ ID NO: 12)

PELI2-R:

5′-tcccaagcccacgttttcct-3′

5. PLEKHF2 (NT_—008046); Pleckstrin homology domain containing, family F (with FYVE domain) member 2

Amplicon size: 203 bp

(SEQ ID NO: 13)

aaggg ctggtcggag tcaggaaagt caggtaaggc gcctcacgtg

cacctcaacg cgtcgcggga gcgcgtcccg acctcacaca

tggacaagct ccgcccgcgg ccggcctgag tgggtgtggc

ctccgccaaa ggccccgccc ctagagcgcg tcgcgagggg

cgcgaggggc ggggcgaggg aactggcaag aaagggcg

(SEQ ID NO: 14)

PLEKHF2-F:

5′-aagggctggtcggagtcagg-3′

(SEQ ID NO: 15)

PLEKHF2-R:

5′-cgccctttcttgccagttcc-3′

6. RERG (NT_—009714); RAS-like, estrogen-regulated, growth inhibitor

Amplicon size: 226 bp

(SEQ ID NO: 16)

gg agcctggagg cttggaaata accagtgaaa aagggaagcc

cgtcttgggt gcagcacgtt aaagacccaa gctcgcaagc

ctgggaggca gcgcggcggg aggagcctgc ccctgccccc

agtagggggc gccgaagcgc cgcactgcag catcctggcc

gctgagcgca gcggccttgg ccgggctcag ctcgcgtcct

gccgcagtcc ctccgccgct agtc

(SEQ ID NO: 17)

RERG-F:

5′-ggagcctggaggcttggaaa-3′

(SEQ ID NO: 18)

RERG-R:

5′-gactagcggcggagggactg-3′

7. THBD (NT_—011387); Thrombomodulin

Amplicon size: 182 bp

(SEQ ID NO: 19)

cgccag ggcagggttt actcatcccg gcgaggtgat cccatgcgcg

agggcgggcg caagggcggc cagagaaccc agcaatccga

gtatgcggca tcagcccttc ccaccaggca cttccttcct

tttcccgaac gtccagggag ggagggccgg gcacttataa

actcgagccc tggccg

(SEQ ID NO: 20)

THBD-F:

5′-cgccagggcagggtttactc-3′

(SEQ ID NO: 21)

THBD-R:

5′-cggccagggctcgagtttat-3′

8. TP53INP1 (NT_—008046); Tumor protein p53 inducible nuclear protein 1

Amplicon size: 229 bp

(SEQ ID NO: 22)

ctccca gcaccctggc tacacgtcta accctaggct gaccaggtgg

ggctctcgga ggcgttagcc ccagccctcc caggagtctt

aatgttcctc tcacaggaaa aaacgttctg cgccctttgt

gccccaaacc ctcgaccctt cactcggcga gaggggtcgc

tgagggagag atccacctct gcccttcccg ttgcccgccg

gttcttcctc gcccgcctct tac

(SEQ ID NO: 23)

TP53INP1-F:

5′-ctcccagcaccctggctaca-3′

(SEQ ID NO :24)

TP53INP1-R:

5′-gtaagaggcgggcgaggaag-3′

9. MGC11324 (NT_—016354); Hypothetical protein MGC11324

Amplicon size: 236 bp

(SEQ ID NO: 25)

ggtggcttc agcccagacc tgggcagcca gcggagaaag

agttaactgg caggggcgag gaggagccca gggaggaagg

aaggatattg ccgtaattct gaaagttttt ttccttcctc

tcttcccttc gcagaggtga gtgccgggct cggcgctctg

ctcctggagc tcccgcggga ctgcctgggg acagggactg

ctgtggcgct cggccctcca ctgcggacct ctcctga

(SEQ ID NO: 26)

MGC11324-F:

5′-ggtggcttcagcccagacct-3′

(SEQ ID NO: 27)

MGC11324-R:

5′-tcaggagaggtccgcagtgg-3′

10. ZFHX1B (NT_—005058); Zinc finger homeobox 1b

Amplicon size: 215 bp

(SEQ ID NO: 28)

cctgcctccc gacactcttg gcgaggtttt tgtacagttt

gctccgggag ctgtttcttc gcttccacct ttttctcccc

cacacttcgc ggcttcttca tgctttttct tctcaccatt

tctggccaaa actacaaaca agacttcgca ggtaggtttt

ttttcctccc cttttctctc tttttatccc tttttggtgt

gctcgtcctc catcc

(SEQ ID NO: 29)

