# Patent

## Title
Methods and compositions for diagnosing lung cancer with specific DNA methylation patterns

## Abstract

The present invention provides a method for identification of differentially methylated genomic CpG dinucleotide sequences within genomic target sequences that are associated with cancer in an individual by comparing the level of methylated genomic CpG dinucleotide sequences in the sample to a reference level of methylated genomic CpG dinucleotide sequences.

## Background
This application is a continuation-in-part of U.S. Ser. No. 10/845,667, filed May 14, 2004, now abandoned which is based on, and claims the benefit of, U.S. Provisional Application No. 60/471,488, filed May 15, 2003, entitled METHODS AND COMPOSITIONS FOR DIAGNOSING CANCER, each of which is incorporated herein by reference.


This invention was made with government support under grant numbers 1 R43 CA097851-01 and 2 R44 CA097851-02 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.


FIELD OF THE INVENTION
The present invention relates to conditions characterized by differentially methylated genomic CpG dinucleotide sequences and, in particular, to diagnostic and prognostic methods that exploit the presence of such genomic DNA sequences that exhibit altered CpG methylation patterns.
BACKGROUND OF THE INVENTION
Methylation of DNA is widespread and plays a critical role in the regulation of gene expression in development, differentiation and diseases such as multiple sclerosis, diabetes, schizophrenia, aging, and cancers. Methylation in particular gene regions, for example in their promoters, can inhibit the expression of these genes. Recent work has shown that the gene silencing effect of methylated regions is accomplished through the interaction of methylcytosine binding proteins with other structural components of chromatin. which, in turn, makes the DNA inaccessible to transcription factors through histone deacetylation and chromatin structure changes. Differentially methylated CpG islands have long been thought to function as genomic imprinting control regions (ICRs).
Deregulation of imprinting has been implicated in several developmental disorders. Identification of the ICRs in a large number of human genes and their regulation patterns during development can shed light on genomic imprinting as well as other fundamental epigenetic control mechanisms. Moreover, rapid advances in genomics, both in terms of technology, for example, high-throughput low-cost capillary sequencers and microarray technologies, as well as in terms of availability of information, for example, information gained by virtue of whole genome sequencing, bioinformatics tools and databases, have paved the way for new opportunities in epigenetic studies. For example, it is known that random autosomal inactivation is one of the mechanisms that mammals use to achieve gene dosage control, in addition to random X-chromosome inactivation in females and genomic imprinting. However, genes belonging to this category are just now emerging, with only a few identified so far. Technologies are needed that can provide a systematic survey for identification of genes regulated by this kind of random monoallelic expression control, and determination of when and how such genes are regulated through a wide screening of samples from different tissues or different disease stages.
Changes in DNA methylation have been recognized as one of the most common molecular alternations in human neoplasia. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes are now firmly established as the most frequent mechanisms for gene inactivation in cancers. In contrast, a global hypomethylation of genomic DNA and loss of IGF imprinting are observed in tumor cells; and a correlation between hypomethylation and increased gene expression has been reported for many oncogenes. In addition, monitoring global changes in methylation pattern has been applied to molecular classification of cancers. Most recently, gene hypermethylation has been associated with clinical risk groups in neuroblastoma and hormone receptor status and response to tamoxifen in breast cancer.
Lung cancer is the second most common cancer among both men and women and is the leading cause of cancer death in both sexes. There is no established early detection test for the disease, and only 15% of lung cancer cases are diagnosed when the disease is localized. The ability to accurately detect malignant cells in a wide range of clinical specimens including sputum, blood, or tissue would provide significant implications for screening high-risk individuals for this cancer.
A Human Epigenome Consortium was formed in 1999 with a mission to systematically map and catalogue the genomic positions of distinct methylation variants. It is likely that large-scale discovery of methylation patterns through de novo DNA sequencing of bisulfite-treated DNA is be carried out in the near future. This would provide a resource for methylation studies analogous to SNP databases for genetic studies, and would be expected to greatly increase the demand for high-throughput, cost-effective methods of carrying out site-specific methylation assays. A publicly accessible database, which carries information about methylation patterns in various biologically significant samples would be the first outcome of these efforts. There is a need for methods for analysis of large sample sets useful for discovering such associations.
Presently, the analysis of DNA methylation patterns in genomic DNA has been significantly hampered by the fact that methylation information is not retained during standard DNA amplification steps such as PCR or biological amplification by cloning in bacteria. Therefore, DNA methylation analysis methods generally rely on a methylation-dependent modification of the original genomic DNA before any amplification step. A battery of DNA methylation detection methods has been developed, including methylation-specific enzyme digestion (Singer-Sam, et al., Nucleic Acids Res. 18(3): 687 (1990), Taylor, et al., Leukemia 15(4): 583-9 (2001)), bisulfite DNA sequencing (Frommer, et al., Proc Natl Acad Sci USA. 89(5): 1827-31 (1992), Feil, et al., Nucleic Acids Res. 22(4): 695-6 (1994)), methylation-specific PCR (MSP) (Herman, et al., Proc Natl Acad Sci USA. 93(18): 9821-6 (1996)), methylation-sensitive single nucleotide primer extension (MS-SnuPE) (Gonzalgo, et al., Nucleic Acids Res. 25(12): 2529-31 (1997)), restriction landmark genomic scanning (RLGS) (Kawai, Mol Cell Biol. 14(11): 7421-7 (1994), Akama, et al., Cancer Res. 57(15): 3294-9 (1997)), and differential methylation hybridization (DMH) (Huang, et al., Hum Mol Genet. 8(3): 459-70 (1999)). However, a need exists to use the methylation pattern to classify and predict different types and stages of cancer, cancer therapeutic outcomes and patient survival through analysis of large sample sets required to discover such associations. This invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides a method for identification of differentially methylated genomic CpG dinucleotide sequences associated with cancer in an individual by obtaining a biological sample comprising genomic DNA from the individual measuring the level or pattern of one or more methylated genomic CpG dinucleotide sequences in two or more of the genomic targets in the sample, and comparing the level of said one or more methylated genomic CpG dinucleotide sequences in the sample to a reference level of methylated genomic CpG dinucleotide sequences, wherein a difference in the level or pattern of methylation of the genomic CpG dinucleotide sequences in the sample compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with cancer.
The methods of the invention enable detection of differentially methylated genomic CpG dinucleotide sequences associated with lung cancer, for example, adenocarcenomas and sqamous cell carcinomas of the lung. In a particular embodiment, the present invention provides genomic targets that are differentially methylated in adenocarcinoma of the lung. In a further embodiment, the present invention provides genomic targets that are differentially methylated in sqamous cell carcinoma of the lung. Notably, the invention also discloses a novel cancer marker. As disclosed herein, the methods of the invention have numerous diagnostic and prognostic applications. The methods of the invention can be combined with a miniaturized array platform that allows for a high level of assay multiplexing and scalable automation for sample handling and data processing.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an assembly of a randomly ordered fiber optic array. Panel A shows a collection of bead types, each with a distinct oligonucleotide capture probe, is pooled. An etched fiber optic bundle is dipped into the bead pool, allowing individual beads to assemble into the microwells at the bundle's end. Panel B shows a scanning electron micrograph of an assembled array containing 3 micron diameter silica beads with 5 micron core-to-core spacing between features. The beads are stably associated with the wells under standard hybridization conditions.
FIG. 2 shows a photograph of a 96-array matrix. Each array is located on the end of an optical fiber bundle containing  ̃50,000 individual fibers. The spacing of the arrays matches that of a 96-well plate, allowing 96 separate samples to be processed simultaneously.
FIG. 3 shows Illumina's SNP genotyping format.
FIG. 4 shows an oligonucleotide design scheme.
FIG. 5 shows plasmid controls used in the assay development. Unmethylated (green), semi-methylated (yellow) and fully-methylated (red) plasmid loci can be correctly scored in the human genomic DNA background.
FIG. 6 shows bisulfite conversion of DNA monitored with internal controls. Top panel: unconverted DNA; bottom panel: DNA after bisulfite conversion. Query oligonucleotides for converted plasmid loci (yellow) and unconverted genomic loci (green) are present in both assays. After bisulfite conversion, signal corresponding to unconverted loci disappears, and signal from converted loci becomes detectable.
FIG. 7 shows methylation assay development and data processing. Left panel: unmethylated, semi-methylated, and fully-methylated loci on the plasmids can be distinguished in the human genomic DNA background. These plasmid DNAs were spiked into human genomic DNA at a 1:1 molar ratio. Right panel: each data point is represented in a red/green/yellow plot, where red indicates a methylated state, green—unmethylated, and yellow—semi-methylated. The whole left panel (bar graph) is represented by one column on a red/green/yellow plot.
FIG. 8 shows reproducible methylation detection in two human reference DNAs: unmethylated (left panel) and methylated (right panel). The red color indicates a methylated state, green—unmethylated, and yellow—semi-methylated. White squares represent the loci with low intensity values, for which the methylation status call could not be made. In the case of amplified gDNA (left panel), some genomic loci may become underrepresented after amplification procedure.
FIG. 9 shows methylation measurement in 15 Coriell genomic DNAs and the reference DNAs.
FIG. 10 shows methylation status of any particular locus determined using a clustering algorithm. Panel A shows raw intensity data (of each bead) for one locus across all 96 replicates. Panel B shows analyzed clusters. Unmethylated, methylated and semi-methylated loci can be distinguished and called correctly by this algorithm.
FIG. 11 shows a schematic overview of a methylation assay that incorporates bisulfite conversion and a bead array format.
FIG. 12 shows reference samples for a methylation assay encompassing amplified genomic DNA in Panel A and corresponding in vitro methylated genomic DNA in Panel B.
FIG. 13 shows correlation in methylation status between replicates of lung cancer clinical samples containing 389 loci across four independent arrays.
FIG. 14 shows reproducibility of the methylation assay between technical replicates as observed in 46 lung cancer clinical samples containing 389 loci across four independent arrays.
