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WO2011133935A2 - Procédés et kits pour évaluation des risques de la progression néoplasique de barrett - Google Patents

Procédés et kits pour évaluation des risques de la progression néoplasique de barrett Download PDF

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WO2011133935A2
WO2011133935A2 PCT/US2011/033671 US2011033671W WO2011133935A2 WO 2011133935 A2 WO2011133935 A2 WO 2011133935A2 US 2011033671 W US2011033671 W US 2011033671W WO 2011133935 A2 WO2011133935 A2 WO 2011133935A2
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methylation
gene
progression
loh
dna
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WO2011133935A3 (fr
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Stephen J. Meltzer
Zhe Jin
Yula Chen
Sarah Margaret Jacobs-Helber
Thomas Robert Reynolds
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AMERICAN INTERNATIONAL BIOTECHNOLOGY SERVICES
Johns Hopkins University
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AMERICAN INTERNATIONAL BIOTECHNOLOGY SERVICES
Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates generally to prediction of a patient's risk for progression of Barrett's esophagus (BE), and more specifically to methods and kits for predicting a patient's risk for progression of Barrett's esophagus (BE) using genetic markers at regions of loss of heterozygosity (LOH) in combination with genetic markers for gene methylation.
  • BE Barrett's esophagus
  • LH loss of heterozygosity
  • BE Barrett's esophagus
  • GERD chronic gastroesophageal reflux disease
  • EAC esophageal adenocarcinoma
  • HGDs cancers or advanced high-grade dysplasias
  • a means of stratifying patients into groups at high, average, and low risk of neoplastic progression would be highly beneficial.
  • Such a system would benefit BE patients in two ways: 1 ) by decreasing the frequency at which low-risk individuals undergo surveillance endoscopy, eliminating unnecessary anxiety, expense, and diminishing procedure-related complications: and 2) by identifying the small group of high-risk BE patients for more frequent, intensive surveillance, resulting in earlier and more accurate detection of HGDs and EACs. More accurate tissue-based biomarkers capable of predicting the risk of progression to HGD or EAC would be extremely useful.
  • TSGs tumor suppressor genes
  • TSGs Abnormalities involving the pl6 (chromosome 9p21) and p53 (17pl3) TSGs are among the most common somatic genetic lesions in human cancers. LOH is the predominant mechanism for inactivating these genes, and these loci are the two most common regions of LOH in esophageal
  • adenocarcinoma adenocarcinoma and are lost early during Barrett's neoplastic progression.
  • Other loci implicated in EAC are 5q, retinoblastoma (Rb) (13ql4), lp, 8p, and 18q.
  • the present invention is based on the seminal discovery of a set of biomarker assays to predict the future risk of esophageal cancer or advanced precancerous esophageal lesions in patients with Barrett's esophagus (BE).
  • the present invention includes, but is not restricted to biomarkers based on gene hypermethylation (CHS), as well as biomarkers based on loss of heterozygosity (LOH), both occurring in Barrett's esophagus tissue biopsy samples.
  • CHS gene hypermethylation
  • LH loss of heterozygosity
  • the present invention provides a method for predicting risk of progression of BaiTett's esophagus (BE) in a subject.
  • the method includes (a) detecting gene- specific methylation and loss of heterozygosity (LOH) of at least one marker in a sample from the subject; wherein the marker includes at least one marker a t a region of LOH in
  • the risk is progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC).
  • the risk is progression of BaiTett's esophagus (BE) to high grade dysplasia (HGD).
  • the region of LO H includes at least one short tandem repeat (STR) region.
  • the gene-specific methylation includes gene promoter methylation.
  • the at least one marker includes at least about one to six markers at regions of LOH.
  • the at least one marker includes at least about one to eight markers for gene-specific methylation, In another aspect, the markers are selected from the group consisting of at least one of D4S243, FGA, Dl 78695, MBP, and MBPa. In another aspect, the markers are selected from the group consisting of at least one of D4S243, FGA, D9S747, D17S654, D17S695, MBP, MBPa, D16S310, D9S162, THOl, and IFN-A.
  • the markers are selected from the group consisting of at least one of D4S243, FGA, D9S747, D17S654, D17S695, MBP, MBPa, D16S310, D9S162, THOl, I FN- A, D5S346a, D5S82, and D9S171.
  • the markers are selected from the group consisting of at least one of D4S243, FGA, D9S747, D17S654, D17S695, MBP, MBPa, D 16S310, D9S162, THOl , IFN-A, D21S 1245, D20S48, D9S171, D16S476, D 13S134, D5S346, and D5S82.
  • the at least one marker includes six markers at regions of LOH. In another aspect, the at least one marker includes eight markers for gene-specific methylation. In another aspect, the markers include makers listed in Table 1-1, 1-2, 2-1, 2-2, 5, 9, Figure 3, or Figure 5 as provided herein,
  • microsatellite analysis is used in the method described herein.
  • STRs short tandem repeats
  • qualitative real-time polymerase chain reaction (PGR) is used in the method described herein.
  • LOH is detected by calculating ratios of allele heights.
  • methylation specific PGR is used in the method described herein.
  • the sample is selected from the group consisting of tissue, blood, ascites, saliva, urine, perspiration, tears, semen, serum, plasma, amniotic fluid, pleural fluid, cerebrospinal fluid, a cell line, a xenograft, a tumor, pericardial fluid, and a combination thereof.
  • the sample is embedded in paraffin.
  • the subject is a mammalian. In another aspect, the subject is human.
  • the present invention provides a kit for predicting risk of progression of Barrett's esophagus (BE) in a subject.
  • the kit includes DNA primers for detecting at least one marker at a region of loss of heterozygosity (LOH); and reagents for real-time methylation specific PGR for at least one marker for gene-specific methylation,
  • LHO loss of heterozygosity
  • reagents for real-time methylation specific PGR for at least one marker for gene-specific methylation
  • the risk is progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC).
  • EAC esophageal adenocarcinoma
  • the risk is progression of Barrett's esophagus (BE) to high grade dysplasia (HGD).
  • the region of LOH includes at least one short tandem repeat (STR) region.
  • the gene-specific methylation includes gene promoter methylation.
  • the method includes DNA primers for short tandem repeat (STR) markers to identify loss of heterozygosity (LOH).
  • the method includes DNA primers for real-time methylation specific PCR for gene-specific methylation.
  • Figure 1 shows a substantial increment in the area under the receiver operating characteristic ROC curve (AUROC) for both the uncorrected (original) and overfttting- corrected ROC curves based on the 8-marker pane! plus age versus age alone.
  • AUROC receiver operating characteristic ROC curve
  • Figure 2 shows an example of loss of heterozygosity on chromosome 9 in EAC. Shown is an electropherogram of an STR. marker located at 9q32 from a matched blood and EAC tumor DNA pair. Two copies of me locus are present in the blood, but only one copy is present in the tumor. Quantitation of LOH (or the degree of allelic imbalance) using capillar ⁇ ' electrophoresis is calculated by determining the ratio of tumor peak height to blood peak height at each locus using the following formula:
  • Ratio (Peak Height of Tumor Allele 1/Peak Height of Tumor Allele 2)
  • Figure 3 shows exemplar ⁇ ' microsatellite analysis of matched blood and EAC (blood-tumor DNA pairs) and matched blood and normal mucosa (normal DNA pairs). Loci with ratios of blood/ tumor peak heights ⁇ 0.70 or >1.3 are considered to have LOH (black boxes).
  • Figure 4 shows ROC curves for the detection of progression of BE patients using eight methylation markers (Figure 4 A, AUC 0.7619: Methylation markers alone) or eight methylation markers + six LOH markers (Figure 4B, AUC 0.9524; Methylation markers plus LOH markers).
  • Figure 5 shows another exemplar ⁇ ' microsatellite analysis of blood/tissue DNA pairs from BE patients who do not progress to E AC or HGD (Nonprogressors) versus BE patients who progressed to EAC or HGD (Progressors). Loci with ratios of blood-to-tumor peak height ⁇ 0.70 or > 1.3 are considered to have LOH (black boxes).
  • Figure 6 shows results of PAM classification of progressors and nonprogressors based on methylation array data. All 9 specimens are classified correctly except for sample E530, which is consistently misclassified as a nonprogressor (it is actually a progressor). Samples 1 -4 are nonprogressors; samples 5-9 are progressors.
  • Figure 7 shows correlation of combined Hazard ratio with time to progression to HGD or EAC.
  • a combined Cox's hazard ratio (HR) index is calculated from the multivariate Cox model, taking into account normalized methylation values (NMV) of 6 genes, age, and BE segment length when each sample is obtained. Solid squares are specimens from progressors (P); open triangles are nonprogressors (NP). At 2 years or less before progression (the endpoint), HR indices of all Ps exceed 5, while HR indices of most NPs remain below 5. The data suggest that HR index begins to predict progression approximately 2 years before it occurs.
  • NMV normalized methylation values
  • Figure 8 shows sample ("slice") of the temporal epigenomic program of Barrett's pre-progression, focusing on hypomethylation.
  • X-axis shows pre -progression interval or NP.
  • Back row genes such as RAX, are consistently and significantly less heavily methylated in progressors than in nonprogressors, except for outlier sample E530.
  • Figure 9 shows sample ("slice") of the temporal epigeiiomic program of Barrett's pre-progression, focusing on hypermethylation.
  • X-axis shows pre-progression interval or NP.
  • Gray loci such as Intra PSCD3, are consistently and significantly more heavily methylated in progressors than in nonprogressors, except for outlier sample E530.
  • Figure 10 shows a treeview of Early versus Late Barrett's Progressors. This hierarchical clustering analysis shows that outlier index sample E530 is located in 1 major branch of the dendrogram, while the remaining 4 progressors constitute the other remaining branch. This finding is consistent with the result that the methylation program of E530 significantly differed from that of the other progressors, possibly due to its longer pre- progression interval of 40 months.
  • This invention relates to methods for identifying genetic markers at regions of loss of heterozygosity (LOH) that can predict a patient's risk for progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) or advanced high-grade dysplasia (HGD) with high sensitivity and specificity.
  • LOH loss of heterozygosity
  • BE Barrett's esophagus
  • EAC esophageal adenocarcinoma
  • HSD advanced high-grade dysplasia
  • analysis of LOH can be done in combination with the methylation markers described herein.
  • BE Barrett's esophagus
  • GSD chronic gastroesophageal reflux disease
  • EAC esophageal adenocarcinoma
  • ail patients with BE must undergo periodic surveillance endoscopy with biopsies, and need for as well as proper time interval between these endoscopies is a matter of considerable debate.
  • a molecular method for stratifying the risk of future BE neoplastic progression would assist physicians in determining the appropriate frequency of endoscopic biopsy.
  • chromosomes are unstabl e in cancerous cells and are deleted, causing a l oss of heterozygosity (LOH) in the tumor sample.
  • LOH heterozygosity
  • This LOH is detected using a method known as microsatellite analysis (MSA).
  • MSA microsatellite analysis
  • PCR polymerase chain reaction
  • comparison of the normal to the tumor DNA can detect the genetic changes indicative of cancerous lesions. While this method is sensitive, it is time consuming and requires many reagents. A faster, more sensitive assay would be more cost effective and would also be useful in a clinical setting.