ZFHX1B-F:

5′-cctgcctcccgacactcttg-3′

(SEQ ID NO: 30)

ZFHX1B-R:

5′-ggatggaggacgagcacacc-3′

11. ADRB2 (NT_—029289); Adrenergic, beta-2-, receptor, surface

amplicon size; 261 bp

gagctgggagggtgtgtctcagtgtctatggctgtggttcggtata
(SEQ ID NO: 31)

agtctgagcatgtctgccagggtgtatttgtgcctgtatgtgcgtgcctcggtgggcact

ctcgtttccttccgaatgtggggcagtgccggtgtgctgccctctgccttgagacctcaa

gccgcgcaggcgcccagggcaggcaggtagcggccacagaagagccaaaagctcccgggt

tggctggtaaggacaccacctccagctttagccct

Forward; 5′-gagctgggagggtgtgtctcag-3′
(SEQ ID NO: 32)

Reverse; 5′-agggctaaagctggaggtggtg-3′
(SEQ ID NO: 33)

12. AR (NT_—011669); Androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease)

amplicon size: 195 bp

(SEQ ID NO: 34)

ggacccga ctcgcaaact gttgcatttg ctctccacct

cccagcgccc cctccgagat cccggggagc cagcttgctg

ggagagcggg acggtccgga gcaagcccac aggcagagga

ggcgacagag ggaaaaaggg ccgagctagc cgctccagtg

ctgtacagga gccgaaggga cgcaccacgc cag

(SEQ ID NO: 35)

Forward;

5′- cag gca acc cag acg tcc aga

g -3′

(SEQ ID NO: 36)

Reverse;

5′- gct ggc gtg gtg cgt ccc t-3′

13. BLVRB (NT_—011109); Biliverdin reductase B (flavin reductase (NADPH)

amplicon size; 256 bp

tttctccccttgctggttctgggaggccctggggcttagagt
(SEQ ID NO: 37)

gcgccccagatccgctccaggctccgggagagggggcgtgagctacgagaggccgtgggt

gaggctcgcctggccccgcccctctccgggaggtggagcgcggaggcagggcgctgagtg

acaagttatctcggctccgtccactctatttgttgctatgtggaggcgtggccttctgtg

gccccgccttccagtctaagtggcgcgcagagag

Forward; 5′-tttctccccttgctggttctgg-3′
(SEQ ID NO: 38)

Reverse; 5′-ctctctgcgcgccacttagact-3′
(SEQ ID NO: 39)

14. CALCR (NT_—007933); Calcitonin receptor

amplicon size: 229 bp

(SEQ ID NO: 40)

cct gtgtttacgc ggcgctttag tctcccggac

tcgcagggtg agccccagcc ctgactggag cgagacagca

gccgcgagcg cagccccact cgcgggccgg ggcgactggg

gctggcgcga ggctcacgga gctcaccagc tcgcccctcc

ctctcctggg acaggagggg gctgactggg gtggcggggt

ccgggaaggg gggctggctc tcatcaattc tgctgc

(SEQ ID NO: 41)

Forward;

5′- cct gtg ttt acg cgg cgc ttt-3′

(SEQ ID NO: 42)

Reverse;

5′- gca gca gaa ttg atg aga gcc

a-3′

15. CDH2 (T_—010966); Cadherin 2, type 1, N-cadherin (neuronal)

amplicon size; 203 bp

tctcaaactcccagaggggacaaa
(SEQ ID NO: 43)

agaaaacaaaacaagaacacaaaaacttgggctgttccagtacatcctcaagggtgggag

ctgaaggtgcgagctccagagaggagccgcggcctccgccctcccccgcccgcaggtggc

tcccggcgagcgcctcagacaacaatagctaggatgagcttggcctgcgtccttagttt

Forward; 5′-tctcaaactcccagaggggaca-3′
(SEQ ID NO: 44)

Reverse; 5′-aaactaaggacgcaggccaagc-3′
(SEQ ID NO: 45)

16. CKAP4 (NT_—019546); Cytoskeleton-associated protein 4

Amplicon size; 214 bp

ggctggattttggacagccttttttgtctccgccttcaaacccaaggcaaaggaca
(SEQ ID NO: 46)

aggcccaaccccatggcgggctgggagagtgaaagcagggggagtcccccaattcccagc

ggaaaggaagggcgatctgttcccacccgctgactcccactcccggggccagggctcctt

gggcgccccccttcatttctctcctctccgcacaggtc

Forward; 5′-ggctggattttggacagccttt-3′
(SEQ ID NO: 47)