FIG. 15 shows methylation status of two housekeeping genes located on the X chromosome. In females X inactivation correlates with promoter methylation, and methylation pattern determined for both genes in 46 samples (in duplicates) allows to match the methylation status of the promoter with the gender of the sample source.
FIG. 16 shows correlation between methylation levels and gender based on methylation status of 6 genes located on the X-chromosome as monitored in 46 samples.
FIG. 17 shows methylation profiling in 46 lung cancer and matched normal tissues based on interrogation of 162 CpG sites. Unmethylated (green), semi-methylated (yellow), methylated (red).
FIG. 18 shows distinct methylation patterns observed for 14 markers in sqamous cell carcinoma versus normal matching tissue.
FIG. 19 shows cluster analysis of methylation profiles in 46 lung cancer samples which demonstrates good separation of cancer samples from normal matching pairs.
FIG. 20 shows internal methylation assay controls. Methylated, unmethylated or semi-methylated (generated by mixing the methylated and unmethylated at 1:1 ratio) plasmid DNAs were spiked into human genomic DNA at 1:1 molar ratio. Methylation states were determined in 46 such samples at 29 CpG sites from 3 plasmids: Unmethylated (Green), semi-methylated (Yellow) and fully-methylated (Red).
FIG. 21 shows a comparison of methylation profiles among lung cancer and matching normal tissues. The β-value (i.e. the methylation ratio measured for all the functional CpG sites) obtained from one replicate experiment is plotted against that obtained from another replicate experiment. The upper panels show the reproducibility of replicate assays on DNA derived from squamous cell carcinoma G12005 (left) and its matching normal tissue (center), and a comparison between the normal and the squamous cell carcinoma (right). The lower panels show the reproducibility of replicate assays on DNA derived from lung adenocarcinoma G12012 (left) and its matching normal tissue (center), and a comparison between the normal and the adenocarcinoma (right).
FIG. 22 shows methylation profiles of 6 housekeeping genes located on the X-chromosome. Data are shown for 14 CpG loci in 16 males and 6 females, which include adenocarcinomas and their matching normal tissue samples, and the two reference samples. Replicate measurements are shown in adjacent for each sample.
FIG. 23 shows methylation profiles of the lung adenocarcinoma markers and the squamous cell carcinoma markers in the training set of samples. Panel 23A shows 11 adenocarcinomas and 11 matching normal tissues. Panel 23B shows 14 squamous cell carcinomas and 10 matching normal tissues. Replicate measurements are shown in adjacent for each sample.
FIG. 24 shows a cluster analysis of lung cancer samples based on methylation profiles. Panel 24A shows adenocarcinoma (training set). Panel 24B shows squamous cell carcinoma (training set). Panel 24C shows adenocarcinoma (test set).
FIG. 25 shows box plots of the array data for selected cancer-specific methylation markers. β-values (y-axis) were calculated for the six adenocarcinoma cancer markers. For the training set (yellow plots), 11 adenocarcinoma and 21 normal tissues were used. For the validation set (grey plots), 13 adenocarcinoma and 12 normal tissues were used. The black bar represents the mean β-value. The box defines quartiles (25% and 75%, respectively). The error bars are upper and lower adjacent limits (median +/−1.5*IQR). Dots represent the outliers.



DETAILED DESCRIPTION OF THE INVENTION
This application file contains drawings executed in color. Copies of this patent or application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The invention disclosed herein provides diagnostic and prognostic methods for a condition that is characterized by differential methylation of genomic CpG dinucleotide sequences. Also provided are populations of genomic targets and corresponding nucleic acid probes that useful for the detection of differentially methylated genomic CpG dinucleotide sequences that can be correlated to the presence of or susceptibility to cancer in an individual.
In particular, the present invention provides genomic targets that are differentially methylated in adenocarcinoma of the lung. The following 14 genomic targets are disclosed herein as differentially methylated in adenocarcinoma: ADCYAP1—2, CDH13—3, GDF10—2, GDF10—3, HOXA5—2, MAGEA1—3, RUNX3—1, SCGB3A1—3, SERPINB5—1, SFN—2, SFTPA1—2, TERT—1, TERT—2, and TNF—2. The 14 genomic targets correspond to eleven gene targets.
In a further embodiment, the present invention provides genomic targets that are differentially methylated in sqamous cell carcinoma of the lung. The following 21 genomic targets are disclosed herein as differentially methylated in squamous cell carcinoma: ADCYAP1—1, ADCYAP1—2, ADCYAP1—3, BCR—2, CALCA—1, GDF10—3, HOXA5—1, HOXA5—2, HOXA5—3, MAGEA1—1, MAGEA1—2, PGR—2, PRKCDBP—3, SCGB3A1—3, SERPINB5—1, SFN—2, SFTPC—3, TERT—1, TERT—2, TERT—3, TNF—2 (FIG. 23B). The 21 genomic targets correspond to 14 gene targets.
Notably, the invention also discloses a novel cancer marker. As described herein, the gene target adenylate cyclase activating polypeptide 1 (ADCYAP1) provided by the present invention and shown to be methylated in adenocarcinoma and squamous cell carcinoma represents a novel cancer marker based on methylation status. Genomic targets designated SEQ ID NOS: 264, 265 and 266 correspond to gene target adenylate cyclase activating polypeptide 1 (ADCYAP1) and are shown herein to be methylated in squamous cell carcinoma, while SEQ ID NO: 265 is shown to be methylated in adenocarcinoma (FIGS. 23A and B).
The methods of the invention are directed to methods for diagnosing an individual with a condition that is characterized by a level and/or pattern of methylated genomic CpG dinucleotide sequences distinct from the level and/or pattern of methylated genomic CpG dinucleotide sequences exhibited in the absence of the particular condition. This invention also is directed to methods for predicting the susceptibility of an individual to a condition that is characterized by a level and/or pattern of methylated genomic CpG dinucleotide sequences that is distinct from the level and/or pattern of methylated genomic CpG dinucleotide sequences exhibited in the absence of the condition.
In various distinct embodiments, the present invention is based, in part, on the identification of reliable CpG methylation markers for the improved prediction of susceptibility, diagnosis and staging of lung cancer. The invention provides a population of reliable genomic target sequences or genomic targets for use in the diagnostic and prognostic methods provided by the present invention, which have been designated herein as SEQ ID NOS: 57, 58, 59, 72, 76, 139, 140, 141, 174, 264, 265, 266, 293, 294, 305, 309, 313, 314, 315, 345, 350, 353, 360, 364, 371 and 373. The genomic targets provided by the invention are associated with the following gene targets: ADCYAP1, BCR, CALCA, CDH13, GDF 10, HOXA5, MAGEA1, PGR, PRKCDBP, RUNX3, SCGB3A1, SERPINB5, SFNT, SFTPA1, SFTPC, TERT and TNF (Table 1). The genomic targets provided by the invention thus represent gene targets for methylation of genomic CpG dinucleotide sequences associated with lung cancer. Also provided are nucleic acid probes that correspond to the genomic target sites of the invention and that can be used to detect differential methylation of selected genomic CpG dinucleotide sequences that serve as markers associated with lung cancer.
It is understood that the genomic target sequences provides the context for the one or more selected genomic CpG dinucleotide sequences being measured within a particular genomic target sequence. Furthermore, according to the invention, any fraction of the total genomic CpG dinucleotide sequences within a genomic target sequence can be measured, including one or more, two or more three or more, four or more, five or more or all of the genomic CpG dinucleotide sequences within a genomic target sequence. In addition, since it is understood that the genomic target sequence provides the context for the one or more selected genomic CpG dinucleotide sequences being measured, the invention encompasses measurement of the particular genomic CpG dinucleotide sequences encompassed within any of the genomic target sequences designated as SEQ ID NOS: 1-366, regardless of the particular nucleic probes that may be used for detecting the methylation of the particular genomic CpG dinucleotide sequence. Althought Table 1 sets forth particular nucleic acid probes provided by the invention that correspond to the genomic targets of SEQ ID NOS: 1-366, the skilled person can practice the invention using any desired nucleic acid probe capable of detecting the methylation status of one or more genomic CpG dinucleotide sequences within a particular genomic target sequence.
The genomic targets and nucleic acid probes provided by the present invention are set forth in Table 1, below, and provide diagnostic and prognostic tools based on their ability to detect differential methylation of selected genomic CpG dinucleotide sequences associated with cancer. In the methods provided by the invention, the genomic targets and nucleic acid probes capable of detecting markers located within the genomic targets can be employed to detect altered levels of methylation of genomic CpG dinucleotide sequences in a biological sample compared to a reference level. Furthermore, the methods of the invention allow for use of the genomic markersand nucleic acid probes for the determination of methylation patterns, which are represented by differential methylation of selected genomic CpG dinucleotide sequences that serve as markers in particular sets or subsets of genomic targets. In embodiments directed to the detection of methylation patterns, it is possible to diagnose or predict the susceptibility of an individual to a specific tumor-type based on the correlation between the pattern and the tumor type.
DNA methylation is a mechanism for changing the base sequence of DNA without altering its coding function. DNA methylation is a heritable, reversible and epigenetic change. Yet, DNA methylation has the potential to alter gene expression, which has profound developmental and genetic consequences. The methylation reaction involves flipping a target cytosine out of an intact double helix to allow the transfer of a methyl group from S adenosylmethionine in a cleft of the enzyme DNA (cystosine-5)-methyltransferase (Klimasauskas et al., Cell 76: 357-369, 1994) to form 5-methylcytosine (5-mCyt). This enzymatic conversion is the most common epigenetic modification of DNA known to exist in vertebrates and is essential for normal embryonic development (Bird, Cell 70: 5-8, 1992; Laird and Jaenisch, Human Mol. Genet. 3: 1487-1495, 1994; and Bestor and Jaenisch, Cell 69: 915-926, 1992). The presence of 5-mCyt at CpG dinucleotides has resulted in a 5-fold depletion of this sequence in the genome during vertebrate evolution, presumably due to spontaneous deamination of 5-mCyt to T (Schoreret et al., Proc. Natl. Acad. Sci. USA 89: 957-961, 1992). Those areas of the genome that do not show such suppression are referred to as “CpG islands” (Bird, Nature 321: 209-213, 1986; and Gardiner-Garden et al., J. Mol. Biol. 196: 261-282, 1987). These CpG island regions comprise about 1% of vertebrate genomes and also account for about 15% of the total number of CpG dinucleotides. CpG islands are typically between 0.2 to about 1 kb in length and are located upstream of many housekeeping and tissue-specific genes, but may also extend into gene coding regions. Therefore, the methylation of cytosine residues within CpG islands in somatic tissues can modulate gene expression throughout the genome (Cedar, Cell 53: 3-4, 1988; Nature 421: 686-688, 2003).