  • MSP Methylation specific-PCR
  • Subsequent PCR amplification may only amplify methylated sequences, thereby identifying the methylation status of the gene or promoter of interest.
  • MSP MSP
  • the present invention provides that certain tumor suppressor genes undergo methylation in BE and can function as biomarkers, predicting whether BE patients will or will not develop HGD or EAC in the future.
  • the present invention provides illustrative examples showing that the genes NELL1, TAC1, SST, AKAP 12, and CDH13 are methylated early and often in Barrett's neoplasia and correlated with clinical risk factors, such as degree of dysplasia and length of Barrett's segment.
  • Ps progressors
  • non-progressors are defined as patients who had undergone at least 3 surveillance endoscopic examinations with index biopsies but did not progress to an endpoint more neoplastically advanced than LGD. Furthermore, progressors are considered both as one group (combined) and in two tiers: progression within 2 years or within 4 years
  • the present invention provides a microsatellite genotyping based assay for detection of bladder cancer.
  • a fluorescence-based multiplex assay has been devel oped to detect loss of heterozygosity (LOH) in DNA isolated from patients with bladder cancer.
  • the original assay used 15 STR markers in 5 PCR reactions, 3 sets of cycling parameters and analysis in 5 lanes of a gel based system.
  • the assay can be adopted into high-throughput and more user- friendly: 3 multiplex PCR reactions and one singleplex reaction, 1 set of cycling parameters and 2 wells in a capillary electrophoresis unit.
  • This assay provides a quantitative measure of LOH by measuring changes in peak height between the blood (normal) and urine sediment (abnormal) DNA,
  • the present invention provides at least the following embodiments: (1) real-time PCR assays to short tandem repeat (STR) markers that have been previously identified as undergoing LOH during BE cancer progression (for example a panel of PCR assays that can be optimized); (2) real-time PC R assay for its ability to determine the copy number of the target marker in normal human DNA (for example a panel of real-time PCR assays that can determine the copy number of the loci in one sample relative to another); and (3 ) assays to detect LOH using purified genomic DNA from normal and tumor specimens (for example a panel of assays that increases the specificity and sensitivity of the methylation biomarker panel for detecting risk of progression from BE to AEC).
  • STR short tandem repeat
  • CHS markers to predict future neoplasia risk in BE patients have not been validated previously.
  • the unique CH3 gene panel described herein has been extensively validated for accuracy in studies performed.
  • LOH markers have been studied to some extent in BE, they have not been combined previously with methylation markers for the purpose of predicting future neopl astic progression.
  • previous assays of LOH have depended on high-quality, high-molecular weight (MW) DNA from both BE tissues and matching normal tissues (the latter of which are not normal ly obtained during endoscopy ) to produce data.
  • the LOH assay disclosed herein obviates the need 1) for high-MW DNA, as well as 2) for matching normal tissues.
  • the methods provided herein can be successfully performed on very tiny bits of tissue, such as those obtained from EGD biopsies. Also the methods provided herein can be carried out on archival (i.e., old or stored) tissue samples from EGDs that have been performed in the past.
  • HDAC histone deacetylase
  • hypermethylation i.e., methylation silenced
  • others with unmethylated promoters for which increased expression is produced by ED AC inhibition.
  • hypermethylation in regions of high frequency loss of heterozygosity (LOH) and throughout the genome have all proven to have utilities for identification of tumor specifically hyperraethylated CpG islands, However, each suffers from either identifying some sites not associated with gene promoters, potential bias of utilized methylation sensitive restriction sites for CpG island subsets or lack of the site in numerous islands, and/or the need to laboriously search for nearby genes once the altered locus is identified.
  • epigeneticaliy silenced or "epigenetic silenced”, when used in reference to a gene, means that the gene is not being transcribed, or is being transcribed at a level that is decreased with respect to the level of transcription of the gene in a corresponding control cell (e.g., a normal cell), due to a mechanism other titan a genetic change.
  • Epigenetic mechanisms of gene silencing are well known and include, for example, hypermethylation of CpG dinucleotides in a CpG island of the 5' regulatory region of a gene, and structural changes in chromatin due, for example, to histone acetylation, such that gene transcription is reduced or inhibited.
  • Methods for detecting epigenetic silencing of a gene include, for example, detecting re-expression (reactiva tion) of the gene following contact of a cell with an agent that relieves the epigenetic silencing, for example, with a demethylating agent where the silencing is due to hypermethylation.
  • gene-specific methylation includes, for example, promoter methylation, intron methylation, and/or exon methylation.
  • gene-specific methylation may include CpG island methylation.
  • methylation or “hypermethylation”, when used in reference to a gene, means that cytosine residues of CpG dinucleotides (for example in a CpG island) associated with the gene are methylated at the 5 '-position, i.e., 5 '-methylcytosine.
  • methylation status is used herein to refer to a relative abundance, including the presence or absence, of methylated cytosine residues of CpG dinucleotides for example in a CpG island.
  • cytosine residues in a CpG island are not methylated in a transcriptionally ac tive gene and, therefore, the detection of methylated cytosine residues in a CpG island indicates that expression of the gene is reduced or inhibited.
  • reference herein to a "methylation silenced" gene means that the gene is not being transcribed, or is being transcribed at a level that is decreased with respect to the level of transcription of the gene in a corresponding control cell (generally a normal cell) due to hypermethylation of CpG dinucleotides in a CpG island of the 5' regulatory region of the gene.
  • a consequence of methylation silenced gene expression is that a cell containing the gene has reduced levels of, or completely lacks, a polypeptide encoded by the gene (i.e., the gene product) such that any function normally attributed to the gene product in the cell is reduced or absent.
  • a method of identifying an epigeneticaliy silenced gene associated with a cancer can be performed, for example, by contacting an array of nucleotide sequences representative of a genome with nucleic acid subtraction products (i.e., nucleic acid molecules corresponding to RNA expressed in cancer cells contacted with at least one agent that reactivates expression of epigenetical iy silenced genes, but not RNA expressed in normal cells corresponding to the cancer cells) under conditions suitable for selective hybridization of nucleic acid subtraction products to complementary nucleotide sequences of the array; and detecting selective hybridization of nucl eic acid subtraction products to a subpopulation of nucleotide sequences of the array, wherein nucleic acid molecules corresponding to RNA.
  • nucleic acid subtraction products i.e., nucleic acid molecules corresponding to RNA expressed in cancer cells contacted with at least one agent that reactivates expression of epigenetical iy silenced genes, but not RNA expressed in normal cells corresponding to the
  • nucleic acid molecules corresponding to RNA of a cell means RN A such as mRNA or polyA+ RNA, cDNA generated using RNA from the cell as a template, or cRNA generated using RNA or cDNA as a template.
  • the nucleic acid molecules corresponding to RNA of a cell generally can be detectably labeled, for example, with a radioisotope, a paramagnetic isotope, a luminescent compound, a chemiiuminescent compound, a fluorescent compound, a metal chelate, an enzyme, a substrate for an enzyme, a receptor, or a iigand for a receptor; or are capable of being detected, for example, using a detectably labeled probe, such that hybridization of the nucleic acid molecuies to nucleotide sequences of the array can be detected,
  • array of nucleotide sequences representative of a genome means an organized group of nucleotide sequences that are linked to a sol id support, for example, a microchip or a glass slide, wherein the sequences can hybridize specifically and selectively to nucleic acid molecules expressed in a cell.
  • the array is selected based on the organism from which the cells to be examined are derived and/or on a tissue or tissues that are to be examined.
  • the array is representative of the genome of a eukaryotic ceil or cell type, particularly a mammalian cell or cell type, and preferably a human cell, including a cell of one or more tissues, as desired (e.g., colorectal epithelial cells).
  • an array of probes that is "representative" of a genome may identify at least about 10% of the expressed nucleic acid molecules in a ceil, generally at least about 20% or 40%, usually about 50% to 70%, typically at least about 80% or 90%, and particularly 95% to 99% or more of the expressed nucleic acid molecules of a cell or organism.
  • I t should be recognized that the grea ter the representa tion, the more likely that a method of the invention can identify all genes that are epigeneticaily silenced in a cancer.
  • Arrays containing nucleotide sequences representative of specified genomes can be prepared using well known methods, or obtained from a commercial source (e.g., Invitrogen Corp.; Affymetrix),
  • the agent that reactivates expression of epigeneticaily silenced genes can be a methyltransferase inhibitor (e.g., 5 -aza-2 ' -deoxycytidine, DAC), a histone deacetylase inhibitor (e.g., trichostatin A; TSA), or a combination of agents such as a combination of DAC and TSA.
  • a methyltransferase inhibitor e.g., 5 -aza-2 ' -deoxycytidine, DAC
  • a histone deacetylase inhibitor e.g., trichostatin A; TSA
  • TSA histone deacetylase inhibitor
  • a combination of agents such as a combination of DAC and TSA.
  • RNA can be isolated from cells such as cancer cells treated with such an agent or agent, and the RNA, or a cDNA product of the RNA can be contacted with RNA molecules from corresponding cells (e.g., cancer cells) that are not treated with the agent(s) under conditions such that RNA (or cDNA) expressed only in the treated cells can be isolated, thus obtaining nucleic acid subtraction products.
  • Methods for performing a nucleic acid subtraction reaction are well known (Hedrick et al., Nature 308:149-155, 1984, which is incorporated herein by reference), and kits for performing such methods are available from commercial sources (e.g., Gibco/BRL),
  • retinoblastoma (Rb) gene has been demonstrated in a small fraction of retinoblastomas (Sakai et al., Am. J. Hum. Genet. 48:880, 1991), and aberrant methylation of the VHL gene was found in a subset of sporadic renal cell carcinomas (Herman et al., Proc. Natl. Acad. Sci. US A 91 :9700-9704, 1994), Expression of a tumor suppressor gene can also be abolished by de novo DNA methylation of a normally unmethylated 5 ' CpG island (see, for example, Issa et al, Nature Genet. 7:536, 1994; Merlo et al,, Nature Med. 1 :686, 1995; Herman et al.. Cancer Res. 56:722, 1 996).
  • Methylati on-silenced transcription of the SOCS-1 gene is associated with various cancers, including hepatocellular carcinoma, multiple myeloma, and acute leukemias (Yoshikawa et al., Nat. Genet. 28:29-35, 2001 , which is incorporated herein by reference).
  • Biological sample includes sections of tissues such as biopsy (e.g., esophageal tissue) or autopsy samples, and frozen sections taken for histologic purposes.
  • Biological samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells, e.g., primary cultures, explants, and transformed cells; stool, urine, ejaculate, or other biological fluids.
  • a biological sample also includes a surgical sample taken from a patient during a surgery, for example, from a tumor or tumor margin, or other tissue,
  • test cell examined according to a method of the invention also can be a primary ceil that has been obtained from a subject and placed in culture, for example, for the purpose of es tablishing a primary cell culture that exhibits substantially the same growth
  • colon epithelial cells can be obtained from a cancer patient, wherein the cells exhibit methylation silenced expression of one or more genes associated with the cancer.
  • the ceils can be treated in culture using one or more agent to be tested for an ability to restores expression of the silenced gene(s), thus providing a means to identify an agent that can be useful for treating the cancer patient, or another patient characterized by methylation silencing of one or more of the same genes.