Reverse; 5′-gacctgtgcggagaggagagaa-3′
(SEQ ID NO: 48)

17. CYBRD1 (T_—005403); Cytochrome b reductase 1

amplicon size; 221 bp

ggagacagccccaagaagtcgacgccccggtcccgccgccc
(SEQ ID NO: 49)

ggccactacccagagggctgccgccgcctctccaagttcttgtggcccccgcggtgcgga

gtatggggcgctgatggccatggagggctactggcgcttcctggcgctgctggggtcggc

actgctcgtcggcttcctgtcggtgatcttcgccctcgtctgggtcctccactaccgaga

Forward; 5′-ggagacagccccaagaagtcg-3′
(SEQ ID NO: 50)

Reverse; 5′-tctcggtagtggaggacccagac-3′
(SEQ ID NO: 51)

18. GFPT1 (NT_—022184); Glutamine-fructose-6-phosphate transaminase 1

amplicon size; 200 bp

tcctcctctttcctc
(SEQ ID NO: 52)

gcttcgtgcagtgctggcgcctggggcctggggctgcacggggcacgcacccgggctatt

gctttgctaacaattccatccttctctttccgtcattccctgccaggtgctgagtttctc

cctccctctcggctcggtccctcccgcggctcgccccggggcgggcgtctccaggaactc

caggc

Forward; 5′-tcctcctctttcctcgcttcgt-3′
(SEQ ID NO: 53)

Reverse; 5′-gcctggagttcctggagacg-3′
(SEQ ID NO: 54)

19. GOLPH2 (T_—023935); Golgi phosphoprotein 2

amplicon size; 208 bp

acggcctcatagggcttctcaaacatca
(SEQ ID NO: 55)

gtgcgcctgagcgtcatctgaggcgctgtttcaaatgcagctgcccgggctataagatca

cacccgaaggcgtccgggaatcttcactttttccgttgctagcagtggaagggtcacaga

ccaaacactaaggcctgagcggtgacaaccgaggcgagatgatggtcaacagggaatgcc

Forward; 5′- acggcctcatagggcttctcaa-3′
(SEQ ID NO: 56)

Reverse; 5′- ggcattccctgttgaccatcat-3′
(SEQ ID NO: 57)

20. HHEX (NT_—030059); Hematopoietically expressed homeobox

amplicon size; 209 bp

gcagggaagtctttccatttccatgattaatggaggaccttagcaga
(SEQ ID NO: 58)

aacggctcaccgcgaggtgtgacgaccgcaggagggcgtcaatcaagaaaatgccccctt

gccccggaggcgagtggagccgcacagagccgaggccatacgggccagcagtacggactg

cttcaatagaaaacacgtgcaaaaccagagagccagactgcg

Forward; 5′- gcagggaagtctttccatttcca-3′
(SEQ ID NO: 59)

Reverse; 5′- cgcagtctggctctctggtttt-3′
(SEQ ID NO: 60)

21. LAMA2 (NT_—025741); laminin, alpha 2 (merosin, congenital muscular dystrophy)

amplicon size: 378 bp

(SEQ ID NO: 61)

cctagctgt ttgcatttcc cagaaataat gttttccatg

tgtagggatt ttacagattt caaagtgctt tcatgtcaat

tactttcttt aatttaaaag aagttcagat accaggtcaa

gctaggaatg atccggtctc agagggaagg agcgctctag

gaaaggagga tcctttaata gagggccgtc ctggggccgc

gtgcccatgg aaggcgagag tggaggagtg tcctctttct

cccccaccct caggcggcgg cccggccaaa gccagagggg

gctgtctcct cctcttcccc agcagctgct gctcgctcag

ctcacaagcc aaggccaggg gacagggcgg cagcgactcc

tctggctccc gagaagtgg

(SEQ ID NO: 62)

Forward:

5′-cct agc tgt ttg cat ttc cca

g-3′

(SEQ ID NO: 63)

Reverse:

5′-cca ctt ctc ggg agc cag ag-3′

22. CPEB3 (NT_—030059); cytoplasmic polyadenylation element binding protein 3

amplicon size; 216 bp

taagggggtcattcccctgaaagataacccggttcttgccagtgacatcgctga
(SEQ ID NO: 64)

caaacacttcacaagttggggagggggaagggggaggggaagagggtaaggaaaactaat

tttgtggattaacaggagtccctctcaggtcaaaacggggttccaaggaaaccagacgcc

actccttagctaagtttcacccaaagtctacgacccaggccg

Forward; 5′-taagggggtcattcccctgaaa-3′
(SEQ ID NO: 65)