Methylation of cytosine residues contained within CpG islands of certain genes has been inversely correlated with gene activity. Thus, methylation of cytosine residues within CpG islands in somatic tissue is generally associated with decreased gene expression and can be the effect a variety of mechanisms including, for example, disruption of local chromatin structure, inhibition of transcription factor-DNA binding, or by recruitment of proteins which interact specifically with methylated sequences indirectly preventing transcription factor binding. Despite a generally inverse correlation between methylation of CpG islands and gene expression, however, most CpG islands on autosomal genes remain unmethylated in the germline and methylation of these islands is usually independent of gene expression. Tissue-specific genes are usually unmethylated at the receptive target organs but are methylated in the germline and in non-expressing adult tissues. CpG islands of constitutively-expressed housekeeping genes are normally unmethylated in the germline and in somatic tissues.
Abnormal methylation of CpG islands associated with tumor suppressor genes can cause decreased gene expression. Increased methylation of such regions can lead to progressive reduction of normal gene expression resulting in the selection of a population of cells having a selective growth advantage. Conversely, decreased methylation (hypomethylation) of oncogenes can lead to modulation of normal gene expression resulting in the selection of a population of cells having a selective growth advantage.
The present invention harnesses the potential of genomic methylation of CpG islands as indicators of the presence of a condition in an individual and provides a reliable diagnostic and/or prognostic method applicable to any condition associated with altered levels or patterns of genomic methylation of CpG islands. CpG islands are contiguous regions of genomic DNA that have an elevated frequency of CpG dinucleotides compared to the rest of the genome. CpG islands are typically, but not always, between about 0.2 to about 1 kb in length, and may be as large as about 3 Kb in length. Generally, for the methods provided by the invention at least two or more, at least three or more, at least four or more CpG dinucleotide sequences are selected that are located within a genomic marker so as to allow for determination of co-methylation status in the genomic DNA of a given tissue sample. Preferably the primary and secondary CpG dinucleotide sequences are co-methylated as part of a larger co-methylated pattern of differentially methylated CpG dinucleotide sequences in the genomic marker. The size of such context regions varies, but generally reflects the size of CpG islands as described above, or the size of a gene promoter region, including the first one or two exons.
With particular regard to cancer, changes in DNA methylation have been recognized as one of the most common molecular alternations in human neoplasia. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is a well-established and common mechanism for gene inactivation in cancer (Esteller, Oncogene 21(35): 5427-40 (2002)). In contrast, a global hypomethylation of genomic DNA is observed in tumor cells; and a correlation between hypomethylation and increased gene expression has been reported for many oncogenes (Feinberg, Nature 301(5895): 89-92 (1983), Hanada, et al., Blood 82(6): 1820-8 (1993)). Thus, a detailed study of methylation pattern in selected, staged tumor samples compared to matched normal tissues from the same patient offers a novel approach to identify unique molecular markers for cancer classification.
Monitoring global changes in methylation pattern has been applied to molecular classification in breast cancer (Huang, et al., Hum Mol Genet. 8(3): 459-70 (1999)). In addition, many studies have identified a few specific methylation patterns in tumor suppressor genes, for example, p16, a cyclin-dependent kinase inhibitor, in certain human cancer types (Otterson, et al., Oncogene 11(6): 1211-6 (1995), Herman, et al., Cancer Res. 55(20): 4525-30 (1995)). Some of the most recent examples include the discoveries of causal relationship between the loss of RUNX3 expression, due to hypermethylation, and gastric cancer (Li, et al., Cell 109(1): 113-24 (2002)); loss of IGF2 imprinting in colorectal cancer (Cui, et al., Science 299(5613): 1753-5 (2003); and reduced Hic gene expression in several types of human cancer (Chen, et al., Nat Genet. 33(2): 197-202 2003), Fujii, et al., Oncogene 16(16): 2159-64 (1998), Kanai, et al., Hepatology 29(3): 703-9 (1999)).
In one embodiment, the invention provides a method for identification of differentially methylated genomic CpG dinucleotide sequences associated with cancer in an individual by obtaining a biological sample comprising genomic DNA from the individual; measuring the level of one or more methylated genomic CpG dinucleotide sequences in two or more of the genomic target sequences set forth herein and designated as SEQ ID NOS: 1-366 in the sample, and comparing the level of the one or more methylated genomic CpG dinucleotide sequences in the sample to a reference level of methylated genomic CpG dinucleotide sequences, wherein a difference in the level of methylation of said one or more genomic CpG dinucleotide sequences in the sample compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with cancer. In additional embodiments, the level of methylated genomic CpG dinucleotide sequences is measured for one or more, three or more, four or more, five or more, six ore more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelwe or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, fifty or more, of the genomic target sequences set forth herein and designated as SEQ ID NOS: 1-377 in the sample. A subset of the genomic target sequences or nucleic acid probes of the invention can be one ore more nucleic acid sequences.
In a further embodiment, the invention provides a method for identification of differentially methylated genomic CpG dinucleotide sequences associated with lung cancer in an individual by obtaining a biological sample comprising genomic DNA from the individual; measuring the level of one or more methylated genomic CpG dinucleotide sequences in two or more of the genomic target sequences set forth herein and designated as SEQ ID NOS: 57, 58, 59, 72, 76, 139, 140, 141, 174, 264, 265, 266, 293, 294, 305, 309, 313, 314, 315, 345, 350, 353, 360, 364, 371 and 373, in the sample, and comparing the level of the one or more methylated genomic CpG dinucleotide sequences in the sample to a reference level of methylated genomic CpG dinucleotide sequences, wherein a difference in the level of methylation of said genomic CpG dinucleotide sequences in the sample compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with cancer. In additional embodiments, the level of one or more methylated genomic CpG dinucleotide sequences is measured for one or more, three or more, four or more, five or more, six ore more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelwe or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, twenty-two or more, twenty-four or more, of the markers set forth herein and designated as SEQ ID NOS: 57, 58, 59, 72, 76, 139, 140, 141, 174, 264, 265, 266, 293, 294, 305, 309, 313, 314, 315, 345, 350, 353, 360, 364, 371 and 373 in the sample. A subset of the genomic markers or nucleic acid probes of the invention can be one ore more nucleic acid sequences.
In one embodiment, the invention provides a method for identification of differentially methylated genomic CpG dinucleotide sequences associated with adenocarcinoma of the lung in an individual by obtaining a biological sample comprising genomic DNA from the individual; measuring the level of one or more methylated genomic CpG dinucleotide sequences for two or more of the markers set forth herein and designated as SEQ ID NOS: 59, 140, 266, 293, 294, 140, 174, 371, 313, 314, 353, 360, 364 and 373 in the sample, and comparing the level of one or more methylated genomic CpG dinucleotide sequences in the sample to a reference level of methylated genomic CpG dinucleotide sequences, wherein a difference in the level of methylation of said genomic CpG dinucleotide sequences in the sample compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with cancer. In additional embodiments, the level of one or more methylated genomic CpG dinucleotide sequences is measured for one or more, three or more, four or more, five or more, six ore more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelwe or more, thirteen or more, of the markers set forth herein and present in SEQ ID NOS: 59, 140, 266, 293, 294, 140, 174, 371, 313, 314, 353, 360, 364 and 373 in the sample. A subset of the genomic markers or nucleic acid probes of the invention can be one ore more nucleic acid sequences.
In one embodiment, the invention provides a method for identification of differentially methylated genomic CpG dinucleotide sequences associated with squamous cell carcinoma of the lung in an individual by obtaining a biological sample comprising genomic DNA from the individual; measuring the level of one or more methylated genomic CpG dinucleotide sequences for two or more of the markers set forth herein and designated as SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371, in the sample, and comparing the level of one or more methylated genomic CpG dinucleotide sequences in the sample to a reference level of methylated genomic CpG dinucleotide sequences, wherein a difference in the level of methylation of said genomic CpG dinucleotide sequences in the sample compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with cancer. In additional embodiments, the level of one or more methylated genomic CpG dinucleotide sequences is measured for one or more, three or more, four or more, five or more, six ore more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelwe or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, of the markers set forth herein and designated as SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371, in the sample. A subset of the genomic markers or nucleic acid probes of the invention can be one ore more nucleic acid sequences.
The level of methylation of the differentially methylated genomic CpG dinucleotide sequences can provide a variety of information about the cancer and can be used, for example, to diagnose cancer in the individual; to predict the course of the cancer in the individual; to predict the susceptibility to cancer in the individual, to stage the progression of the cancer in the individual; to predict the likelihood of overall survival for the individual; to predict the likelihood of recurrence of cancer for the individual; to determine the effectiveness of a treatment course undergone by the individual.
As described herein, the level of methylation that is detected in a biological sample can be decreased or increased in comparison to the reference level and alterations that increase or decrease methylation can be detected and provide useful prognostic or diagnostic information. For example, hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes have been established as common mechanisms for gene inactivation in cancers (Esteller, Oncogene 21(35): 5427-40 (2002)). Thus, a detailed study of methylation pattern in selected, staged tumor samples compared to matched normal tissues from the same patient can identify unique molecular markers for cancer classification.
In addition to detecting levels of methylation, the present invention also allows for the detection of patterns of methylation. It has been confirmed previously that neoplastic cells can exhibit unusual patterns of gene methylation (Feinberg and Vogelstein, Nature 301: 89-92 (1983)). Previous genetic studies of various conditions, for example, schizophrenia and bipolar disorder, seemed to implicate regions of particular chromosomes 22, but studies failed to identify a susceptibility gene. Analysis of methylation patterns across these chromosome in biological samples from afflicted individuals can reveal epigenetic changes in the form of altered levels of methylation of subsets of genomic CpG dinucleotide sequences that make up a pattern of affected genomic targets that can be correlated with a condition.