  • test ceil can be obtained from a subject in any way typically used in clinical setting for obtaining a sample containing the cells.
  • the test cells can be obtained by a biopsy procedure such as needle biopsy of an organ or tissue containing the cells to be tested.
  • the test cells can be obtained from a gastrointestinal tract sample (e.g., a biopsy of a polyp), a liver sample, a bone marrow sample, a skin sample, a lymph node sample, a kidney sample, a lung sample, a muscle sample, a bone sample, a brain sample, or the like.
  • test cell also can be a component of a biological fluid, for example, blood, lymph, cerebrospinal fluid, saliva, sputum, stool, urine, or ejaculate. If appropriate, the test cells also can be obtained by lavage, for example, for obtaining test cells from the colon, uterus, abdominal cavity, or the like, or using an aspiration procedure, for example, for obtaining a bone marrow sample.
  • a biological fluid for example, blood, lymph, cerebrospinal fluid, saliva, sputum, stool, urine, or ejaculate.
  • the test cells also can be obtained by lavage, for example, for obtaining test cells from the colon, uterus, abdominal cavity, or the like, or using an aspiration procedure, for example, for obtaining a bone marrow sample.
  • a method of the invention also can be practiced using an extract of a test cell, wherein the extract includes nucleic acid molecules of the test ceil, particularly genomic DNA, including all or a CpG island containing portion of the gene or genes to be examined.
  • the extract can be a crude extract including, for example, a freeze-thawed sample of a tissue containing the test cells: can include partially purified genomic DNA, which can include, for example, components of the nuclear matrix; or can include substantially purified genomic DNA, which is obtained, for example, following treatment with a protease and alcohol precipitation.
  • the test cell also can be a component of a histologic sample that is embedded in paraffin.
  • the method for identifying a cell that exhibits or is predisposed to exhibiting unregulated growth is performed by detecting methylation of one or more target genes in the cell.
  • Methylation of a CpG dimicleotide in a CpG island of a gene can be detected using any of various well known methods for detecting CpG methylation of a nucleic acid molecule.
  • Such methods include contacting the gene with one or a series of chemical reagents that selectively modify either unmethylated cytosine residues or methylated cytosine residues, but not both, such that the presence or absence of the modification can be detected; contacting the gene sequence with a methylation sensitive restriction endonuciease, which has a recognition site that includes a CpG dinucleotide, and that cleaves a recognition site either having a methylated cytosine residue of the CpG or lacking a methylated cytosine residue of the CpG, but not both, such that the presence or absence of cleavage of the sequence can be detected: or contacting a nucleic acid molecule including the gene with an oligonucleotide probe, primer, or amplification primer pair that selectively hybridizes to the gene sequence and allows a determination to made as to whether the CpG methylation is present, Examples of such methods are provided herein, and modifications and variations on such methods are well known in the art,
  • Methylation silencing of a gene also can be detected by contacting a 5' regul atory region of the nucleic acid molecule including the gene of the test cell w ith a chemical reagent that selectively modifies either an uiimethylated cytosine residue or a methylated cytosine residue, and detecting a product generated due to said contacting, wherein the product is indicative of methylation of a cytosine residue in a CpG dinucleotide of the gene, thereby detecting methylation silencing of the gene of the test cell.
  • the product can be detected using an electrophoresis method, a chromatography method, a mass spectrometry method, or a combination of such methods.
  • the gene is contacted with hydrazine, which modifies cytosine residues, but not methylated cytosine residues, then the hydrazine treated gene sequence is contacted with a reagent such as piperidine, which cleaves the nucleic acid molecule at hydrazine modified cytosine residues, thereby generating a product including fragments.
  • a reagent such as piperidine
  • piperidine cleaves the nucleic acid molecule at hydrazine modified cytosine residues, thereby generating a product including fragments.
  • a nucleic acid molecule including the target gene is contacted with a chemical reagent including bisulfite ions, for example, sodium bisulfite, which converts unmethylated cytosine residues to bisulfite modified cytosine residues, then the bisulfite ion treated gene sequence is exposed to alkaline conditions, which convert bisulfite modified cytosine residues to uracil residues.
  • bisulfite ions for example, sodium bisulfite
  • alkaline conditions which convert bisulfite modified cytosine residues to uracil residues.
  • Sodium bisulfite reacts readily with the 5,6- double bond of cytosine (but poorly with methylated cytosine) to form a sulfonated cytosine reaction intermediate thai is susceptible to deamination, giving rise to a sulfonated uracil.
  • the sulfonate group can be removed by exposure to alkaline conditions, resulting in the formation of uracil.
  • the DNA then can amplified, for example, by PCR, and sequenced to determine the methylation status of all CpG sites.
  • Uracil is recognized as a thymine by Taq polymerase and, upon PCR, the resultant product contains cytosine only at the position where 5-methylcytosine is present in the starting template DNA.
  • the amount or distribution of uracil residues in the bisulfite ion treated gene sequence of the test cell By comparing the amount or distribution of uracil residues in the bisulfite ion treated gene sequence of the test cell with a similarly treated corresponding unmethylated gene sequence, detection of a decrease in the amount or distribution of uracil residues in the gene from the test cell is indicative of methyla tion of cytosine residues in CpG dinucleotides in the target gene of the test cell.
  • the amount or distribution of uracil residues also can be detected by contacting the bisulfite ion treated target gene sequence, following exposure to alkaline conditions, with an
  • oligonucleotide that selectively hybridizes to a nucleotide sequence of the target gene that either contains uracil residues or that lacks uracil residues, but not both, and detecting selective hybridization (or the absence thereof) of the oligonucleotide.
  • a methylation-specific amplification reaction such as methylation-specific PCR (MSP) is used alone, or in combination with bisulfite treatment, to detect the methylation status of a nucleic acid molecule (see U.S. Pat. Nos. 6,265,171;
  • MSP is a particularly sensitive method that allows detection of low numbers of methylated alleles and the use of small amounts of a nucleic acid sample, including paraffin-embedded materials, and also can be conveniently adapted to a multiplex analysis, including, for example, simultaneous detection of unmethylated and methylated products in a single sample, thus providing an internal control.
  • the amplification primer pairs used in an MSP reaction are designed to specifically distinguish between bisulfite untreated or unmodified DNA, and methylated and
  • MSP primer pairs for unmethylated DN A generally have a thymidine residue in the 3 '-CpG pair to distinguish it from the cytosine residue retained in methylated DNA, and the complement is designed for the antisense primer.
  • MSP primer pairs usually contain relatively few cytosine or guanine residues in the sequence because cytosme is absent in the sense (forward) primer and the guanine is absent in the antisense (reverse) primer; cytosine becomes modified to uracil, which is amplified as thymidine in the amplification product,
  • MSP is used for detecting the amount or distribution of uracil residues in a bisulfite ion treated target genes following alkaline treatment.
  • a method can be performed by contacting the gene sequence with a first amplification primer pair and a second amplification primer pair under conditions suitable for amplification, wherein the first amplification primer pair comprises a forward primer and a reverse primer, and at least one primer of the first primer pair comprises an oligonucleotide that selectively hybridizes to a nucleotide sequence of the target gene that contains uracil residues, and wherein the second amplification primer pair comprises a forward primer and a reverse primer, and both primers of the second primer pair selectively hybridize to a target gene containing cytosine residues, but not to a target gene sequence containing uracil residues, and wherein an amplification product, if any, generated by the first primer pair has a first length, and an amplification product, if any
  • the amount or distribution of uracil residues also can be detected by contacting the 5' regulatory region of the gene with a first amplification primer pair and a second amplification primer pair under conditions suitable for amplification, wherein the first amplification primer pair comprises a forward primer and a reverse primer, wherein at least one primer of the first primer pair comprises an oligonucleotide that selectively hybridizes to a nucleotide sequence of the 5' regulator ⁇ ' region of the gene containing uracil residues, and wherein the second amplification primer pair comprises a forward primer and a reverse primer, wherein both primers of the second primer pair selectively hybridize to a nucleotide sequence of the 5 ' regulatory region of the gene containing cytosine residues, but not to a corresponding nucleotide sequence of the 5' regulator ⁇ ' region of the gene containing uracil residues, and wherein an amplification product, if any, generated by the first primer pair has a first length, and wherein
  • Methylation silencing of a gene in a cell exhibiting or suspected of exhibiting unregulated growth also can be identified by contacting a test cell with a demethylating agent and detecting increased expression of an RNA encoded by the gene as compared to a le vel of expression of the RNA in a test cell not contacted with a demethylating agent.
  • Such a method can further include detecting methylation, if any , of cytosine residues in a CpG dinucleotide in a CpG island of the 5' regulatory region of the gene in a corresponding cell exhibiting regulated growth, or an extract of the corresponding ceil
  • the demethylating agent can be a methyltransferase inhibitor such as DAC.
  • Increased expression of an RN A can be detected using any method for detecting RNA, including, for example, northern blot analysis, a reverse transcript! on-polymerase chain reaction assay, or selective hybridization to an array of nucleotide sequences as disclosed herein.
  • test cell, or extract of the test cell comprises one of a plurality of test cells, or extracts of the test cell s, or a combination thereof; and each of the test, cells, or extracts of the test cells, of the plurality is the same or different, or a combination thereof.
  • test cells, or extracts of the test cell can be arranged in an array, which can be an addressable array, on a solid support such as a microchip, a glass slide, or a bead, and the cells (or extracts) can be contacted serially or in parallel with an oligonucleotide probe or primer (or primer pair) as disclosed herein.
  • Samples arranged in an array or other reproducible pattern can be assigned an address (i.e., a position on the array), thus facilitating identification of the source of the sample.
  • An additional advantage of arranging the samples in an array, particularly an addressable array is that an automated system can be used for adding or removing reagents from one or more of the samples at various times, or for adding different reagents to particular samples.
  • high throughput assays provide a means for examining duplicate, triplicate, or more aliquots of a single sample, thus increasing the validity of the results obtained, and for examining control samples under the same conditions as the test samples, thus providing an internal standard for comparing results from different assays.
  • cells or extracts at a position in the array can be contacted with two or more oligonucleotide probes or primers (or primer pairs), wherein the oligonucleotides are differentially labeled or comprise a reaction that generates distinguishable products, thus providing a means for performing a multiplex assay.
  • Such assays can allow the examination of one or more, particularly 2, 3, 4, 5, 10, 15, 20, or more genes to identify epitoped.
  • the present invention also provides oligonucleotides, which can be useful as probes or primers for identifying an epigeiietic silenced gene (or the absence thereof.
  • oligonucleotide polynucleotide
  • nucleic acid molecule is used broadly to mean a sequence of two or more deoxyribonucieotides or ribonucleotides that are linked together by a phosphodiester bond.
  • gene also is used herein to refer to a polynucleotide sequence contained in a genome, it should be recognized, however, that a nucleic acid molecule comprising a portion of a gene can be isolated from a cell or can be examined as genomic DNA, for example, by a hybridization reaction or a PCR reaction.
  • oligonucleotide is also used herein to refer to a polynucleotide that is used as a probe or primer, whereas the term “polynucleotide” or “nucleic acid molecule” is used more broadly to encompass any sequence of two or more nuc leotides, including an oligonucleotide.