Reverse; 5′-cggcctgggtcgtagactttg-3′
(SEQ ID NO: 66)

23. HTR1B (NT_—007299); 5-hyroxytryptamine (serotonin) receptor 1B gene

Amplicon size: 434 bp

(SEQ ID NO: 67)

gggagcttcc ttggccagga aaggaacagg gcggggggaa

aagagggagg gagagagaaa aagggaaggt gatggggagc

gggcgtccgc acccgccgcg ccgcacgagt tgcactgctc

tggcggaccg gacctggact ctatataaag agcccatctg

ctccgtagct cgcacgcttc tcccgggctg gtgcacgccg

cgtccctcca gctcccccag acacctgccc cttcccagtg

tgccgcgcca ggtcctccag acccgcgcac ccagtggcat

ggctccgagt ggctcccgtg ggaccagggt ggcggtggcg

gcggcgcggc ccgagcagcc gcaactccag cccccgtgtc

ccccctttta tggctccgtc tccgcggggc agctcgtccg

agtggccaga gagtgaaaag agagggaggg cagag

(SEQ ID NO: 68)

Forward:

5′-gggagcttccttggccagga-3′

(SEQ ID NO: 69)

Reverse:

5′-ctctgccctccctctcttttc-3′

The bolded “ccgg” refers to sites of methylation, which are also recognized by a methylation sensitive restriction enzyme HpaII.

Methylation

Any nucleic acid sample, in purified or nonpurified form, can be utilized in accordance with the present invention, provided it contains or is suspected of containing, a nucleic acid sequence containing a target locus (e.g., CpG-containing nucleic acid). One nucleic acid region capable of being differentially methylated is a CpG island, a sequence of nucleic acid with an increased density relative to other nucleic acid regions of the dinucleotide CpG. The CpG doublet occurs in vertebrate DNA at only about 20% of the frequency that would be expected from the proportion of G*C base pairs. In certain regions, the density of CpG doublets reaches the predicted value; it is increased by ten fold relative to the rest of the genome. CpG islands have an average G*C content of about 60%, compared with the 40% average in bulk DNA. The islands take the form of stretches of DNA typically about one to two kilobases long. There are about 45,000 such islands in the human genome.

In many genes, the CpG islands begin just upstream of a promoter and extend downstream into the transcribed region. Methylation of a CpG island at a promoter usually prevents expression of the gene. The islands can also surround the 5′ region of the coding region of the gene as well as the 3′ region of the coding region. Thus, CpG islands can be found in multiple regions of a nucleic acid sequence including upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g., exons), downstream of coding regions in, for example, enhancer regions, and in introns.

In general, the CpG-containing nucleic acid is DNA. However, invention methods may employ, for example, samples that contain DNA, or DNA and RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded, or a DNA-RNA hybrid may be included in the sample. A mixture of nucleic acids may also be employed. The specific nucleic acid sequence to be detected may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be studied be present initially in a pure form; the nucleic acid may be a minor fraction of a complex mixture, such as contained in whole human DNA. The nucleic acid-containing sample used for determination of the state of methylation of nucleic acids contained in the sample or detection of methylated CpG islands may be extracted by a variety of techniques such as that described by Sambrook, et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989; incorporated in its entirety herein by reference).

A nucleic acid can contain a regulatory region which is a region of DNA that encodes information that directs or controls transcription of the nucleic acid. Regulatory regions include at least one promoter. A “promoter” is a minimal sequence sufficient to direct transcription, to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents. Promoters may be located in the 5′ or 3′ regions of the gene. Promoter regions, in whole or in part, of a number of nucleic acids can be examined for sites of CG-island methylation. Moreover, it is generally recognized that methylation of the target gene promoter proceeds naturally from the outer boundary inward. Therefore, early stage of cell conversion can be detected by assaying for methylation in these outer areas of the promoter region.

Nucleic acids isolated from a subject are obtained in a biological specimen from the subject. If it is desired to detect gastric cancer or stages of gastric cancer progression, the nucleic acid may be isolated from gastric tissue by scraping or taking a biopsy. These specimen may be obtained by various medical procedures known to those of skill in the art.