In one embodiment of the invention, an altered level of methylation of genomic CpG dinucleotide sequences is observed only in a subset of the genomic targets set forth in Table 1 and designated SEQ ID NOS: 1-377. In this embodiment, the subset can represent a methylation pattern characteristic of a particular type of cancer. A particular subset provided by the invention encompasses genomic targets that are differentially methylated in lung cancer and set forth herein and designated as SEQ ID NOS: 57, 58, 59, 72, 76, 139, 140, 141, 174, 264, 265, 266, 293, 294, 305, 309, 313, 314, 315, 345, 350, 353, 360, 364, 371 and 373. A further subset provided by the invention encompasses genomic targets that are differentially methylated in adenocarcinoma of the lung and set forth herein and designated as SEQ ID NOS: 59, 140, 266, 293, 294, 140, 174, 371, 313, 314, 353, 360, 364 and 373. An additional subset provided by the invention encompasses genomic targets that are differentially methylated in squamous cell carcinoma of the lung and set forth herein and designated as SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371. Therefore, as described herein with reference to cancer, methylation patterns can be correlated with a particular type, class or origin of a condition and detection and comparison of methylation patterns across samples that share a phenotypic characteristic can be useful to identify further methylation patterns.
In a further embodiment the present invention provides a population of genomic targets comprising nucleic acid sequences designated SEQ ID NOS: 1-377, and set forth in Table 1. Also provided in a distinct, but related embodiment is a population of genomic targets selected from the group consisting of nucleic acid sequences designated SEQ ID NOS: 1-377. In a further embodiment the invention provides a population of genomic targets comprising a subset of the nucleic acid sequences designated SEQ ID NOS: 1-366, and set forth in Table 1. Differential methylation of genomic CpG dinucleotide sequences in a subset of SEQ ID NOS: 1-366 can be characteristic of a particular type, class or origin of cancer. Detection of differential methylation in a subset of genomic targets can be useful to diagnose or predict susceptibility for a particular type, class or origin of cancer.
In one embodiment, a population of genomic targets selected from the group consisting of nucleic acid sequences designated SEQ ID NOS: 1-366 can encompass SEQ ID NOS: 57, 58, 59, 72, 76, 139, 140, 141, 174, 264, 265, 266, 293, 294, 305, 309, 313, 314, 315, 345, 350, 353, 360, 364, 371 and 373. In yet another embodiment, a population of genomic targets selected from the group consisting of nucleic acid sequences designated SEQ ID NOS: 1-377 can encompass SEQ ID NOS: 59, 140, 266, 293, 294, 140, 174, 371, 313, 314, 353, 360, 364 and 373. In a further embodiment, a population of genomic targets selected from the group consisting of nucleic acid sequences designated SEQ ID NOS: 1-377 can encompass SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371. In a further embodiment, a population of genomic targets selected from the group consisting of nucleic acid sequences designated SEQ ID NOS: 1-377 can encompass SEQ ID NOS: 57, 58, 59, 72, 76, 139, 141, 264, 265, 293, 309, 315, 345, 350, 353, 364 and 373. In a further embodiment, a population of genomic targets selected from the group consisting of nucleic acid sequences designated SEQ ID NOS: 1-377 can encompass SEQ ID NOS: 59, 293, 353, 364 and 373. The genomic targets are capable of exhibiting altered levels of methylation of genomic CpG dinucleotide sequences that are predictive of the presence or susceptibility of an individual for cancer.
Also provided by the present invention is a population of nucleic acid probes capable of detecting methylation of genomic CpG dinucleotide sequences of two or more genomic targets selected from the group consisting of the nucleic acid sequences designated SEQ ID NOS: 1-377, and set forth in Table 1. The population of nucleic acid probes provided by the invention consists of two or more nucleic acid sequences selected from the group consisting of SEQ ID NOS: 378-1889, and set forth in Table 1, which sets forth four probes for each genomic target of SEQ ID NOS: 1-377. The nucleic acid probes of the invention are capable of detecting altered levels of methylation of genomic CpG dinucleotide sequences of two or more genomic targets, wherein altered levels are predictive of the presence or susceptibility of an individual for cancer. Based on the observation that adjacent CpG sites tend to be co-methylated or co-de-methylated, a design scheme can be applied in which a “CG” sequence was used for all the CpG sites within the vicinity of the design, in particular for the landing sites for both ASO and LSO, to target any methylated CpG site; while a “TG” sequence was used for all the CpG sites within the vicinity of the design, to target any un-methylated CpG site. This approach requires two separate LSO oligos, but adds better discrimination between the methylated and unmethylated alleles.
In a further embodiment aimed at determination of patterns of DNA methylation, a population of nucleic acid probes is utilized that is capable of detecting altered levels of methylation of genomic CpG dinucleotide sequences of a subset of a population of two or more genomic targets. Thus, the detection of differential methylation of genomic CpG dinucleotide sequences in only a subset of genomic targets can be used to identify a pattern that correlates with a particular type, class or origin of cancer.
As disclosed herein, the present invention provides a subset of genomic targets consisting of nucleic acid sequences set forth in Table 1 and designated SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371 which exhibits differential methylation of genomic CpG dinucleotide sequences associated with lung squamous cell carcinoma. FIG. 23B shows the differential methylation pattern observed for genomic targets designated SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371, which correspond to gene targets MAGEA1 (SEQ ID NOS: 57 and 58), BCR (SEQ ID NO: 72), CALCA (SEQ ID NO: 76), HOXA5 (SEQ ID NO:S:139, 140, and 141), TNF (SEQ ID NO: 174), ADCYAP1 (SEQ ID NOS: 264, 265 and 266), GDF10 (SEQ ID NO: 294), SFN (SEQ ID NO: 305), SFTPC (SEQ ID NO: 309), TERT (SEQ ID NOS: 313, 314 and 315), PRKCDBP (SEQ ID NO: 345), PGR (SEQ ID NO: 350), SERPINB (SEQ ID NO: 360) and SCGB3A1 (SEQ ID NO: 371).
In a further embodiment, the present invention provides a subset of genomic targets consisting of nucleic acid sequences set forth in Table 1 and designated SEQ ID NOS: 59, 140, 265, 293, 294, 140, 174, 371, 313, 314, 353, 360, 364 and 373 which exhibits differential methylation of genomic CpG dinucleotide sequences associated with lung adenocarcinoma. FIG. 23A shows the differential methylation pattern observed for genomic targets designated SEQ ID NOS: 59, 140, 174, 265, 293, 294, 305, 313, 314, 353, 360, 364, 371, and 373, which correspond to gene targets MAGEA1 (SEQ ID NO: 59), HOXA5 (SEQ ID NO: 140), ADCYAP1 (SEQ ID NO: 265), GDF10 (SEQ ID NOS: 293 and 294), TNF (SEQ ID NO: 174), ADCYAP1 (SEQ ID NO: 265), GDF10 (SEQ ID NO: 293, 294), SFN (SEQ ID NO: 305), TERT (SEQ ID NO: 313, and 314), CDH13 (SEQ ID NO: 353), SERPINB (SEQ ID NO: 360) RUNX3 (SEQ ID NO: 364) and SFTPA1 (SEQ ID NO: 373).
As demonstrated in Example IV, the differentially methylated markers identified from squamous cell carcinomas largely overlap with those identified from adenocarcinomas. Methylation of the gene target adenylate cyclase activating polypeptide 1 (ADCYAP1) as shown in the present disclosure for adenocarcinoma and squamous cell carcinoma has not previously been reported. Genomic targets designated SEQ ID NOS: 264, 265 and 266 correspond to gene target adenylate cyclase activating polypeptide 1 (ADCYAP1) and are shown herein to be methylated in squamous cell carcinoma, while SEQ ID NO: 265 is shown to be hypermethylated in adenocarcinoma and squamous cell carcinoma (FIGS. 23A and B). Gene targets ADCYAP1, CDH13, GDF10, HOXA5, SCGB3A1 and TERT had increased methylation levels in adenocarcinoma samples compared to normal (FIGS. 23A & 26), while MAGEA1, RUNX3, SERPINB5, SFN, SFTPA1 and TNF were less methylated in cancer (FIG. 23A). Methylation of CDH13, HoxA5, SCGB3A1 and GDF10 is associated with tumor progression in various types of cancer as described by Hibi et al., Br J Cancer 91: 1139-1142 (2004); Hibi et al., Br J Cancer 90, 1030-3 (2004); Ogama et al. Int J Oncol 25, 685-91 (2004); Chen et al., Am J Pathol 163, 37-45 (2003); Fackler et al., Int J Cancer 107, 970-5 (2003); Aldred et al., Cancer Res 63, 2864-71 (2003). The human telomerase reverse transcriptase gene (TERT) is shown to be inactivated in most differentiated cells, but is reactivated in the majority of cancer cells (Liuet al., Genes Chromosomes Cancer 41, 26-37 (2004)).
Also provided are target nucleic acid probes for detection of a subset of genomic targets consisting of nucleic acid sequences designated SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360, and 371, which exhibits differential methylation of genomic CpG dinucleotide sequences associated with squamous cell carcinoma (FIG. 23 B). In the presence of differentially methylated genomic CpG dinucleotide sequences, the subsets of genomic targets signify a pattern distinctive of a particular type of cancer, for example, adenocarcinoma or sqamous cell carcinoma of the lung tissue. The p value can be calculated for each individual marker. More than one CpG dinucleotide sequence that serves as genomic marker can be selected from the same gene if desired. Thus a gene can provide the context for more than one genomic CpG dinucleotide sequence such that methylation is determined for more than one CpG dinucleotide sequence within a single gene.