  • nucleotide sequence is used to refer to the molecules that are present on an array, As such, it should be recognized that the various terms used herein to conveniently distinguish different nucleic acid molecules. As such, the terms include RNA and DNA, which can be a gene or a portion thereof, a cDNA, a synthetic
  • polynucleotide can be single stranded or double stranded, as well as a DNA/RNA hybrid, although it may be recognized that the strands of a double stranded oligonucleotide that is to be used as a probe or primer can be separated, for example, by heating a solution containing the oligonucleotide above the melting temperature of the particular oligonucleotide.
  • oligonucleotide include naturally occurring nucleic acid molecules, which can be isolated from a cell, as well as fragments thereof as produced, for example, by a restriction endonuclease digestion, and synthetic molecules, which can be prepared, for example, by methods of chemical synthesis or by enzymatic methods such as by PCR.
  • an oligonucleotide or polynucleotide of the invention can contain nucleoside or nucleotide analogs, or a backbone bond other than a phosphodiester bond, for example, a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tarn et al, Nucl. Acids Res. 22:977-986, 1994); Ecker and Crooke, BioTechnology 13:351360, 1995, each of which is incorporated herein by reference).
  • nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the polynucleotide is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium, a cell or in a living subject, since the modified polynucleotides can be designed to be less (or, if desired, more) susceptible to degradation.
  • the nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2'- deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose.
  • a polynucleotide (or oligonucleotide) also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides.
  • nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220- 5234, 1994; jeilinek et al, Biochemistry 34: 11363-11372, 1995; Pagratis et al, Nature Biotechnol. 15:68-73, 1997, each of which is incorporated herein by reference).
  • a polynucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA. methods, using an appropriate polynucleotide as a template.
  • a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally can be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs into a polynucleotide and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jeilinek et al,, supra, 1995).
  • the polynucleotide can be prepared using a method such as
  • a cell proliferative disorder as described herein may be a neoplasm, Such neoplasms are either benign or malignant.
  • neoplasm refers to a new, abnormal growth of cells or a growth of abnormal cells that reproduce faster than normal, A neoplasm creates an unstructured mass (a tumor) which can be either benign or malignant.
  • the neoplasm may be a head, neck, lung, esophageal, stomach, small bowel, colon, bladder, kidney, or cervical neoplasm.
  • benign refers to a tumor that is noncancerous, e.g., its cells do not proliferate or invade surrounding tissues.
  • malignant refers to a tumor thai is metastastic or no longer under normal cellular growth control.
  • allelic imbalance refers to the chromosomal loss or gain that is characteristic of tumor cells. Diploid organisms, including humans, have a pair of
  • chromosomes for each member of the chromosomal set.
  • Tumor cells characteristically lose chromosomes, often resulting in a single chromosome, rather than a pair of chromosomes, for each member of a chromosomal set.
  • Tumor cells also on occasion gain chromosomes, resulting in a two or more chromosomes, rather than a pair of chromosomes, for each member of the chromosomal set.
  • chromosomes with two alleles for a genetic locus is heterozygous. Whether a genetic locus is heterozygous for a subject can readily be determined by analyzing a sample of DNA from the normal (non-tumor) cells of the subject. Because microsatellite DNA is polymorphic, a genetic locus that contains microsatellite DNA may frequently be heterozygous, When a tumor cell loses or gains a chromosome, the result is that the ceil loses or gains an additional copy of one of the alleles, causing an allelic imbalance (loss of heterozygosity),
  • Microsatellite DNA markers that are heterozygous in normal (non-tumor) cell DNA can be used to detect mutations in tumor cell DN A.
  • the loss of one allele identifies chromosomal deletions after gel electrophoresis or other techniques.
  • An imbalance between the two alleles also identifies chromosomal amplifications.
  • To do these analyses by conventional methods requires extensive microdissection of neoplastic cells so that normal (non-tumor) contaminating cells would not disrupt the assay.
  • the method of the invention by contrast, provides a non-invasive sampling technique in which the presence of normal (non- tumor) ceils does not interfere with the assay.
  • a combination of microsatellite DNA A combination of microsatellite DNA.
  • markers may be amplified in a single amplification reaction.
  • the markers are multiplexed in a single amplification reaction, for example, by combining primers for more than one locus,
  • DNA from a urine sample can be amplified with three different randomly labeled primer sets, in the same amplification reaction.
  • the reaction products are separated on a denaturing polyacrylamide gel, for example, and then exposed to film for visualization and analysis.
  • microsatellite DNA refers to mononucleotide, dinucleotide, or trinucleotide sequences where alleles differ by one or more repeat units. Microsatellite DNA is an especially common and highly polymorphic class of genomic elements in the human genome.
  • the microsatellite DNA most preferred in the method of the invention has a sequence (X)n, wherein X is the number of nucleotides in the repeat sequence and is greater than or equal to 1, preferably greater than or equal to 2, and most preferably greater than or equal to 3 and wherein n is the number of repea ts and is greater than or equal to 2, and preferably from 4 to 6. When X is 2, the nucleotide sequence may be TC.
  • the nucleotide sequence may be selected from AGC, TCC, CAG, CAA, and CTG. Two examples of trinucleotide repeats are D1S50 and DRPL A markers. Preferably when X is 4, the nucleotide sequence may be selected from AAAG, AGAT and TCTT. Two examples of tetranucleotide repeats are included in D21 SI 245 and FgA markers.
  • the microsatellite DN A sequence may be genetically linked to a unique locus.
  • sample of microsatellite DNA refers to DNA present in or prepared from any tissue of a subject.
  • the nucleic acid from any specimen, in purified or nonpurified form can be used as the starting nucleic acid or acids, provided it contains, or is suspected of containing, the specific nucleic acid sequence containing the target nucleic acid.
  • the process may employ, for example, DNA or RNA, including messenger RNA (rriRNA).
  • the DNA or RNA may be single stranded or double stranded.
  • enzymes and conditions optimal for reverse transcribing the template to DNA would be used.
  • a DNA-RNA hybrid that contains one strand of each may also be used.
  • a mixture of nucleic acids may also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers may be so used.
  • the mutant nucleotide sequence to be amplified may be a fraction of a larger molecule or can be present initially as a discrete molecule, such that the specific sequence is the entire nucleic acid. It is not necessary' that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as contained in whole human DNA.
  • allelic imbalance may be detected as a decrease in the level of DNA
  • the term "decrease in the level of DNA” refers to the observed difference of the ratio between the two alleles for a genetic locus.
  • a sample of cells can have a ratio approaching 1 : 1 for a subject that lacks the cell proliferative disorder.
  • the actual ratio for a subject can readily be determined by analyzing the DNA from the normal (non-tumor) cells of the tested subject.
  • the level of DNA corresponding to the allele may be less than 50% of the level of DN A of a corresponding allele in a microsatellite DN A sample of a subject that lacks the cell proliferative disorder.
  • An allelic imbalance may also be detected as an increase in the level of DN A corresponding to an allele.
  • the term "increase in the level of DNA” refers to the observed difference of the ratio between the two alleles for a genetic locus.
  • a sampl e of cells may have a ratio that approaches 1 : 1 for a subject that lacks the cell proliferative disorder. Analyzing the DNA from the normal (non-tumor) cells of a test subject can readily determine the actual ratio for the subject,
  • An increase in the level of DNA may be detected as the appearance of a new allele.
  • Presence of a new allele refers both to the observed difference of the ratio between the two alleles for a genetic locus and to genetic instability, the genetic
  • one of the chromosomes may undergo genetic
  • microsatellite DNA sequence is therefore a new allele for that genetic locus, as compared with the normal (non-tumor) cells of the subject.
  • a tumor cell may have three or more different alleles for a genetic locus instead of the two alleles found in the normal (non-tumor) cells.
  • the presence of a new allele may correspond to the loss of an allele found in normal (non-tumor) cells.
  • PGR polymerase chain reaction
  • polymerase chain reaction refers to a method for amplifying a DNA. base sequence using a heat-stable DNA polymerase and two oligonucleotide primers, one complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (-)-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. The polymerase chain reaction is used to detect the existence of the defined sequence in the microsatellite DNA sample.
  • DNA can be subjected to 30 to 35 cycles of amplification in a thermocycler as follows: 95 °C. for 30 seconds, 52 to 60 °C. for 1 minute, and 72 °C. for 1 minute, with a final extension step of 72 °C. for 5 minutes.
  • DNA can be subjected to 35 polymerase chain reaction cycles in a thermocycler at a denaturing
  • Diagnoses and prognoses determined using the subject methods can be confirmed and combined with other means of assessment. These include physical findings, radiological findings, pH determinations, endoscopic determinations, pathological determinations, patient reports of symptoms, and the like.
  • the biological sample tested in the methods of the present invention may be any biological sample from which polynucleotides or proteins may be obtained.
  • the biological sample is one that contains cells or cellular material, proteins or polynucleotides.
  • the biological sample may be a biological fluid, such as lymph, serum, plasma, whole blood, urine, synovial fluid and spinal fluid; a cell type, such as bone marrow, immune, keratinocytes, epithelial cells, hepatocytes.
  • the biological sample is mucosa, esophageal epithelium, or squamous esophageal epithelium.
  • the biological sample is tissue diagnosed as Barrett's esophagus. Samples can be collected by endoscopy, or other collecting means, including surgical spatulas, sponges, balloons, esophageal brush- capsule. See Cancer Cytopathol. 2000; 90: 10-6; see also Cancer. 1997; 80(l l):2047-59. Some of these may be used in conjunction with endoscopy and some may be used independently.
  • nondysplastic Barrett's esophagus markers D5S82 and DSS346 on chromosome 5q and D13S134 on chromosome 13p. These primer pairs are used to compare matched pairs of DNA samples isolated from blood and esophageal tissue. Twelve samples are tested: 10 matched blood and EAC samples previously found to be cancerous by histology, and 2 matched pairs of normal blood and esophageal tissue, Additionally, to determine if allelic imbalance and/or LOH can be detected at other loci implicated in EAC, such as chromosomes 1, 18, 3, 17 and 9, the samples are also tested using microsatellite assay. The blood/tumor or blood/normal DNA samples are amplified using three multiplex panels and three new sets of primers in singleplex, then resolved using capillary electrophoresis on a 3100 genetic analyzer from Applied Biosystems.
  • T he contribution of each marker is assessed in order to determine whether the detection of LOH in the samples can, in combination with changes in methylation status, improve the specificity and sensitivity of the methylation marker panel.
  • Six loci exhibiting LOH are identified in the majority of Ps but not of NPs ( Figure 5).
  • the occurrence/absence of LOH at these 6 loci is combined with the methylation status of the 8 methylation markers and analyzed by receiver-operating characteristic (ROC) curves, the area under the curve (AUC) jumped from 0.7619 to 0.9524 ( Figure 4).
  • ROC receiver-operating characteristic
  • AUC area under the curve
  • the present invention provides that adding allelic deletion markers can improve the sensitivity and specificity of methylation markers alone.
  • the advantage of the method provided herein is that there is a true normal to for comparison to the test sample for determination of allelic imbalance.
  • electrophoresis is not an optimal clinical assay method. Furthermore, certain methylation markers being validated are typically assayed using real-time PCR. Therefore, the present invention provides a quantitative PCR assay to detect LOH in BE.