In one aspect of the invention, the state of methylation in nucleic acids of the sample obtained from a subject is hypermethylation compared with the same regions of the nucleic acid in a subject not having the cellular proliferative disorder of gastric tissue. Hypermethylation, as used herein, is the presence of methylated alleles in one or more nucleic acids. Nucleic acids from a subject not having a cellular proliferative disorder of gastric tissues contain no detectable methylated alleles when the same nucleic acids are examined.

Samples

The present application describes early detection of gastric cancer. Gastric cancer specific gene methylation is described. Applicant has shown that gastric cancer specific gene methylation also occurs in tissues that are adjacent to the tumor region. Therefore, in a method for early detection of gastric cancer, any bodily sample, including liquid or solid tissue may be examined for the presence of methylation of the gastric-specific genes. Such samples may include, but not limited to, serum, or plasma.

Individual Genes and Panel

It is understood that the present invention may be practiced using each gene separately as a diagnostic or prognostic marker or a few marker genes combined into a panel display format so that several marker genes may be detected for overall pattern or listing of genes that are methylated to increase reliability and efficiency. Further, any of the genes identified in the present application may be used individually or as a set of genes in any combination with any of the other genes that are recited in the application. For instance, a criteria may be established where if for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and so forth of the 23 or so gastric-specific genes are methylated, it indicates a certain level of likelihood of developing cancer. Or, genes may be ranked according to their importance and weighted and together with the number of genes that are methylated, a level of likelihood of developing cancer may be assigned. Such algorithms are within the purview of the invention.

Methylation Detection Methods

Detection of Differential Methylation—Methylation Sensitive Restriction Endonuclease

Detection of differential methylation can be accomplished by contacting a nucleic acid sample with a methylation sensitive restriction endonuclease that cleaves only unmethylated CpG sites under conditions and for a time to allow cleavage of unmethylated nucleic acid. In a separate reaction, the sample is further contacted with an isoschizomer of the methylation sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG-sites under conditions and for a time to allow cleavage of methylated nucleic acid. Specific primers are added to the nucleic acid sample under conditions and for a time to allow nucleic acid amplification to occur by conventional methods. The presence of amplified product in the sample digested with methylation sensitive restriction endonuclease but absence of an amplified product in sample digested with an isoschizomer of the methylation sensitive restriction enzyme endonuclease that cleaves both methylated and unmethylated CpG-sites indicates that methylation has occurred at the nucleic acid region being assayed. However, lack of amplified product in the sample digested with methylation sensitive restriction endonuclease together with lack of an amplified product in the sample digested with an isoschizomer of the methylation sensitive restriction enzyme endonuclease that cleaves both methylated and unmethylated CpG-sites indicates that methylation has not occurred at the nucleic acid region being assayed.

As used herein, a “methylation sensitive restriction endonuclease” is a restriction endonuclease that includes CG as part of its recognition site and has altered activity when the C is methylated as compared to when the C is not methylated. Preferably, the methylation sensitive restriction endonuclease has inhibited activity when the C is methylated (e.g., SmaI). Specific non-limiting examples of methylation sensitive restriction endonucleases include Sma I, BssHII, or HpaII, BSTUI, and NotI. Such enzymes can be used alone or in combination. Other methylation sensitive restriction endonucleases will be known to those of skill in the art and include, but are not limited to SacII, and EagI, for example. An “isoschizomer” of a methylation sensitive restriction endonuclease is a restriction endonuclease that recognizes the same recognition site as a methylation sensitive restriction endonuclease but cleaves both methylated and unmethylated CGs, such as for example, MspI. Those of skill in the art can readily determine appropriate conditions for a restriction endonuclease to cleave a nucleic acid (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989).

Primers of the invention are designed to be “substantially” complementary to each strand of the locus to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to perform. Primers of the invention are employed in the amplification process, which is an enzymatic chain reaction that produces exponentially increasing quantities of target locus relative to the number of reaction steps involved (e.g., polymerase chain reaction (PCR)). Typically, one primer is complementary to the negative (−) strand of the locus (antisense primer) and the other is complementary to the positive (+) strand (sense primer). Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA Polymerase I (Klenow) and nucleotides, results in newly synthesized + and − strands containing the target locus sequence. Because these newly synthesized sequences are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the target locus sequence) defined by the primer. The product of the chain reaction is a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed.