The p-value can be calculated based on a t-test of the level of methylation [i.e. methylation allele intensity/(methylation allele intensity+un-methylation allele intensity)] as is shown among 9 lung squamous cell carcinoma and 5 matching normal samples or 14 other normal samples in Table 2 and among 16 lung adenocarcinoma and 11 normal samples in Table 3.
As demonstrated in Example IV, a data matrix of β values can be used to identify CpG loci which show differential methylation in cancer. A p-value cutoff can be chosen by the skilled person to minimize the number of false positives and additional filters can be incorporated to select markers which have the largest difference between cancer and normal tissues. As shown in FIG. 23A for adenocarcinoma, a list of 14 differentially methylated markers was obtained: ADCYAP1—2, CDH13—3, GDF10—2, GDF10—3, HOXA5—2, MAGEA1—3, RUNX3—1, SCGB3A1—3, SERPINB5—1, SFN—2, SFTPA1—2, TERT—1, TERT—2, and TNF—2. Utilizing the same methylation analysis with the normal and squamous cell carcinoma samples, a list 21 differentially methylated markers was obtained: ADCYAP1—1, ADCYAP1—2, ADCYAP1—3, BCR—2, CALCA—1, GDF10—3, HOXA5—1, HOXA5—2, HOXA5—3, MAGEA1—1, MAGEA1—2, PGR—2, PRKCDBP—3, SCGB3A1—3, SERPINB5—1, SFN—2, SFTPC—3, TERT—1, TERT—2, TERT—3, TNF—2 (FIG. 23B).
The invention also provides subsets of target nucleic acid probes capable of detecting a pattern of methylation of genomic CpG dinucleotide sequences that is associated with a particular type of cancer. Thus, the invention provides a subset of genomic targets consisting of nucleic acid sequences designated SEQ ID NOS: 59, 140, 266, 293, 294, 140, 174, 371, 313, 314, 353, 360, 364 and 373, which exhibits differential methylation of genomic CpG dinucleotide sequences associated with adenocarcinoma (FIG. 23A). The population of nucleic acid probes capable of detecting altered levels of methylation of genomic CpG dinucleotide sequences of a subset of said two or more genomic targets associated with adenocarcinoma are set forth in Table 1 and designated as SEQ ID NOS: 59, 140, 266, 293, 294, 140, 174, 371, 313, 314, 353, 360, 364 and 373.
In a further embodiment, the invention provides a subset of genomic targets consisting of nucleic acid sequences set forth in FIG. 23B and designated SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371, which exhibits differential methylation of genomic CpG dinucleotide sequences associated with sqamous cell carcinoma. The population of nucleic acid probes capable of detecting altered levels of methylation of genomic CpG dinucleotide sequences of a subset of said two or more genomic targets associated with sqamous cell carcinoma are set forth in Table 1 and designated as SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371.
In one embodiment, this invention provides diagnostic markers for cancer. The markers of the invention are genomic sequences having methylation states that are diagnostic or prognostic of the presence or severity of cancer. A list of exemplary genes for which methylation state can be used to determine the presence or severity of cancer is provided in Table 1. Cancer diagnosis or prognosis in a method of the invention can be made in a method of the invention based on the methylation state of particular sequence regions of the gene including, but not limited to, the coding sequence, the 5′-regulatory regions, or other regulatory regions that influence transcription efficiency.
The prognostic methods of the invention are useful for determining if a patient is at risk for recurrence. Cancer recurrence is a concern relating to a variety of types of cancer. For example, of patients undergoing complete surgical removal of colon cancer, 25-40% of patients with stage II colon carcinoma and about 50% of patients with stage III colon carcinoma experience cancer recurrence. One explanation for cancer recurrence is that patients with relatively early stage disease, for example, stage II or stage III, already have small amounts of cancer spread outside the affected organ that were not removed by surgery. These cancer cells, referred to as micrometastases, cannot typically be detected with currently available tests.
The prognostic methods of the invention can be used to identify surgically treated patients likely to experience cancer recurrence so that they can be offered additional therapeutic options, including preoperative or postoperative adjuncts such as chemotherapy, radiation, biological modifiers and other suitable therapies. The methods are especially effective for determining the risk of metastasis in patients who demonstrate no measurable metastasis at the time of examination or surgery.
The prognostic methods of the invention also are useful for determining a proper course of treatment for a patient having cancer. A course of treatment refers to the therapeutic measures taken for a patient after diagnosis or after treatment for cancer. For example, a determination of the likelihood for cancer recurrence, spread, or patient survival, can assist in determining whether a more conservative or more radical approach to therapy should be taken, or whether treatment modalities should be combined. For example, when cancer recurrence is likely, it can be advantageous to precede or follow surgical treatment with chemotherapy, radiation, immunotherapy, biological modifier therapy, gene therapy, vaccines, and the like, or adjust the span of time during which the patient is treated. As described herein, the diagnosis or prognosis of cancer state is typically correlated with the degree to which one or more of the genes in Table I is methylated. Thus, the invention can include a determination made based on the methylation state for the entire set of genes in Table I or a subset of the genes.
Furthermore, as the list in Table I is exemplary, the methylation state of other genes or genomic sequences can also be used in a method of the invention to determine the presence or severity of cancer. Exemplary cancers that can be evaluated using a method of the invention include, but are not limited to hematoporetic neoplasms, Adult T-cell leukemia/lymphoma, Lymphoid Neoplasms, Anaplastic large cell lymphoma, Myeloid Neoplasms, Histiocytoses, Hodgkin Diseases (HD), Precursor B lymphoblastic leukemia/lymphoma (ALL), Acute myclogenous leukemia (AML), Precursor T lymphoblastic leukemia/lymphoma (ALL), Myclodysplastic syndromes, Chronic Mycloproliferative disorders, Chronic lymphocytic leukemia/small lymphocytic lymphoma (SLL), Chronic Myclogenous Leukemia (CML), Lymphoplasmacytic lymphoma, Polycythemia Vera, Mantle cell lymphoma, Essential Thrombocytosis, Follicular lymphoma, Myelofibrosis with Myeloid Metaplasia, Marginal zone lymphoma, Hairy cell leukemia, Hemangioma, Plasmacytoma/plasma cell myeloma, Lymphangioma, Glomangioma, Diffuse large B-cell lymphoma, Kaposi Sarcoma, Hemanioendothelioma, Burkitt lymphoma, Angiosarcoma, T-cell chronic lymphocytic leukemia, Hemangiopericytoma, Large granular lymphocytic leukemia, head & neck cancers, Basal Cell Carcinoma, Mycosis fungoids and sezary syndrome, Squamous Cell Carcinoma, Ceruminoma, Peripheral T-cell lymphoma, Osteoma, Nonchromaffin Paraganglioma, Angioimmunoblastic T-cell lymphoma, Acoustic Neurinoma, Adenoid Cystic Carcinoma, Angiocentric lymphoma, Mucoepidermoid Carcinoma, NK/T-cell lymphoma, Malignant Mixed Tumors, Intestinal T-cell lymphoma, Adenocarcinoma, Malignant Mesothelioma, Fibrosarcoma, Sarcomotoid Type lung cacer, Osteosarcoma, Epithelial Type lung cancer, Chondrosarcoma, Melanoma, cancer of the gastrointestinal tract, olfactory Neuroblastoma, Squamous Cell Carcinoma, Isolated Plasmocytoma, Adenocarcinoma, Inverted Papillomas, Carcinoid, Undifferentiated Carcinoma, Malignant Melanoma, Mucoepidermoid Carcinoma, Adenocarcinoma, Acinic Cell Carcinoma, Gastric Carcinoma, Malignant Mixed Tumor, Gastric Lymphoma, Gastric Stromal Cell Tumors, Amenoblastoma, Lymphoma, Odontoma, Intestinal Stromal Cell tumors, thymus cancers, Malignant Thymoma, Carcinids, Type I (Invasive thymoma), Malignant Mesethelioma, Type II (Thymic carcinoma), Non-mucin producing adenocarcinoma, Squamous cell carcinoma, Lymph epithelioma, cancers of the liver and biliary tract, Squamous Cell Carcinoma, Hepatocellular Carcinoma, Adenocarcinoma, Cholangiocarcinoma, Hepatoblastoma, papillary cancer, Angiosarcoma, solid Bronchioalveolar cancer, Fibrolameller Carcinoma, Small Cell Carcinoma, Carcinoma of the Gallbladder, Intermediate Cell carcinaoma, Large Cell Carcinoma, Squamous Cell Carcinoma, Undifferentiated cancer, cancer of the pancreas, cancer of the female genital tract, Squamous Cell Carcinoma, Cystadenocarcinoma, Basal Cell Carcinoma, Insulinoma, Melanoma, Gastrinoma, Fibrosarcoma, Glucagonamoa, Intaepithelial Carcinoma, Adenocarcinoma Embryonal, cancer of the kidney, Rhabdomysarcoma, Renal Cell Carcinoma, Large Cell Carcinoma, Nephroblastoma (Wilm's tumor), Neuroendocrine or Oat Cell carcinoma, cancer of the lower urinary tract, Adenosquamous Carcinoma, Urothelial Tumors, Undifferentiated Carcinoma, Squamous Cell Carcinoma, Carcinoma of the female genital tract, Mixed Carcinoma, Adenoacanthoma, Sarcoma, Small Cell Carcinoma, Carcinosarcoma, Leiomyosarcoma, Endometrial Stromal Sarcoma, cancer of the male genital tract, Serous Cystadenocarcinoma, Mucinous Cystadenocarcinoma, Sarcinoma, Endometrioid Tumors, Speretocytic Sarcinoma, Embyonal Carcinoma, Celioblastoma, Choriocarcinoma, Teratoma, Clear Cell Carcinoma, Leydig Cell Tumor, Unclassified Carcinoma, Sertoli Cell Tumor, Granulosa-Theca Cell Tumor, Sertoli-Leydig Cell Tumor, Disgerminoma, Undifferentiated Prostatic Carcinoma, Teratoma, Ductal Transitional carcinoma, breast cancer, Phyllodes Tumor, cancer of the bones joints and soft tissue, Paget's Disease, Multiple Myeloma, Insitu Carcinoma, Malignant Lymphoma, Invasive Carcinoma, Chondrosacrcoma, Mesenchymal Chondrosarcoma, cancer of the endocrine system, Osteosarcoma, Adenoma, Ewing Tumor, endocrine Carcinoma, Malignant Giant Cell Tumor, Meningnoma, Adamantinoma, Cramiopharlingioma, Malignant Fibrous Histiocytoma, Papillary Carcinoma, Histiocytoma, Follicular Carcinoma, Desmoplastic Fibroma, Medullary Carcinoma, Fibrosarcoma, Anoplastic Carcinoma, Chordoma, Adenoma, Hemangioendothelioma, Memangispericytoma, Pheochromocytoma, Liposarcoma, Neuroblastoma, Paraganglioma, Histiocytoma, Pineal cancer, Rhabdomysarcoms, Pineoblastoma, Leiomyosarcoma, Pineocytoma, Angiosarcoma, skin cancer, cancer of the nervous system, Melanoma, Schwannoma, Squamous cell carcinoma, Neurofibroma, Basal cell carcinoma, Malignant Periferal Nerve Sheath Tumor, Merkel cell carcinoma, Sheath Tumor, Extramamary Paget's Disease, Astrocytoma, Paget's Disease of the nipple, Fibrillary Astrocytoma, Glioblastoma Multiforme, Brain Stem Glioma, Cutaneous T-cell lymphoma, Pilocytic Astrocytoma, Xanthorstrocytoma, Histiocytosis, Oligodendroglioma, Ependymoma, Gangliocytoma, Cerebral Neuroblastoma, Central Neurocytoma, Dysembryoplastic Neuroepithelial Tumor, Medulloblastoma, Malignant Meningioma, Primary Brain Lymphoma, Primary Brain Germ Cell Tumor, cancers of the eye, Squamous Cell Carcinoma, Mucoepidermoid Carcinoma, Melanoma, Retinoblastoma, Glioma, Meningioma, cancer of the heart, Myxoma, Fibroma, Lipoma, Papillary Fibroelastoma, Rhasdoyoma, or Angiosarcoma among others.