  • a panel of quMSA markers can be developed for the early detection and prediction of BE progression using quMSA and the high-throughput 7900HT real-time PCR analyzer from Applied Biosystems. The advantage of this approach is that these LOH markers can be combmed with methylation markers to develop a unified diagnostic test to determine a patient's risk from progression from BE to HGD or EAC.
  • Advantages of this method over the capillary electrophoresis method include:
  • the assay is rapid: less than four hours required from sample setup to data generation.
  • the assay is sensitive: copy number can be detected in as little as 2 ng of genomic UNA, thereby saving precious clinical samples and allowing analysis of samples containing limited genetic material (such as paraffin-embedded endoscopic biopsies).
  • test sample is compared to an internal control, precluding the need for a normal genomic DNA sample for comparison to the test article.
  • the assay is capable of ultra-high throughput, with 96- and 384-well sample formats.
  • heterozygosity it may be able to detect changes in homozygous as well as heterozygous alleles.
  • Table 1-1 shows a list of loci tested for loss of heterozygosity (LOH). Chart indicates chromosome number and region corresponding to the loci currently or previously tested for a Loss of Heterozygosity. There loci are tested in the present example.
  • a high-throughput quMSA assay can be developed to determine copy number of STR markers in esophageal biopsies in order to detect LOU.
  • the method described herein has the added advantage of using the same detection method as the methylation specific PCR markers (real-time PCR). In conjunction with a methylation marker panel, therefore, this assay can serve as an early surveillance algorithm to determine the risk of future BE neoplastic progression.
  • the present invention provides a kit that uses real-time PCR technology to amplify genetic and epigenetic markers and identify the risk of neoplastic progression in BE patients.
  • the kit can be comprised of reagents to measure gene-specific methylation using real-time methylation-specific PCR, as well as primers that can amplify short tandem repeat (STR) markers that undergo LOH during cancer progression. Combined data from these tests can determine the risk for developing EAC and assist the physician in selecting the appropriate frequency of endoscopy for monitoring BE patients.
  • Table 1-2 shows a list of additional loci and Chromosomal regions for
  • Tables 2-1 [(non-progressors (NP)] and Table 2-2 [progressors (P)] show that LOH differ in Non-Progressors Patients and Progressors Patients, respectively. Frozen tissue samples of patients with known outcomes are examined for LOH at multiple loci, The lower and upper limits in Table 2-2 are calculated from both Tables 2-1 and 2-2,
  • Tissue from a bank of normal tissue is sampled for controls.
  • Each numbered column contains allele ratio data at multiple selected chromosomal loci for patients identified as having a pre-malignant Barrett's Esophagus condition.
  • Allele height is measured for both alleles and a height ratio is calculated.
  • an upper limit (UL) ratio and lower limit (LL) ratio is determined. Normal ratios of two alleles are usual ly slightly less than 1.0 (-0,8), because two alleles are not exactly the same size. Ratios that exceeded the UL and LL by more than 1 -2 standard deviations at a particular locus are identified as positive for LOH at that locus.
  • Ratios that did not exceed the UL and LL by more than 1-2 standard deviations at a particular locus are identified as negative for LOH at that locus. Patients identified as Progressors had more ratios that exceeded the UL or LL than patients identified as Non- progressors.
  • LOH can be detected using quantitative PGR.
  • This method known as quantitative microsatellite analysis (quMSA)
  • quMSA quantitative microsatellite analysis
  • a Taqman® probe complementary to the STR region such as CA(n)
  • a sequence directly flanking the STR region is designed vvith a 5 ' fluorescent label (such as VIC or FAM) and contains a 3 ' quencher (such as TAMRA).
  • the SYBR® Green ⁇ dye chemistry uses SYBR® Green I dye, a highly specific, double-stranded DNA binding dye, to detect PGR product as it accumulates during PGR cycles.
  • SYBR® Green I dye chemistry can detect all double-stranded DNA, including non-specific reaction products. A well-optimized reaction is therefore essential for accurate results. Previous studies have shown that both technologies are able to distinguish between one and two copies at a particular locus, permitting detection of LOH.
  • Detection of copy number using real-time PGR is based on the principle of relative quantitation (RQ), which determines the change in copy number or expression of a nucleic acid sequence (target) in a test sample relative to the same sequence in a calibrator sample (Described in ABI Bulletin P/N 4371095).
  • RQ provides accurate comparisons between initial levels of template in each sample without requiring the exact copy number of the template. Also, the relative levels of templates in samples can be determined without the use of standard curves.
  • RQ requires four components: (1) A target: the nucleic acid that is to be studied, e.g., the D5S346 STR marker; (2) A calibrator: the sample used as the basis for comparative results (in this case, normal human genomic DNA).
  • the endogenous control may serve as the calibrator; (3) An endogenous control: a gene present at consistent levels in all experimental sample sets. By using an endogenous control as an active reference, the DNA may be normalized for differences in the amount of DNA added to each reaction.
  • the endogenous control and the target assay are performed in a multiplex assay for Taqman® assays and as singlepiex assays for SYBR® Green I assays; (4) Replicate wells: Each sample can be tested in quadruplicate to insure statistical significance.
  • the threshold cycle (Ct) of a sample is defined as cycle number (in qPCR) at which the fluorescence generated within a reaction well exceeds a defined threshold.
  • Relative copy number (RCN) is determined as 2 x 2 - ⁇ Ct and is calculated by the 7900HT software. A RCN of 1 indicates no LOB in the test sample (total of two copies in the genome).
  • the target can be the STR marker to detect LOH in the biopsy DNA sample;
  • the calibrator can be genomic DNA isolated from esophageal tissue shown to be normal by cytology; and (3)
  • the endogenous control can be the Applied Biosystems ⁇ -actin assay. This assay is designed to amplify unique genomic sequences in the reference genome assembly (NCBI Build 36) and is required for relative quantitation of copy number targets.
  • SYBR ⁇ GREEN I or Taqman® ⁇ based assay There are a number of factors to consider when deciding which qPCR assay can be used. Advantages and disadvantages of each technology are listed in Table 3.
  • SYBR® Green I employs the same chemistry used in the MSP assay, enabling the development of a simple, singie-mastermix kit to amplify the entire combined panel.
  • the Taqman chemistry is more specific than the SYBR® Green I, and SYBR® Green I cannot be performed in multiplex, thus doubling the number of reactions necessary for each locus.
  • the present invention pro vides that either one of these chemistries can be used for the method described herein.
  • a Taqman ⁇ assay is designed to compare the copy number of an 8TR marker from matched head and neck squamous cancer (HNSCC)-normal DNA sample pairs.
  • the primers and probe for this marker, D9S 162 include a forward primer 5 '- ACCTAGGCCATGTTCACAGC-3 ' (SEQ ID NO: 1); a reverse primer 5 '- GAGCAGAATGAGAGGCCAAG-3 ' (SEQ ID NO: 2); and a probe 5 '- TGT GTG TGT GTG TGT GTG TGT-3 ' (SEQ ID NO: 3).
  • the 5 ' end of the probe can be modified with FAM and the 3 ' end of the probe can be modified with MGBNFQ.
  • Quantitative PCR for D9S 162 detects LOH in 3 of 10 normal-HNSCC sample pairs shown in Figure 3.
  • the forward and reverse primers are specific for the D9S 162 marker, while the probe is complementary to the CA(n) repeat region within the marker.
  • blood DNA is used as the calibrator, while sputum DNA is used as the target assay, and Applied Biosystems ⁇ -actin control reagents (P/N 4333760F) are used as an endogenous control to normalize the signal of the test marker and allow comparison of blood DNA to test sample DNA.
  • the present example provides not only that quMSA assays can be successfully performed, but also that it can serve as an alternative method to test the ability of new assays to detect LOH.
  • qPCR assays are feasible, and changes in copy number can be verified at heterozygous loci using traditional capillar) ' MSA.
  • STR short tandem repeat
  • assays of the present example focus on genomic regions shown to undergo LOH early during neoplastic progression in BE, viz., regions 9p21 and 17p 13. Assays can be developed at multiple loci within these regions, thereby increasing the likelihood that successful assays are developed rapidly. Results from Figure 3 also show those loci in regions 5q21, 18q, and 13 exhibit LOH in EAC and may therefore also be useful for the detection of neoplastic progression.
  • PCR Primer Design Standard real-time assays can be developed for each STR locus. A single PCR primer pair is used in both the SYBR® Green ⁇ and Taqman® assays for a direct side-by side comparison of the two technologies. The typical real-time PCR assay is designed to amplify a region of 100-200 base pair (bp); smaller amp 1 icons are preferred. PCR primers are designed between 20-30 bases in length, have approximately 40-60% GC content, have a 55-60% melting temperature and have a G or C as the last base (a "GC clamp").
  • Primers are designed to 5' and 3' to the STR of interest and are BLASTed against the
  • PCR primers are tested in a standard PCR reaction to ensure that only one amplification product is generated.
  • the amplification products are resolved on the Agilent 2100 capillary electrophoresis bioanalyzer, which can detect amplification products from 50 bp to 12,000 bp. The presence of a single PCR product from each primer pair can ensure the specificity of the primers for the target sequence,
  • Probe Design Taqman ⁇ Probes are designed at each STR. The probe has the 6- FAM dye as its 5' detection dye and the MGBNFQ (Molecular-Groove Binding Non- Fluorescent Quencher) at its 3' end. Probes are 20-30 bases in length, and do not have a guanosine ("G") as the first base (since G's can quench the fluorescence of the FAM dye), and may have a melting temperature of about 65-67 °C,
  • Endogenous control design The beta-aetin endogenous control is used for both the methylation and STR markers.
  • a VIC-labeied Taqman® probe is used so that it can be multiplexed with the FAM-labeled target probe.
  • a new probe can be designed that includes unique flanking non-repetitive sequence, if these problems occur, the assays are re-designed accordingly.
  • assays for other markers within regions of LOH e.g., D9S 135, D9S32, D17S974, D17S 1303 can also be used.
  • the target assay and ⁇ -aetin assay is combined into a duplex reaction and optimized for primer concentration according Applied Biosystems recommended procedures (Applied Biosystems User Bulletin #5). Quadruplicate runs require a total of 8 reactions.
  • a tolerance index is generated for each marker from the norma] human genomic DNA data.
  • TI tolerance index
  • Each SYBR® Green I and Taqman® assay is tested on DNAs isolated from 50 histologically normal esophageal biopsies. The TI is calculated based on the mean and standard deviation of ACt values from each marker and the calibrator. If the RQ falls below the TI for each marker, this marker is scored as showing significantly reduced relative copy number (RCN) and therefore as a candidate locus for LOH.
  • the PCR primer pairs used in the Taqman® assay are configured for CE, consisting of a iluorescentiy labeled 5' PCR. primer and unlabeled PCR. primer.
  • the PCR. primer pairs are tested on DNA isolated from 50 histologically normal esophageal biopsies described in the previous section using conventional capillary electrophoresis methods currently in use. If there are too many repeats such that the PCR product is >300 bp, the primers can be redesigned.
  • a copy number assay is designed directly proximal to a locus shown to exhibit LOH in the BE progressors.
  • the target, D4S243 is identified as a region deleted in the BE progressors (see Figure 5).