Preferably, the method of amplifying is by PCR, as described herein and as is commonly used by those of ordinary skill in the art. However, alternative methods of amplification have been described and can also be employed such as real time PCR or linear amplification using isothermal enzyme. Multiplex amplification reactions may also be used.

Detection of Differential Methylation—Bisulfite Sequencing Method

Another method for detecting a methylated CpG-containing nucleic acid includes contacting a nucleic acid-containing specimen with an agent that modifies unmethylated cytosine, amplifying the CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and non-methylated nucleic acid and detecting the methylated nucleic acid. The amplification step is optional and although desirable, is not essential. The method relies on the PCR reaction itself to distinguish between modified (e.g., chemically modified) methylated and unmethylated DNA. Such methods are described in U.S. Pat. No. 5,786,146, the contents of which are incorporated herein in their entirety especially as they relate to the bisulfite sequencing method for detection of methylated nucleic acid.

Substrates

Once the target nucleic acid region is amplified, the nucleic acid can be hybridized to a known gene probe immobilized on a solid support to detect the presence of the nucleic acid sequence.

As used herein, “substrate,” when used in reference to a substance, structure, surface or material, means a composition comprising a nonbiological, synthetic, nonliving, planar, spherical or flat surface that is not heretofore known to comprise a specific binding, hybridization or catalytic recognition site or a plurality of different recognition sites or a number of different recognition sites which exceeds the number of different molecular species comprising the surface, structure or material. The substrate may include, for example and without limitation, semiconductors, synthetic (organic) metals, synthetic semiconductors, insulators and dopants; metals, alloys, elements, compounds and minerals; synthetic, cleaved, etched, lithographed, printed, machined and microfabricated slides, devices, structures and surfaces; industrial polymers, plastics, membranes; silicon, silicates, glass, metals and ceramics; wood, paper, cardboard, cotton, wool, cloth, woven and nonwoven fibers, materials and fabrics.

Several types of membranes are known to one of skill in the art for adhesion of nucleic acid sequences. Specific non-limiting examples of these membranes include nitrocellulose or other membranes used for detection of gene expression such as polyvinylchloride, diazotized paper and other commercially available membranes such as GENESCREEN™, ZETAPROBE™ (Biorad), and NYTRAN™. Beads, glass, wafer and metal substrates are included. Methods for attaching nucleic acids to these objects are well known to one of skill in the art. Alternatively, screening can be done in liquid phase.

Hybridization Conditions

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows: 2×SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2×SSC/0.1% SDS at about 42.degree. C. (moderate stringency conditions); and 0.1.times.SSC at about 68° C. (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. In general, conditions of high stringency are used for the hybridization of the probe of interest.

Label

The probe of interest can be detectably labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.

Kit

Invention methods are ideally suited for the preparation of a kit. Therefore, in accordance with another embodiment of the present invention, there is provided a kit useful for the detection of a cellular proliferative disorder in a subject. Invention kits include a carrier means compartmentalized to receive a sample therein, one or more containers comprising a first container containing a reagent which sensitively cleaves unmethylated cytosine, a second container containing primers for amplification of a CpG-containing nucleic acid, and a third container containing a means to detect the presence of cleaved or uncleaved nucleic acid. Primers contemplated for use in accordance with the invention include those set forth in SEQ ID NOS:1-69, and any functional combination and fragments thereof. Functional combination or fragment refers to its ability to be used as a primer to detect whether methylation has occurred on the region of the genome sought to be detected.

Carrier means are suited for containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. In view of the description provided herein of invention methods, those of skill in the art can readily determine the apportionment of the necessary reagents among the container means. For example, one of the container means can comprise a container containing methylation sensitive restriction endonuclease. One or more container means can also be included comprising a primer complementary to the locus of interest. In addition, one or more container means can also be included containing an isoschizomer of the methylation sensitive restriction enzyme.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to theose skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.