This invention provides methods for determining a prognosis for survival for a cancer patient. One method involves (a) measuring a level of methylation for one or more of the genes listed in Table 1 in a neoplastic cell-containing sample from the cancer patient, and (b) comparing the level of methylation in the sample to a reference level of methylation for the gene, wherein a low level of methylation for the gene in the sample correlates with increased survival of the patient.
In a particular embodiment, the invention provides methods for determining a prognosis for survival for a lung cancer patient. In this embodiment, the method involves (a) measuring a level of methylation for one or more of the genes listed in Table 1 and described herein to be associated with genomic targets differentially methylated in lung cancer in a neoplastic cell-containing sample from the lung cancer patient, and (b) comparing the level of methylation in the sample to a reference level of methylation for the one or more genes, wherein a low level of methylation for the one or more genes in the sample correlates with increased survival of the patient. In this embodiment, the level of methylation can be measured for one or more gene targets, including, for example, ADCYAP1, BCR, CALCA, CDH13, GDF10, HOXA5, MAGEA1, PGR, PRKCDBP, RUNX3, SCGB3A1, SERPINB5, SFN, SFTPA1, SFTPC, TERT, and TNF. Genomic markers associated with each of these genes are described throughout this application and are set forth in Table 1 along with their corresponding proble sequences.
Another method involves (a) measuring a level of methylation for one or more of the genes listed in Table 1 in a neoplastic cell-containing sample from the cancer patient, and (b) classifying the patient as belonging to either a first or second group of patients, wherein the first group of patients having low levels of methylation for a gene is classified as having an increased likelihood of survival compared to the second group of patients having high level of methylation for a gene.
In a particular embodiment, the invention provides methods for determining a prognosis for survival for a lung cancer patient. In this embodiment, the method involves (a) measuring a level of methylation for one or more of the genes listed in Table 1 and described herein to be associated with genomic targets differentially methylated in lung cancer in a neoplastic cell-containing sample from the cancer patient, and (b) classifying the patient as belonging to either a first or second group of patients, wherein the first group of patients having low levels of methylation for a gene is classified as having an increased likelihood of survival compared to the second group of patients having high level of methylation for a gene. In this embodiment, the level of methylation can be measured for one or more gene targets, including, for example, ADCYAP1, BCR, CDH13, CALCA, GDF10, HOXA5, PRKCDBP, SCGB3A1 SFTPC, TERT. Genomic markers associated with each of these genes are described throughout this application and are set forth in Table 1 along with corresponding probe sequences.
Another method involves (a) measuring a level of methylation for one or more of the genes listed in Table 1 in a neoplastic cell-containing sample from the cancer patient, and (b) classifying the patient as belonging to either a first or second group of patients, wherein the first group of patients having high levels of methylation for a gene is classified as having an increased likelihood of survival compared to the second group of patients having low level of methylation for a gene.
In a particular embodiment, the invention provides methods for determining a prognosis for survival for a lung cancer patient. In this embodiment, the method involves (a) measuring a level of methylation for one or more of the genes listed in Table 1 and described herein to be associated with genomic targets differentially methylated in lung cancer in a neoplastic cell-containing sample from the cancer patient, and (b) classifying the patient as belonging to either a first or second group of patients, wherein the first group of patients having high levels of methylation for a gene is classified as having an increased likelihood of survival compared to the second group of patients having low level of methylation for a gene. In this embodiment, the level of methylation can be measured for one or more gene targets, including, for example, MAGEA1, PGR, RUNX3, SERPINB5, SFN, SFTPA1, and TNF. Genomic markers associated with each of these genes are described throughout this application and are set forth in Table 1 along with their corresponding proble sequences.
The invention also provides a method for monitoring the effectiveness of a course of treatment for a patient with cancer. The method involves (a) determining a level of one or more of the genes listed in Table 1 in a neoplastic cell containing sample from the cancer patient prior to treatment, and (b) determining the level of methylation for the gene in a neoplastic cell-containing sample from the patient after treatment, whereby comparison of the level of methylation for the gene prior to treatment with the level of methylation for the gene after treatment indicates the effectiveness of the treatment.
In a particular embodiment, the invention also provides a method for monitoring the effectiveness of a course of treatment for a patient with lung cancer. The method involves (a) determining a level of one or more of the genes listed in Table 1 and described herein to be associated with genomic targets differentially methylated in lung cancer in a neoplastic cell containing sample from the lung cancer patient prior to treatment, and (b) determining the level of methylation for the gene in a neoplastic cell-containing sample from the patient after treatment, whereby comparison of the level of methylation for the gene prior to treatment with the level of methylation for the gene after treatment indicates the effectiveness of the treatment. In this embodiment, the level of methylation can be measured for one or more gene targets, including, for example, ADCYAP1, BCR, CALCA, CDH13, GDF10, HOXA5, MAGEA1, PGR, PRKCDBP, RUNX3, SCGB3A1, SERPINB5, SFN, SFTPA1, SFTPC, TERT, and TNF. Genomic markers associated with each of these genes are described throughout this application and are set forth in Table 1 along with their corresponding proble sequences.
For adenocarcinoma, the level of methylation is elevated compared to normal tissue samples for genomic targets corresponding to gene targets, including, for example, ADCYAP1, CDH13, GDF10, HOXA5, SCGB3A1, SERPINB5, SFN, SFTPA1, TERT; and decreased compared to normal tissue samples for genomic targets corresponding to gene targets, including, for example, MAGEA1, RUNX3, SERPINB5, SFN, SFTPA1, and TNF (Table 23 A). For squamous cell carcima, the level of methylation is elevated compared to normal tissue samples for genomic targets corresponding to gene targets, including, for example, ADCYAP1, BCR, CALCA, GDF10, HOXA5, PRKCDBP, SCGB3A1, SFTPC, and TERT; and decreased compared to normal tissue samples for genomic targets corresponding to gene targets, including, for example, TNF, MAGEA1, PGR, SERPINB5 and SFN (Table 23 B). Based on discovery of these differential methylation states associated with lung cancer, the skilled person can design various application of the present invention by selecting whether to measure for elevated or decreased methylation within one or more gene targets.
As used herein, the term “reference level” refers to a control level of expression of a marker used to evaluate a test level of expression of a biomarker in a neoplastic cell-containing sample of a patient. For example, when the level of methylation of one or more genes, referred to herein as “genomic targets,” in the neoplastic cells of a patient are higher than the reference level of methylation for the genes, the cells are considered to have a low level of expression of the gene. Conversely, when the level of methylation of one or more genes in the neoplastic cells of a patient are lower than the reference level, the cells are considered to have a low level of expression, of the gene. It is also possible that the level of methylation of one or more genes in the neoplastic cells of a patient can be higher than the reference level and the cells are considered to have a high level of expression, of the gene. Furthermore, the level of methylation of one or more genes in the neoplastic cells of a patient can be lower than the reference level and the cells are considered to have a low level of expression, of the gene.
A reference level can be determined based on reference samples collected from age-matched normal classes of adjacent tissues, and with normal peripheral blood lymphocytes. The reference level can be determined by any of a variety of methods, provided that the resulting reference level accurately provides a level of a marker above which exists a first group of patients having a different probability of survival than that of a second group of patients having levels of the biomarker below the reference level. The reference level can be determined by, for example, measuring the level of expression of a biomarker in non-tumorous cells from the same tissue as the tissue of the neoplastic cells to be tested. The reference level can also be a level of a biomarker of in vitro cultured cells which can be manipulated to simulate tumor cells, or can be manipulated in any other manner which yields expression levels which accurately determine the reference level. The reference level can also be determined by comparison of the level of a biomarker, such as methylation of one or more genes, in populations of patients having the same cancer. This can be accomplished, for example, by histogram analysis, in which an entire cohort of patients are graphically presented, wherein a first axis represents the level of the biomarker, and a second axis represents the number of patients in the cohort whose neoplastic cells express the biomarker at a given level.