  • the assay is designed 208 bp upstream of the STR region in the D4S243 locus.
  • the endogenous control js the Applied Biosysterns RNAseP assay.
  • This assay is designed to amplify unique genomic sequences in the reference genome assembly and is required for relative quantitation of copy number targets.
  • the D4S243 locus is chosen and combined with 10 ng of genomic DNA in quadruplicate in a standard copy number assay from Applied Biosysterns.
  • the assay is tested on a group of 12 progressors and 10 non-progressors.
  • the real-time PCR. data is analyzed using the
  • CopyCailer program Table 6 shows copy number (Copy #) analysis of Non-Progressor (Non-Prog) and Progressor (Prog) BE patients at the D4S243 Locus. The average copy number of each group and the standard deviation (StD) are shown, P value to determine significance is calculated using a two-tailed student's T-test. Table 6 shows that the copy number assay is able to detect a significant loss of copy number in progressors as compared to non-progressors when analyzed with a two tailed student's T-Test (P value ⁇ 0.001).
  • An assay can be optimized or redesigned according to the principles described herein. This process is accomplished by a) redesigning the less-optimal assay and repeating the validation experiment and/or b) modifying the primer and probe concentrations and re-performing the validation experiment. If neither of these strategies yields a valid result, assays based on alternative STR markers within the region of LOH can be developed. 2) If the RCN result in normal genomic DNA is ⁇ 0,75 or >] ,45, the assay may require further optimization. This optimization is accomplished by modifying the primer and probe concentrations and performing the validation experiment again, If this modification does not result in a valid result, assays for alternative STR markers within the region of LOH can be developed.
  • the optimized panel of markers is assessed for their ability to detect LOH using purified genomic DNA from 50 normal, 50 BE, and 50 EAC specimens from patients biopsied for these conditions.
  • the present invention provides that the panel of markers can correctly detect the presence of tumor DNA by accurately sensing a reduction in copy number.
  • the assay of the present example is qualified using the foll owing parameters. Sensitivity and specificity: the ability of the panel to detect reduction in copy number in EAC samples is tested. Sensitivity can reflect the percentage of tumor samples in which LOH is detected. As a positive control for these studies, the twenty pairs of bl ood/tumor specimens are subjected to traditional capillary electrophoresis for each optimized marker to confirm the presence of LOH detected by quMSA and determine whether the quMSA assay is as sensiti ve as CE in detecting LOH. The present invention provides that the CE method can detect LOH in the tumor specimens tested (see Figure 3).
  • the present invention provides that ail of the markers together, taken in combination with the methylation markers, are able to detect LOH with higher sensitivity than the methylation markers alone.
  • the sensitivity of the assay is calculated as the percentage of tumor samples that have a positive result, i.e., the true positives/(true positives + false negatives).
  • the specificity of the assay is calculated by the percentage of negati ve samples that have a negati ve result, i.e., true negatives/(true negatives + false positives).
  • the data are combined and plotted as an ROC curve (as in Figure 1 ). Feasibility is obtained where the addition of new markers to the panel increases the accuracy of the assay, evinced by an increase in the area under the ROC curve (AUROC) from its current value of 0.745 (43% sensitivity/90% specificity).
  • Table 7 shows an exemplary evaluation of sensitivity of methylation and quMSA marker panels, where the sensitivity and specifi city of the new marker panel are evaluated as demonstrated.
  • Top theoretical ability of the methylation marker panel to detect changes in methylation patterns in 20 tumor samples and 20 norma! samples. Below is the theoretical ability of bo th the methylation markers and the quMSA assay to detect the tumor and normal samples. The assay is deemed feasible where the sensitivity of the assay increases due to addition of the quMSA markers.
  • a clinical diagnostic test is provided for the early detection of BE progression.
  • This assay can detect LOH during BE progression and can also establish the panel of markers and conditions to be used.
  • a blinded pre-validation study is performed to determine if the combined methylation/quMSA assay can predict the progression of BE to HGD and EAC.
  • the LOH status of the 195 BE patient samples tested in the methylation pre- validation study described above (50 progressors and 145 non-progressors) can be evaluated with the quMSA panel.
  • An increase in the accuracy of the assay by the addition of the quMSA markers is indicative of a clinically relevant diagnostic assay.
  • a IVD kit is created as a prototype to the final product.
  • the MSP and the quMSA assays can be performed separately.
  • the present invention also provides integration of the two assays into a simple procedure that can amplify all targets under similar conditions,
  • the prototype IVD kit includes the following reagents: (I) Locus-specific primers for the MSP; (2) Locus-specific primers for the qMSA; (3) Endogenous control: ⁇ -actin primers (for the MSP) and Probe (for qMSA); (4) Controls: MSP Positive control: universal methylated DNA (commercially available) and quMSA Positive control: normal human genomic DNA.; (5) 2X SYBR.® Green I amplification buffer; and (6) Kit protocol outlining procedures.
  • the assay is tested to determine the specificity and sensiti vity of the assay in a prospecti ve manner. Exemplary markers are shown in Tabl e 9.
  • the currently accepted marker for cancer risk is histologic dysplasia, with high- grade dysplasia (HGD) being considered more accurate than low-grade dysplasia (LCD.
  • HGD high- grade dysplasia
  • LCD low-grade dysplasia
  • confirmed HGD is treated in the same manner as is early-stage EAC, by endoscopic mucosal ablation, photodynamic therapy, or surgical esophagectomy.
  • the predictive value of LGD for cancer risk assessment is controversial.
  • poor reproducibility (high inter-observer variation) in histologic assessment often makes clinical risk assessment problematic.
  • more accurate tissue-based biomarkers capable of predicting the risk of progression to HGD or E AC are extremely useful.
  • BE Due to frequent endoscopic surveillance, BE has become, by default, a de facto human model of early human preneoplastic events. Unlike colorectal adenomas, the premalignant lesions at the other end of the GI tract, the at-risk organ is left in place for repea t serial observations, often for 30 or 40 years.
  • This BE model lends itself quite readily to molecular genetic studies in which "tissue is the issue.” In human diseased tissue-based studies, there is no problem with clinical relevance, and one does not need to worry about being "led down the (proverbial) garden path" by the sometimes irrelevant findings (traps) that often crop up in noiihumaii or in vitro models of human disease,
  • TSGs tumor suppressor genes
  • LOH point mutation
  • homozygous deletion and hypermethylation.
  • BN Barrett's neoplastic progression
  • the present invention provides that an unbiased, epigenome-wide approach to this aspect of BN molecular genetics can shift the paradigm in several ways: 1) the predominant epigenomic change in progression appears to be hypomethylation, rather than hypermethylation, implying the activation or unmasking of growth -stimulator ⁇ ' genes; 2) some genes change their methylation levels late during the runup to progression, while others change earlier; this finding implies that by using arrays, methods pro vided herein can a) find better early predicti ve biomarkers of progression for future clinical coiTelative studies; and b) for the current proposal, dissect out the temporal epigenomic program of Barrett's neoplastic development, [0130]
  • the present invention provides that the global methylation profile of Barrett's esophagus is in a constant state of flux and changes continuously as Barrett's evolves from early pre-progression, to later pre-progression, to LGD, to HGD, and finally to EAC.
  • BE The precise size of the BE community is not known, but it has been estimated at between 1% and 5% of the general population. Thus, in the United States, it is widely assumed that as many as 3 million people may harbor BE. BE is a highly
  • DNA methylation in BE and EAC has been studied previously. Aberrant DNA methylation occurs early in this process, specifically in BE, and methylation increases in frequency in LGD and HGD, becoming most common in EAC.
  • the present invention provides that certain tumor suppressor genes that undergo methylation in BE can function as biomarkers, predicting whether BE patients will or will not develop HGD or EAC.
  • the present invention provides that the genes NELLl, TACl, SST, AKAP12, and CDH13 are methylated early and often in Barrett's neoplasia and correlated with clinical risk factors, such as degree of dysplasia and length of Barrett's segment.
  • This unbiased, comprehensive approach is not only likely to ultimately provide better predictive biomarkers than are previously a vailable or possible; but can provide biologic insights into the progression process by identifying cellular and biochemical pathways previously unsuspected of involvement in this process. By shedding light on such novel pathways, this pangenomic approach can also suggest novel etiologic insights into, as well as potential interventions in, what has until now existed as a virtual "black box," the Barrett's neoplastic progression process.
  • Surgical and endoscopic esophageal tissue samples have been collected for the past 20 years.
  • the current sample bank contains fresh-frozen tissue specimens and/or DNAs in the following quantities: esophageal tumor, 688 (437 of which have matc hed normal esophagus [NE], and 84 of which also have matched BE); BE without EAC, 778 (269 of which have matched NE): low-grade dysplasia (LGD), 23; and high-grade dysplasia (HGD), 18.
  • DNA samples from 195 cases are also obtained (50 progressors and 145
  • nonprogressors that are enrolled in the validation study of 8 methylation markers.
  • a large cohort is assembled consisting of nonprogressor DNAs, 300 timepoints; progressor DNAs, 63 timepoints.
  • Valuable DNA samples are obtained at multiple timepoints in the same patients, for example 118 timepoint-specimens from 35 progressors and 27 nonprogressors, are obtained with a greater number of timepoints per subject in the progressor group.
  • Temporal epigenomic program mapping strategy To identify the epigenome of patients with Barrett's metaplasia with known outcomes, those known to have progressed to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), termed “progressors;” and those known to have not progressed to HGD or EAC, termed “nonprogressors” are studied, in addition, this epigenomic understanding is extended to the later stages of BN progression (LGD, HGD, and EAC). In fact, in many cases, matching "endpoint" progression tissues are obtained that correspond to the same patients' "index" preprogression biopsies that are studied as “progressor” and “nonprogressor” specimens.
  • Methylation array are studied for 20 BE-P timepoints (ranging from 60 to 6 months pre-progression), 40 BE-NP timepoints (with intervals varying from 40 to 100 months before censoring or nonprogression), 10 LGDs, 10 HGDs, and 10 frank EACs.
  • This cohort is well within the sample size available from the ongoing EDRN tissue methylation biomarker validation study.
  • Each array is co-hybridized to fully methylated DNA, labeled with Cy3, as a universal control; and to a single experimental (BE. L/HGD, or EAC specimen) DNA, labeled with Cy5.
  • Data are expressed as ratios of Cy5 to Cy3, or sample DNA to fully methylated DNA. These data can be displayed in both log and linear terms.
  • MCAM approach A methylation-sensitive restriction enzyme-based method is used to prepare the DNA. The principle of this strategy is based on serial digestion of genomic DNAs, first with the methylation-sensitive restriction enzyme, Smal, to create blunt ends and destroy only unmethylated CCCGGG recognition sites; and then with its
  • methylation-insensitive isoschizomer Xmal
  • Xmal methylation-insensitive isoschizomer
  • Xmal-digested sticky- ended i.e., methylated genomic DNA.
  • PCR is performed using primers that anneal to the linkers. Since only unmethylated DNA is cut during the first digest by Smal, while methylated DNA is not, CCCGGG recognition sites are lost only in unmethylated DNA and not available for subsequent Xmal digestion and linker ligation, Thus, only methylated DNA is amplified.