EXAMPLES
Example 1
Identification of Genes Repressed in Gastric Cancer

To identify genes repressed in gastric cancer, microarray hybridization experiments were carried out. Microarray hybridizations were performed according to standard protocol (Schena et al, 1995, Science, 270: 467-470). Total RNA was isolated from paired tumor-adjacent tissues (4 samples) and tumor tissues (4 samples) of gastric cancer patients. To compare relative difference in gene expression level between paired tumor-adjacent and tumor tissues indirectly, we prepared common reference RNA (indirect comparison). Total RNA was isolated from 11 human cancer cell lines. Total RNA from cell lines and gastric tissues was isolated using Tri Reagent (Sigma, USA) according to manufacturer's instructions. To make common reference RNA, equal amount of total RNA from 11 cancer cell lines was combined. The common reference RNA was used as an internal control. To compare relative difference in gene expression levels in paired tumor-adjacent and tumor tissues, RNAs isolated from non-tumor and tumor tissues were indirectly compared with common reference RNA. 100 ug of total RNA was labeled with Cy3-dUTP or Cy5-dUTP. The common referene RNA was labeled with Cy3 and RNA from gastric tissues was labeled with Cy5, respectively. Both Cy3- and Cy5-labeled cDNA were purified using PCR purification kit (Qiagen, Germany). The purified cDNA was combined and concentrated at a final volume of 27 ul using Microcon YM-30 (Millipore Corp., USA).

Total 80 ul of hybridization mixture contained: 27 ul labeled cDNA targets, 20 ul of 20×SSC, 8 ul of 1% SDS, 24 ul of formamide (Sigma, USA) and 20 ug of human Cotl DNA (Invitrogen Corp., USA). The hybridization mixtures were heated at 100° C. for 2 min and immediately hybridized to human 22K oligonucleotide (GenomicTree, Inc) microarrays. The arrays were hybridized at 42° C. for 12-16 h in the humidified HybChamber X (GenomicTree, Inc., Korea). After hybridization, microarray slides were imaged using Axon 4000B scanner (Axon Instruments Inc., USA). The signal and background fluorescence intensities were calculated for each probe spot by averaging the intensities of every pixel inside the target region using GenePix Pro 4.0 software (Axon Instruments Inc., USA). Spots were excluded from analysis due to obvious abnormalities. All data normalization, statistical analysis and cluster analysis were performed using GeneSpring 7.3 (Agilent, USA).

To determine relative difference in gene expression levels between non-tumor and tumor tissues, statistical analysis (ANOVA (p<0.05) for indirect comparison was performed. From the results of statistical analysis, a total of 818 genes down regulated in tumor compared with paired tumor-adjacent tissues by indirect comparisons (FIG. 1).

Example 2
Identification of Methylation Controlled Gene Expression

To determine whether the expression of any of the genes identified in Example 1 is controlled by promoter methylation, gastric cancer cell lines MKN1, MKN28 and SNU484 were treated with demethylation agent, 5-aza-2′ deoxycytidine (DAC, Sigma, USA) for three days at a concentration of 200 nM. Cells were harvested and total RNA was isolated from treated and untreated cell lines using Tri reagent. To determine gene expression changes by DAC treatment, transcript level between untreated and treated cell lines was directly compared. From this experiment, 3,036 genes have been identified that show elevated expression when treated with DAC compared with the control group which was not treated with DAC. 61 common genes between the 818 tumor repressed genes and the 3,036 reactivated genes were identified (FIG. 1).

Example 3
Confirmation of Methylation of Identified Genes
Example 3.1
In Silico Analysis of Cpg Island in Promoter Region

The promoter regions of the 61 genes were scanned for the presence of CpG islands using MethPrimer (http://itsa.ucsf.edu/˜urolab/methprimer/indexl.html). Twenty one genes did not contain the CpG island and were dropped from the common gene list.

Example 3.2
Biochemical Assay for Methylation

To biochemically determine the methylation status of the remaining 40 genes, methylation status of each promoter was detected using the characteristics of restriction endonucleases, HpaII (methylation-sensitive) and MspI (methylation-insensitive) followed by PCR (FIG. 2). Both enzymes recognize the same DNA sequence, 5′-CCGG-3′. HpaII is inactive when internal cytosine residue is methylated, whereas MspI is active regardless of methylated or not. In the case that the cytosine residue at the CpG site is unmethylated, both enzymes can digest the target sequence. To determine the methylation status of a specific gene, PCR targets containing one or more HpaII sites from CpG islands in the promoter region were selected. 100 ng of genomic DNA from gastric cancer cell lines AGS, MKN1, MKN28 and SNU484 were digested with 5 U of HpaII and 10 U of MspI, respectively and purified using Qiagen PCR purification kit. Specific primers were used to amplify regions of interest. 5 ng of the purified genomic DNA was amplified by PCR using gene-specific primer sets. DNA from undigested control sample was amplified to determine PCR adequacy. The PCR was performed as follows: 94° C., 1 min; 66° C., 1 min; 72° C., 1 min (30 cycles); and 72° C., 10 min for final extension. Each amplicon was separated on a 2% agarose gel containing ethidium bromide. If the band density of HpaII amplicon is 1.5-fold greater than that of MspI amplicon, the target region was considered to be methylated, while less than 1.5-fold was considered to be unmethylated. From this, it was discovered that 17 genes were not methylated, leaving 23 confirmed candidate genes that fit the criteria of being down regulated in tumor, up regulated under demethylation conditions, contains a CpG island in its promoter and is actually methylated in the cancer cell lines. See FIG. 3 and FIG. 4.