Two or more separate groups of patients can be determined by identification of subset populations of the cohort which have the same or similar levels of the biomarker. Determination of the reference level can then be made based on a level which best distinguishes these separate groups. A reference level also can represent the levels of two or more markers. Two or more markers can be represented, for example, by a ratio of values for levels of each biomarker. The reference level can be a single number, equally applicable to every patient, or the reference level can vary, according to specific subpopulations of patients. For example, older individuals might have a different reference level than younger individuals for the same cancer. In another example, the reference level might be a certain ratio of a biomarker in the neoplastic cells of a patient relative to the biomarker levels in non-tumor cells within the same patient. Thus the reference level for each patient can be proscribed by a reference ratio of one or moregenomic markers, such as methylation of one or more genes, wherein the reference ratio can be determined by any of the methods for determining the reference levels described herein.
It is understood that the reference level has to correspond to the level of one or more methylated genomic CpG dinucleotide sequences present in a corresponding sample that allows comparison to the desired phenotype. For example, in a diagnostic application a reference level can be based on a sample that is derived from a cancer-free origin so as to allow comparison to the biological test sample for purposes of diagnosis. In a method of staging a cancer it can be useful to apply in parallel a series of reference levels, each based on a sample that is derived from a cancer that has been classified based on parameters established in the art, for example, phenotypic or cytological characteristics, as representing a particular cancer stage so as to allow comparison to the biological test sample for purposes of staging. In addition, progression of the course of a condition can be determined by determining the rate of change in the level or pattern of methylation of genomic CpG dinucleotide sequences by comparison to reference levels derived from reference samples that represent time points within an established progression rate. It is understood, that the user will be able to select the reference sample and establish the reference level based on the particular purpose of the comparison.
As used herein, the term “neoplastic cell” refers to any cell that is transformed such that it proliferates without normal homeostatic growth control. Such cells can result in a benign or malignant lesion of proliferating cells. Such a lesion can be located in a variety of tissues and organs of the body. Exemplary types of cancers from which a neoplastic cell can be derived are set forth above.
As used herein, the term “cancer” is intended to mean a class of diseases characterized by the uncontrolled growth of aberrant cells, including all known cancers, and neoplastic conditions, whether characterized as malignant, benign, soft tissue or solid tumor. Specific cancers include digestive and gastrointestinal cancers, such as anal cancer, bile duct cancer, gastrointestinal carcinoid tumor, colon cancer, esophageal cancer, gallbladder cancer, liver cancer, pancreatic cancer, rectal cancer, appendix cancer, small intestine cancer and stomach (gastric) cancer; breast cancer; ovarian cancer; lung cancer; renal cancer; CNS 30 cancer; leukemia and melanoma. By exemplification, a list of known cancers is set forth above.
As used herein, the term “sample” is intended to mean any biological fluid, cell, tissue, organ or portion thereof, that contains genomic DNA suitable for methylation detection via the invention methods. A test sample can include or be suspected to include a neoplastic cell, such as a cell from the colon, rectum, breast, ovary, prostate, kidney, lung, blood, brain or other organ or tissue that contains or is suspected to contain a neoplastic cell. The term includes samples present in an individual as well as samples obtained or derived from the individual. For example, a sample can be a histologic section of a specimen obtained by biopsy, or cells that are placed in or adapted to tissue culture. A sample further can be a subcellular fraction or extract, or a crude or substantially pure nucleic acid molecule or protein preparation. A reference sample can be used to establish a reference level and, accordingly, can be derived from the source tissue that meets having the particular phenotypic characteristics to which the test sample is to be compared.
A sample may be obtained in a variety of ways known in the art. Samples may be obtained according to standard techniques from all types of biological sources that are usual sources of genomic DNA including, but not limited to cells or cellular components which contain DNA, cell lines, biopsies, bodily fluids such as blood, sputum, stool, urine, cerebrospinal fluid, ejaculate, tissue embedded in paraffin such as tissue from eyes, intestine, kidney, brain, heart, prostate, lung, breast or liver, histological object slides, and all possible combinations thereof. A suitable biological sample can be sourced and acquired subsequent to the formulation of the diagnostic aim of the marker. A sample can be derived from a population of cells or from a tissue that is predicted to be afflicted with or phenotypic of the condition. The genomic DNA can be derived from a high-quality source such that the sample contains only the tissue type of interest, minimum contamination and minimum DNA fragmentation. In particular, samples should be representative of the tissue or cell type of interest that is to be handled by the diagnostic assay. It is understood that samples can be analyzed individually or pooled depending on the purpose of the user. In addition, a population or set of samples from an individual source can be analyzed to maximize confidence in the results and can be a sample set size of 10, 15, 20, 25, 50, 75, 100, 150 or sample set sizes in the hundreds.
In subsequent steps of the method, the methylation levels of CpG positions are compared to a reference sample, to identify differentially methylated CpG positions. Each class may be further segregated into sets according to predefined parameters to minimize the variables between the at least two classes. In the following stages of the method, all comparisons of the methylation status of the classes of tissue, are carried out between the phenotypically matched sets of each class. Examples of such variables include, age, ethnic origin, sex, life style, patient history, drug response etc.
As used herein, the term “disease-free survival” refers to the lack of tumor recurrence and/or spread and the fate of a patient after diagnosis, for example, a patient who is alive without tumor recurrence.
The phrase “overall survival” refers to the fate of the patient after diagnosis, regardless of whether the patient has a recurrence of the tumor. As used herein, the term “risk of recurrence” refers to the probability of tumor recurrence or spread in a patient subsequent to diagnosis of cancer, wherein the probability is determined according to the process of the invention. Tumor recurrence refers to further growth of neoplastic or cancerous cells after diagnosis of cancer. Particularly, recurrence can occur when further cancerous cell growth occurs in the cancerous tissue. Tumor spread refers to dissemination of cancer cells into local or distant tissues and organs, for example during tumor metastasis. Tumor recurrence, in particular, metastasis, is a significant cause of mortality among patients who have undergone surgical treatment for cancer. Therefore, tumor recurrence or spread is correlated with disease free and overall patient survival.
The methods of the invention can be applied to the characterization, classification, differentiation, grading, staging, diagnosis, or prognosis of a condition characterized by a pattern of one or more methylated genomic CpG dinucleotide sequences that is distinct from the pattern of one or more methylated genomic CpG dinucleotide sequences exhibited in the absence of the condition. A condition that is suitable for practicing the methods of the invention can be, for example, cell proliferative disorder or predisposition to cell proliferative disorder; metabolic malfunction or disorder; immune malfunction, damage or disorder; CNS malfunction, damage or disease; symptoms of aggression or behavioural disturbance; clinical, psychological and social consequences of brain damage; psychotic disturbance and personality disorder; dementia or associated syndrome; cardiovascular disease, malfunction and damage; malfunction, damage or disease of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as an abnormality in the development process; malfunction, damage or disease of the skin, the muscles, the connective tissue or the bones; endocrine and metabolic malfunction, damage or disease; headache or sexual malfunction, and combinations thereof.
Methylation of CpG dinucleotide sequences can be measured using any of a variety of techniques used in the art for the analysis of specific CpG dinucleotide methylation status. For example, methylation can be measured by employing a restriction enzyme based technology, which utilizes methylation sensitive restriction endonucleases for the differentiation between methylated and unmethylated cytosines. Restriction enzyme based technologies include, for example, restriction digest with methylation-sensitive restriction enzymes followed by Southern blot analysis, use of methylation-specific enzymes and PCR, restriction landmark genomic scanning (RLGS) and differential methylation hybridization (DMH).
Restriction enzymes characteristically hydrolyze DNA at and/or upon recognition of specific sequences or recognition motifs that are typically between 4- to 8-bases in length. Among such enzymes, methylation sensitive restriction enzymes are distinguished by the fact that they either cleave, or fail to cleave DNA according to the cytosine methylation state present in the recognition motif, in particular, of the CpG sequences. In methods employing such methylation sensitive restriction enzymes, the digested DNA fragments can be separated, for example, by gel electrophoresis, on the basis of size, and the methylation status of the sequence is thereby deduced, based on the presence or absence of particular fragments. Preferably, a post-digest PCR amplification step is added wherein a set of two oligonucleotide primers, one on each side of the methylation sensitive restriction site, is used to amplify the digested genomic DNA. PCR products are not detectable where digestion of the subtended methylation sensitive restriction enzyme site occurs. Techniques for restriction enzyme based analysis of genomic methylation are well known in the art and include the following: differential methylation hybridization (DMH) (Huang et al., Human Mol. Genet. 8, 459-70, 1999); Not I-based differential methylation hybridization (see e.g., WO 02/086163 A1); restriction landmark genomic scanning (RLGS) (Plass et al., Genomics 58: 254-62, 1999); methylation sensitive arbitrarily primed PCR (AP-PCR) (Gonzalgo et al., Cancer Res. 57: 594-599, 1997); methylated CpG island amplification (MCA) (Toyota et. al., Cancer Res. 59: 2307-2312, 1999). Other useful methods for detecting genomic methylation are described, for example, in U.S. Pat. App. pub. No. 2003/0170684 or WO 04/05122.
Methylation of CpG dinucleotide sequences also can be measured by employing cytosine conversion based technologies, which rely on methylation status-dependent chemical modification of CpG sequences within isolated genomic DNA, or fragments thereof, followed by DNA sequence analysis. Chemical reagents that are able to distinguish between methylated and non methylated CpG dinucleotide sequences include hydrazine, which cleaves the nucleic acid, and bisulfite treatment. Bisulfite treatment followed by alkaline hydrolysis specifically converts non-methylated cytosine to uracil, leaving 5-methylcytosine unmodified as described by Olek A., Nucleic Acids Res. 24: 5064-6, 1996 or Frommer et al., Proc. Natl. Acad. Sci. USA 89: 1827-1831 (1992). The bisulfite-treated DNA can subsequently be analyzed by conventional molecular techniques, such as PCR amplification, sequencing, and detection comprising oligonucleotide hybridization.