  • the amplified methylated DNA is then hybridized to human CpG island microarrays (Agilent), each containing 244K probes corresponding to 27,800 CpG islands, Probe-based signal data produced by Agilent scanning software is normalized using the LOWESS-based intra- and inter-slide method.
  • the human genome contains approximately 28 million CpGs and 27,639 CpG islands. Smal/Xmal sites are found in 374,000 locations in the human genome, and 15.3% of Smal/Xmal sites are located within 500 bps up- or downstream of CpG islands. The total bps comprise by CpG islands plus CpGs up to 500 bps beyond (upstream or downstream of) the most conservatively defined circumscriptions of these islands are 21,101,376 (0.685% of the genome) and 48,740,376 (1.58% of the genome), respectively. Thus, this 15.3% CpG-related fraction of Smal/Xmal sites is concentrated in only 1.58% of the human genome, a considerable enrichment, possibly because the
  • Smal/Xmal recognition sequence CCCGGG occurred nonrandomiy, being much more abundant in CpG islands and their immediate surrounding regions.
  • the actual distribution of numbers of oligonucleotide probes per Smal/Xmal-digested fragment on the Agilent microarray is calculated to estimate the number of CpG islands analyzable by this microarray platform. Based on this analysis, 57.2% of all CpG islands in the entire human genome are assessable with this microarray chip,
  • S/X Smal/Xmal
  • methylated DNA known as methylated DNA.
  • immunoprecipitation a monoclonal antibody raised against 5 -methyl cytidine (5mC) is used to isolate methylated DNA. Genomic DNA is sheared via sonication to produce random fragments. After fragmentation, DNA is denatured at 95 °C to yield single-stranded DNA fragments, because the anti-5mC antibody has a higher affinity for this form of 5mC-containing DNA.
  • the remainder of the assay is very similar to a classical immunoprecipitation protocol and invol ves the use of protein G coupl ed to standard or magnetic beads, as wel l as multiple washes following incubation with the anti-5mC antibody, Immunoprecipitated DNA can then be hybridized to high-resolution genomic microarrays.
  • Methylation array data are used to detect differences in global methylation pattern that occur between adjacent temporal intervals and stages during BN evolution and progression. These adjacent categories comprise
  • nonprogressors vs. progressors, early versus late pre-progressors, late progressors versus LCDs, LGDs versus HGDs, and HGDs versus EACs.
  • Student's t-test parametric testing
  • Mann- Whitney U-test nonparametric testing
  • the means of the 2 categories studied for each S/X fragment are used to calculate the inter-category ratio for each S/X fragment.
  • the most significant of these fragments by t-test, sorted from lowest to highest p-value, can be tabulated and further analyzed.
  • the fragments can also be sorted from lowest to highest by Mann- Whitney U-test.
  • classifier methods can include prediction analysis of microarrays (PAM) and artificial neural networks (ANNs), among others. Outliers can be identified using these methods. Unsupervised methods, such as hierarchical clustering, can be used to look for natural groupings in the specimens and among the methylation loci.
  • PAM microarrays
  • ANNs artificial neural networks
  • Unsupervised methods such as hierarchical clustering, can be used to look for natural groupings in the specimens and among the methylation loci.
  • the pre-progression intervals of the progressors can be evaluated. Progression intervals and other clinical parameters of all progressors are tabulated and further analyzed. In particular, samples with longer pre-progression intervals (early pre- progression samples) are compared to those with shorter pre-progression intervals (late pre- progression samples), using tools such as significance analysis of microarrays (SAM ).
  • SAM significance analysis of microarrays
  • Similar supervised bioinformatic comparisons can be performed between adjacent stages of progression, viz.: late pre-progression versus LGD, LGD versus HGD, and HGD versus EAC. Again, outliers that crop up during these comparisons can be identified.
  • Various clinical parameters of outlier timepoint specimens and patients can be evaluated, including distance from malignant or premalignant lesions, time interval between date specimen obtained and diagnosis of next neoplastic progression stage, anatomic location, length of Barrett's segment, concomitant inflammation or ulceration, age, body mass index (BMI), use of aspirin or nonsteroidal anti-inflammatory drugs (NS AIDs), use of acid-inhibitory drugs, sex, and race.
  • PCA principal components analysis
  • SAM is performed on these data, and SAM scores are assigned to all loci on the array (rather than just to the most significantly different loci, as is customary with SAM analyses). Similar comparisons are made between NPs and Ps and between adjacent stages of progression. SAM-processed data can then be sorted by corrected SAM q-vaiue. By using the q-value, not just the p-value of the difference between Ps and NPs (or LGD and HGD, or HGD and EAC) is considered, but also the magnitude of the difference in means between groups. This enables us to later choose genes that may offer future potential utility as biomarkers, since biomarkers generally require greater mean differences and less confidence interval overlap than do genes that are to be pursued as leads to a deeper biological understanding of BN progression.
  • Ontology group exploration To broaden analysis of the gene ontology of temporal epigenomic programming in BN evolution, studies are performed with 20 progressor- timepoints, 40 nonprogressor-timepoints, 10 LGDs, 10 HGDs, and 10 frank EACs, but also by applying diverse strategies to discover the gene ontology of the temporal BN epigenome.
  • Differential methylation in this analysis is defined by the following 2 criteria: a) the difference in the iog2 intensity ra tio of the mean of category I samples vs. the mean of category I I samples for each comparison must be greater than 0.5 (equivalent to 1.4-fold nonlog change), and b) the log2 intensity ratio of category I must lie outside the 99% confidence interval range of the mean for category II samples.
  • GOstat can then be used to identify the Gene Ontologies that are significantly overrepresented in sample Category I relative to all genes whose promoter regions are analyzed in the MCAM analysis. The 30 most significantly under- or over-represented ontologies are tabulated and further analyzed.
  • Ontologies that change significantly in NPs versus Ps, early versus late progressors, late Ps versus LGDs, LGDs versus HGDs, and HGDs versus EACs can be identified. These two-way comparisons are performed with each specimen class represented by "Category I" or Category II.” Parameters analyzed include the enrichment ratio for ontologies over- represented by genes that are differentially methylated in Category I versus Category II specimens. Significantly different ontology categories identified by GOstat are inspected for thematic characteristics of potential relevance to BE or neoplastic progression.
  • a global epigenomic survey of esophageal cancer (EC) is assembled based on functional reactivation by 5 -aza-2 ' -deoxy cytidine (5-AzaC) and trichostatin A, using mRNA arrays.
  • This approach identifies 58 genes epigenetically silenced in EC cells and included several known candidate TSGs, among which 43 (74%) harbored dense CpG islands in their promoters. Promoter hypermethylation are confirmed for 3 genes (cytokine-like factor- 1, apolipoprotein D, and neuromedin U) in EC cells and of 2 genes in primary EC ' s, which exerted growth-suppressive activity in EC cells.
  • pharmacologic reversal of epigenetic silencing is a powerful strategy for the comprehensive identification of TSGs in human EC.
  • ANNs artificial neural networks
  • SAM microarrays
  • An ANN trained on 12 samples using these 160 genes and tested against the remaining 10 samples learned to correctly diagnose all 22 samples.
  • gene expression signatures and, by implication, their control by epigenetic signatures) contain unique fingerprints of BN stages.
  • Tissue methylation biomarkers have been provided for predicting responsiveness to chemoradiotherapy from EAC patients in a uniform treatment protocol. 13 (37%) of 35 patients are responders (R), while 22 (63%) of 35 patients are non-responders (NR.), The combined mean level of promoter methylation of 9 genes is lower in R than NR.
  • the present invention provides potential clinical application of Reprimo or the 9-geiie panel in defining EAC prognosis and management.
  • a qMSP study of Reprimo is performed on endoscopic biopsy specimens and EC cells before versus after treatment with 5-AzaC.
  • HGD (7 (64%) of 11 samples, p 0.003 )
  • EAC 47 (63%) of 75 samples, p 0.002 ).
  • the le vel and frequency of Reprimo methylation are significantly higher than in NE (0 of .19 samples).
  • Area under the curve (AUROC) for Reprimo methylation in EAC versus NE is 0.812 (p 0.0001 ⁇ .
  • 5-AzaC treatment of OE33 EAC cells reactivates Reprimo expression.
  • the present invention provides that Reprimo methylation is an early with event and a potential biomarker.
  • Multivariate analyses reveal that methylation levels of pi 6, RUNX3, and HPP l are independently associated with increased progression risk, whereas age, BE segment length, and methylation of ⁇ 3, APC, or CRBP1 are not. Increased risk is detectable up to 2 years preceding neoplastic progression.
  • the present invention provides that methylation of p 16, RUNX3 and HPPl in BE or LGD represents independent risk biomarkers for progression to HGD or EAC.
  • a 3-tiered risk stratification strategy is developed, based on systematically selected epigenetic and clinical parameters, to improve BE surveillance efficiency.
  • Progressors are defined as BE patients with either LGD or no dysplasia who later developed HGD or EAC, where 4 epigenetic and 3 clinical parameters in 118 BE tissue timepoints from 35 Ps and 27 NPs are analyzed, Based on 2-year and 4-year prediction models built using linear discriminant analysis (area under the ROC curve: 0.8386 and 0.7910, respectively), BE specimens are stratified into high-risk (HR), intermediate-risk (IR), or low-risk (LR) groups.
  • HR high-risk
  • IR intermediate-risk
  • LR low-risk
  • nel-like 1, tachykinin- 1, somatostatin, A-kinase anchoring protein 12, and H-cadherin genes are methylated early and often in BN, and that their methylation levels correlate with clinical parameters of increased progression risk, such as BE segment length and histology.
  • a progression prediction model based on the single-gene methylation markers HPP1 , pi 6, and R.UNX3 is provided. Further, 5 more methylation markers are later added in order to refine and strengthen the original panel. Both the 3 -marker and 8-marker models are tested on a large cohort of 195 BE patients. 50 Ps are defined as patients with index (i.e., experimental, study) biopsies showing either no dysplasia, indefinite for dysplasia, or low-grade dysplasia (LGD) at endoscopy performed at least 6 months before diagnosis of either high-grade dysplasia (HGD) or EAC. 145 NPs are defined as patients who had undergone at least 3 surveillance endoscopic examinations with index biopsies but did not progress beyond LGD.
  • index i.e., experimental, study
  • LGD low-grade dysplasia
  • 145 NPs are defined as patients who had undergone at least 3 surveillance endoscopic examinations with index biopsies but did not progress beyond LGD.
  • Ps are considered both as 1 group (combined) and in 2 tiers: progression within 2 years or within 4 years.
  • Ps versus NPs does not differ significantly with regard to BM1, length of BE segment, gender, percentage with LGD, family history of LGD/HGD/EAC, cigarette smoking, alcohol use, or use of acid-inhibitory drugs.
  • Ps are significantly older than NPs (70.6 vs. 62,5 y; p ⁇ 0.001, Student's t-test),
  • A. linear combination of the 8 markers is evaluated, using coefficients from a multivariate logistic regression analysis in the 2-, 4- year, and combined models. Overfitting can be corrected by cross-validation.
  • the performance of age alone can be used, since it differs so markedly between Ps and NPs and is itself a fairly good predictor of progression.