Example 3.3
Bisulfite Sequencing of Methylated Promoter

To further confirm the methylation status of the 23 identified genes, the inventors performed bisulfite sequencing of the individual promoters. Upon treatment of the DNA with bisulfite, unmethylated cytosine is modified to uracil and the methylated cytosine undergoes no change. The inventors performed the bisulfite modification according to Sato, N. et al., Cancer Research, 63:3735, 2003, the contents of which are incorporated by reference herein in its entirety especially regarding the use of bisulfite modification method as applied to detect DNA methylation. The bisulfite treatment was performed on 1 μg of the genomic DNA of the gastric cancer cell lines AGS, MKN1, MKN28 and SNU484 using MSP (Methylation-Specific PCR) bisulfite modification kit (In2Gen, Inc., Seoul, Korea). After amplifying the bisulfite-treated AGS, MKN1, MKN28 and SNU484 genomic DNA by PCR, the nucleotide sequence of the PCR products was analyzed. The results confirmed that the genes were all methylated.

Example 4
Gene expression profile of the identified genes

FIG. 5 shows the gene expression profiles of the 23 genes that were identified. As shown in FIG. 5, gene expression was repressed in the tumor compared with paired tumor-adjacent tissues.

Example 5
Reactivation of 23 identified genes by treatment of demethylating agent

FIG. 6 shows reactivation of the 23 genes that were identified. As shown in FIG. 6, gene expression was reactivated in the gastric cancer cells treated with demethylating agent (DAC) compared with untreated cells.

Example 6
Promoter methylation assay on clinical samples

To determine the clinical applicability of the methylated promoters of the 23 selected genes of the present invention, methylation assay was performed with normal tissues from non-patients, paired tumor-adjacent tissues and early gastric cancer tissues clinical samples. Methylation assay was performed as described supra using restriction enzyme/PCR.

FIG. 7 shows the results of the methylation assay on early gastric cancer. As shown in FIG. 7, none of the genes are methylated in the normal tissues from non-patient samples (Biochain). However, all of the genes are methylated in gastric cancer tissues as well as in paired tumor-adjacent tissues. All of the genes are methylated in cancer samples but not in normal cells as predicted. Moreover, as shown in FIG. 7, since the 23 identified genes were methylated in paired tumor-adjacent tissues, the results indicate that these 23 identified genes are useful for early detection of gastric cancer. FIG. 7B shows methylation frequency of identified markers in tumor tissue and paired tumor-adjacent tissue.

The methylation frequency of identified markers is obtained by dividing the total number of samples tested, which include the tumor tissue and the paired tumor-adjacent tissue samples, into either the number of marker methylated tumor tissue samples to obtain frequency of marker methylation in tumor tissue, or dividing the total number of samples into the number of marker methylated paired tumor-adjacent tissue samples to obtain frequency of methylation of the markers in paired tumor-adjacent tissue. This is expressed in percentages.

Example 7
Promoter Methylation Assay on Advanced Gastric Cancer Clinical Samples

To determine the clinical applicability of the methylated promoters of the 23 selected genes of the present invention, methylation assay was performed with advanced gastric cancer tissues clinical samples, as compared with normal samples. Methylation assay was performed as described supra using restriction enzyme/PCR. FIG. 8 shows the results of the methylation frequency (%) of 23 selected genes on advanced gastric cancer. As shown in FIG. 8, all of the genes are highly methylated in the advanced gastric cancer tissues. All of the genes are methylated in cancer samples but not in normal cells as predicted.

The methylation frequency of identified markers is obtained by dividing the total number of samples tested, which include the tumor tissue and normal tissue, into the number of marker methylated tumor tissue samples to obtain frequency of methylation in an identified marker in tumor tissue. This is expressed in percentages.

All of the references cited herein are incorporated by reference in their entirety.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.

GASTRIC CANCER BIOMARKER DISCOVERY

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Abstract

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Provisional Applications (1)