  • Incremental value above age is derived by comparing a model based on age alone to one based on a panel consisting of the 8 markers plus age, after o verfitting correction. Performance can be evaluated using ROC curves.
  • Figure 1 shows a substantial increment in AUC for both the uncorrected (original) and overfitting-corrected ROC curves based on the 8-marker panel plus age vs. age alone.
  • the precise magnitudes of these incremental (delta-) AUCs are shown in Table 10, which shows that delta-AUCs contributed by the 8-marker panel versus age alone are substantial in all 3 model tiers.
  • delta-AUCs for prostate-specific antigen (PSA) versus other clinical prostate cancer parameters range from 0.003 to 0.015.
  • Table 10 shows logistic regression (8-marker panel plus age) versus age alone: incremental value above age.
  • AUC area under the ROC curve
  • AUCl AUC of age alone
  • AUC2 AUC of age plus biomarkers
  • AUC3 overfitting-corrected AUC of age plus biomarkers
  • AUC3-AUC1 incremental value above age
  • CI confidence interval.
  • this 8-gene methylation biomarker panel performs substantially better in stratifying patients according to their future risk of developing HGD and EAC.
  • the present invention provides that a genome-wide, unbiased, comprehensive survey of all CpG islands can not only provide us with insights into the epigenomic biology and temporal program of Barrett's neoplastic progression, but can also afford us a greater aptitude to stratify neoplastic progression risk in patients with BE.
  • HGD high-grade dysplasia
  • EAC esophageal adenocarcinoma
  • nonprogressors those known to have not progressed to HGD or EAC.
  • Methylation array studies of a small cohort containing 5 known progressors and 4 known nonprogressors are performed. Each array is co-hybridized to fully methylated DNA, labeled with Cy3, as a universal control; and to a single P or NP experimental (BE specimen) DNA, labeled with Cy5.
  • Data are expressed as ratios of Cy5 to Cy3, or sample DNA to fully methylated DNA. These data are displayed in log and linear terms.
  • A. methylation-sensitive restriction enzyme-based method is used to prepare the DNA. After removing results for fragments greater than 2500 bp in length, data on 34,396 Smal/Xmal (S/X) target fragments remain. Each S/X fragment corresponds to a CpG island within either a gene promoter region, an intragenic region, or an extragenic region. Many promoters or genes are represented by multiple S/X fragments. T-testing and U-testing are performed on the log and non-log versions of these data. The means of the 4 NPs and of the 5 Ps for each S/X fragment are used to calculate the P/NP ratio for each S/X fragment.
  • S/X Smal / Xmal fragment ID
  • NP nonprogressor;
  • Avg, a verage of all NPs or of all Ps;
  • P progressor;
  • ratio ratio of means of Ps to means of NPs;
  • T-test student's t-test of NP group v ersus P group;
  • U-test Mann- Whitney U-test of NP group versus P group. Numbers above NP and P columns signify coded sample ID numbers,
  • IGFIR insulin-like growth factor I receptor
  • t-test p-value ;: 0.00078
  • P/N 0.585
  • This tyrosine kinase receptor is highly overexpressed in most malignant tissues, where it functions as an anti- apoptotic agent by enhancing cell survival (NCBI EntrezGene).
  • a classifier is constructed to distinguish Ps from NPs based on these methylation array data, using prediction analysis of microarrays (PAM), However, PAM consistently misclassitied 1 progressor specimen (sample E530) as a nonprogressor, regardless of the size of the classifica tion centroid (see Figure 6).
  • the clinical data for patient E530 is then evaluated and the time interval between sample collection and progression for patient E530 is 40 months. As shown in Figure 7, the data suggest that HR index begins to predict progression approximately 2 years before it occurs. Thus, sample E530 is obtained before the pre-progression "window" of 2 years, at which methylation marker levels begin to change.
  • the methylation array spreadsheet is recalculated accordingly, this time inspecting specific genes for methylation levels in individual progressor and nonprogressor specimens, and results are illustrated in Tables 12-1 and 12-2, showing representative SAM-processed data, sorted by corrected p- (Le., SAM q-)value, on genes, progressors, nonprogressors, and "outlier" index sample E530,
  • Table 12-1 reveals that many of the gene methylation levels in E530, the sample with the longest pre-progression interval, are intermediate between levels in other progressors and those in the nonprogressor group. Thus, some genes are good early predictors of progression, while others tend to change their methylation levels later, i.e., temporally closer to the progression endpoint. The pre-progression intervals of the remaining progressors are then evaluated. Progression intervals and other clinical parameters of all 5 progressors are displayed in Table 13.
  • the index sample for patient E530 is obtained at 40.13 months prior to progression, while the index samples for the remaining 4 progressors are obtained at 32.27, 18.17, 16.5, and 16.1 months prior to progression.
  • the data in Figure 7 suggest that a predictability "window" opens approximately 2 years before progression, but remains closed until that point. However, these data are based on a single candidate gene approach. By using the paradigm-shifting approach of methylation arrays, genes that became
  • hypomethylated earlier can be identified, even at 40 months prior to progression (i.e., even in sample E530; for example front row gene in Figure 8).
  • E530 is an outlier for loci that became hypennethylated during progression (front row gene in Figure 9)
  • methylation arrays can be used to identify genes that became hypennethylated at in E530, at 40 months prior to progression (back row gene in Figure 9).
  • the present in vention pro vides application of hierarchical clustering to niethylation array data from progressor specimens.
  • This function can be performed in Statistica (v. 7.0, Statsoft, Tulsa, OK).
  • Hierarchical clustering reveals that there are 2 principal branches in these data: 1 main branch, containing samples E653, E343, El 66, and 753; and the other main branch, consisting entirely of sample E530 ( Figure 10).
  • E530 appears to constitute a category of progressor that differed markedly from the other four.
  • the present invention provides that in the transition from early to late preprogression, as neoplasia becomes more imminent, the epigenomic program "switches" to become dominated by aberrant organ development (perhaps related to the ectopic, abnonnal intestinal developmental process that is central to Barrett's epithelium per se).
  • the present invention provides that the most significantly over- represented ontologies include sequence-specific DNA binding, positive regulation of cell proliferation, proteinaceous extracellular matrix, cell-cell signaling, transcription factor activity, extracellular region part, nervous system development, transmembrane receptor activity, multicellular organismal development, system development, organ development, and multicellular organismal process.

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Abstract

La présente invention concerne la découverte importante d'un ensemble de dosages de biomarqueur en vue de prédire le risque d'un cancer de l'œsophage ou de lésions œsophagiennes précancéreuses avancées chez des patients atteints de l'œsophage de Barrett (BE). La présente invention concerne, sans s'y limiter, des biomarqueurs basés sur l'hyperméthylation génique (CH3), ainsi que des biomarqueurs basés sur la perte d'hétérozygosité (LOH), tous deux présents dans des échantillons de biopsie de tissu de l'œsophage de Barrett. Les résultats des dosages permettent de procurer aux patients et à leurs soignants une évaluation de la probabilité d'une future progression néoplasique (cancéreuse ou précancéreuse avancée).
PCT/US2011/033671 2010-04-22 2011-04-22 Procédés et kits pour évaluation des risques de la progression néoplasique de barrett Ceased WO2011133935A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013171508A1 (fr) * 2012-05-18 2013-11-21 Medical Research Council Procédé pour déterminer si un sujet présente une probabilité augmentée de dysplasie de grade élevé ou d'adénocarcinome oesophagien
WO2016160454A1 (fr) 2015-03-27 2016-10-06 Exact Sciences Corporation Détection de troubles de l'œsophage
US9556482B2 (en) 2013-07-03 2017-01-31 The United States Of America, As Represented By The Secretary Of Commerce Mouse cell line authentication
EP3387167A4 (fr) * 2015-12-08 2019-08-07 JS Yoon Memorial Cancer Research Institute, LLC Détection du cancer de la vessie par analyse de microsatellites dans des échantillons appariés d'urine et d'écouvillon buccal
US12188093B2 (en) 2014-09-26 2025-01-07 Mayo Foundation For Medical Education And Research Detecting cholangiocarcinoma

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AKAGI, T. ET AL.: ''Chromosomal abnormalities and novel disease-related regions in progressioni from Barrett's esophagus to esophageal adenocarcinoma'' INTERNATIONAL JOURNAL OF CANCER. vol. 125, no. 10, 15 November 2009, pages 2349 - 2359 *
BARRETT, M. T. ET AL.: 'Evolution of neoplastic cell lineages in Barrett oesophagus' NATURE GENETICS. vol. 22, no. 1, May 1999, pages 106 - 109 *
JIN, Z. ET AL.: ''A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett's esophagus'' CANCER RESEARCH. vol. 69, no. 10, 12 May 2009, pages 4112 - 4115 *
JIN, Z. ET AL.: 'Hypermethylation of the nel-like 1 gene is a common and early event and is associated with poor prognosis in early-stage esophageal adenocarcinoma' ONCOGENE vol. 26, no. 43, 23 April 2007, pages 6332 - 6340 *
KAWAKAMI, K. ET AL.: 'Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma' JOURNAL OF NATIONAL CANCER INSTITUTE. vol. 92, no. 22, 15 November 2000, pages 1805 - 1811 *
KISSEL, H. D. ET AL.: 'Translation of an STR-based biomarker into a clinically compatible SNP-based platform for loss of heterozygosity' CANCER BIOMARKERS. vol. 5, no. 3, 2009, pages 143 - 158 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013171508A1 (fr) * 2012-05-18 2013-11-21 Medical Research Council Procédé pour déterminer si un sujet présente une probabilité augmentée de dysplasie de grade élevé ou d'adénocarcinome oesophagien
US9556482B2 (en) 2013-07-03 2017-01-31 The United States Of America, As Represented By The Secretary Of Commerce Mouse cell line authentication
USRE49835E1 (en) 2013-07-03 2024-02-13 United States Of America As Represented By The Secretary Of Commerce Mouse cell line authentication
US12188093B2 (en) 2014-09-26 2025-01-07 Mayo Foundation For Medical Education And Research Detecting cholangiocarcinoma
WO2016160454A1 (fr) 2015-03-27 2016-10-06 Exact Sciences Corporation Détection de troubles de l'œsophage
EP3274440A4 (fr) * 2015-03-27 2019-03-06 Exact Sciences Corporation Détection de troubles de l' sophage
US10435755B2 (en) 2015-03-27 2019-10-08 Exact Sciences Development Company, Llc Detecting esophageal disorders
US11104960B2 (en) 2015-03-27 2021-08-31 Exact Sciences Development Company, Llc Detecting esophageal disorders
US12319969B2 (en) 2015-03-27 2025-06-03 Exact Sciences Corporation Detecting esophageal disorders
EP3387167A4 (fr) * 2015-12-08 2019-08-07 JS Yoon Memorial Cancer Research Institute, LLC Détection du cancer de la vessie par analyse de microsatellites dans des échantillons appariés d'urine et d'écouvillon buccal
US11401557B2 (en) 2015-12-08 2022-08-02 Hjm Foundation Corporation Bladder cancer detection using microsatellite analysis in paired buccal swab and urine samples
US11976335B2 (en) 2015-12-08 2024-05-07 Hjm Foundation Corporation Bladder cancer detection using microsatellite analysis in paired buccal swab and urine samples

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