Techniques for the analysis of bisulfite treated DNA can employ methylation-sensitive primers for the analysis of CpG methylation status with isolated genomic DNA as described by Herman et al., Proc. Natl. Acad. Sci. USA 93: 9821-9826, 1996, and in U.S. Pat. Nos. 5,786,146 and 6,265,171. Methylation sensitive PCR (MSP) allows for the detection of a specific methylated CpG position within, for example, the regulatory region of a gene. The DNA of interest is treated such that methylated and non-methylated cytosines are differentially modified, for example, by bisulfite treatment, in a manner discernable by their hybridization behavior. PCR primers specific to each of the methylated and non-methylated states of the DNA are used in a PCR amplification. Products of the amplification reaction are then detected, allowing for the deduction of the methylation status of the CpG position within the genomic DNA. Other methods for the analysis of bisulfite treated DNA include methylation-sensitive single nucleotide primer extension (Ms-SNuPE) (Gonzalgo & Jones, Nucleic Acids Res. 25: 2529-2531, 1997; and see U.S. Pat. No. 6,251,594), and the use of real time PCR based methods, such as the art-recognized fluorescence-based real-time PCR technique MethyLight.TM (Eads et al., Cancer Res. 59: 2302-2306, 1999; U.S. Pat. No. 6,331,393 to Laird et al.; and see Heid et al., Genome Res. 6: 986-994, 1996). It is understood that a variety of methylation assay methods can be used for the determination of the methylation status of particular genomic CpG positions. Methods which require bisulfite conversion include, for example, bisulfite sequencing, methylation-specific PCR, methylation-sensitive single nucleotide primer extension (Ms-SnuPE), MALDI mass spectrometry and methylation-specific oligonucleotide arrays and are described, for example, in U.S. patent application Ser. No. 10/309,803 and international application International Patent Application No.: PCT/US03/38582.
In one embodiment, methylation of genomic CpG positions in a sample can be detected using an array of probes. In particular embodiments, a plurality of different probe molecules can be attached to a substrate or otherwise spatially distinguished in an array. Exemplary arrays that can be used in the invention include, without limitation, slide arrays, silicon wafer arrays, liquid arrays, bead-based arrays and others known in the art or set forth in further detail below. In preferred embodiments, the methods of the invention can be practiced with array technology that combines a miniaturized array platform, a high level of assay multiplexing, and scalable automation for sample handling and data processing.
An array of arrays, also referred to as a composite array, having a plurality of individual arrays that is configured to allow processing of multiple samples can be used. Exemplary composite arrays that can be used in the invention are described in U.S. Pat. No. 6,429,027 and U.S. 2002/0102578 and include, for example, one component systems in which each array is located in a well of a multi-well plate or two component systems in which a first component has several separate arrays configured to be dipped simultaneously into the wells of a second component. A substrate of a composite array can include a plurality of individual array locations, each having a plurality of probes and each physically separated from other assay locations on the same substrate such that a fluid contacting one array location is prevented from contacting another array location. Each array location can have a plurality of different probe molecules that are directly attached to the substrate or that are attached to the substrate via rigid particles in wells (also referred to herein as beads in wells).
In a particular embodiment, an array substrate can be fiber optical bundle or array of bundles, such as those generally described in U.S. Pat. Nos. 6,023,540, 6,200,737 and 6,327,410; and PCT publications WO9840726, WO9918434 and WO9850782. An optical fiber bundle or array of bundles can have probes attached directly to the fibers or via beads. Other substrates having probes attached to a substrate via beads are described, for example, in U.S. 2002/0102578. A substrate, such as a fiber or silicon chip, can be modified to form discrete sites or wells such that only a single bead is associated with the site or well. For example, when the substrate is a fiber optic bundle, wells can be made in a terminal or distal end of individual fibers by etching, with respect to the cladding, such that small wells or depressions are formed at one end of the fibers. Beads can be non-covalently associated in wells of a substrate or, if desired, wells can be chemically functionalized for covalent binding of beads. Other discrete sites can also be used for attachment of particles including, for example, patterns of adhesive or covalent linkers. Thus, an array substrate can have an array of particles each attached to a patterned surface.
In a particular embodiment, a surface of a substrate can include physical alterations to attach probes or produce array locations. For example, the surface of a substrate can be modified to contain chemically modified sites that are useful for attaching, either-covalently or non-covalently, probe molecules or particles having attached probe molecules. Chemically modified sites can include, but are not limited to the linkers and reactive groups set forth above. Alternatively, polymeric probes can be attached by sequential addition of monomeric units to synthesize the polymeric probes in situ. Probes can be attached using any of a variety of methods known in the art including, but not limited to, an ink-jet printing method as described, for example, in U.S. Pat. Nos. 5,981,733; 6,001,309; 6,221,653; 6,232,072 or 6,458,583; a spotting technique such as one described in U.S. Pat. No. 6,110,426; a photolithographic synthesis method such as one described in U.S. Pat. Nos. 6,379,895 or 5,856,101; or printing method utilizing a mask as described in U.S. Pat. No. 6,667,394. Accordingly, arrays described in the aforementioned references can be used in a method of the invention.
The size of an array used in the invention can vary depending on the probe composition and desired use of the array. Arrays containing from about 2 different probes to many millions can be made. Generally, an array can have from two to as many as a billion or more probes per square centimeter. Very high density arrays are useful in the invention including, for example, those having from about 10,000,000 probes/cm2 to about 2,000,000,000 probes/cm2 or from about 100,000,000 probes/cm2 to about 1,000,000,000 probes/cm2. High density arrays can also be used including, for example, those in the range from about 100,000 probes/cm2 to about 10,000,000 probes/cm2 or about 1,000,000 probes/cm2 to about 5,000,000 probes/cm2. Moderate density arrays useful in the invention can range from about 10,000 probes/cm2 to about 100,000 probes/cm2, or from about 20,000 probes/cm2 to about 50,000 probes/cm2. Low density arrays are generally less than 10,000 probes/cm2 with from about 1,000 probes/cm2 to about 5,000 probes/cm2 being useful in particular embodiments. Very low density arrays having less than 1,000 probes/cm2, from about 10 probes/cm2 to about 1000 probes/cm2, or from about 100 probes/cm2 to about 500 probes/cm2 are also useful in some applications.
Thus, the invention provides a robust and ultra high-throughput technology for simultaneously measuring methylation at many specific sites in a genome. The invention further provides cost-effective methylation profiling of thousands of samples in a reproducible, well-controlled system. In particular the invention allows implementation of a process, including sample preparation, bisulfite treatment, genotyping-based assay and PCR amplification that can be carried out on a robotic platform.
The methods of the invention can be carried out at a level of multiplexing that is 96-plex or even higher including, for example, as high as 1,500-plex. An advantage of the invention is that the amount of genomic DNA used for detection of methylated sequences is low including, for example, less that 1 ng of genomic DNA per locus. In one embodiment, the throughput of the methods can be 96 samples per run, with 1,000 to 1,500 methylation assays per sample (144,000 data points or more per run). In the embodiment exemplified herein, the system is capable of carrying out as many as 10 runs per day or more. A further object of the invention is to provide assays to survey methylation status the 5′-regulatory regions of at least 1,000 human genes per sample. Particular genes of interest are tumor suppressor genes or other cancer-related genes, as well as genes identified through RNA profiling.
Therefore, the invention makes available diagnostic and/or prognostic assays for the analysis of the methylation status of CpG dinucleotide sequence positions as markers for disease or disease-related conditions. As exemplified herein for lung cancer, the invention provides a systematic method for the identification, assessment and validation of genomic targets as well as a systematic means for the identification and verification of multiple condition relevant CpG positions to be used alone, or in combination with other CpG positions, for example, as a panel or array of markers, that form the basis of a clinically relevant diagnostic or prognostic assay. The inventive method enables differentiation between two or more phenotypically distinct classes of biological matter and allows for the comparative analysis of the methylation patterns of CpG dinucleotides within each of the classes.
A further object of the invention is to provide assays for specific identifying methylation patterns in different cancer types and cancer stages. The invention provides assays for specific methylation patterns characteristic of different cancer types and cancer stages. For example, the invention provides genomic targets that give rise to differential methylation patterns characteristic of lung cancer in general, adenocarcinoma of the lung, squamous cell carcinoma of the lung as well as methylation patterns that can distinguish between adenocarcinoma and squamous cell carcinoma of the lung. As described herein, the genomic targets designated SEQ ID NOS: 57, 58, 59, 72, 76, 139, 140, 141, 174, 264, 265, 266, 293, 294, 305, 309, 313, 314, 315, 345, 350, 353, 360, 364, 371 and 373 show differential methylation patterns characteristic of lung cancer. The subset of genomic targets designated SEQ ID NOS: 59, 140, 266, 293, 294, 140, 174, 371, 313, 314, 353, 360, 364 and 373 shows differential methylation patterns characteristic of adenocarcinoma of the lung, while the genomic targets designated SEQ ID NOS: 57, 58, 72, 76, 139, 140, 141, 174, 264, 265, 266, 294, 305, 309, 313, 314, 315, 345, 350, 360 and 371 show differential methylation patterns characteristic of squamous cell carcinoma of the lung. The genomic targets designated SEQ ID NOS: 59, 293, 353, 364 and 373 show differential methylation patterns in adenocarcinoma of the lung, but not in sqamous cell carcinoma of the lung and can thus be used to distinguish between the two types of lung cancer. The genomic targets designated SEQ ID NOS: 57, 58, 59, 72, 76, 139, 141, 264, 265, 293, 309, 315, 345, 350, 353, 364 and 373 show differential methylation patterns in sqamous cell carcinoma of the lung, but not in adenocarcinoma of the lung and can also be used to distinguish between the two types of lung cancer.
A further object of the invention is to provide software to retrieve and annotate CpG island sequence information, design and analyze primers, track sample information, and analyze and report results obtained from methylation profiling methods of the invention. An advantage of the invention is that it provides a high throughput methylation analysis system that can be commercialized, both through a service business—in which customers can provide samples and a gene list (CpG site list) for analysis in the methods—and through products that can used in standard laboratory conditions.
RLGS profiling of the methylation pattern of 1184 CpG islands in 98 primary human tumors revealed that the total number of methylated sites is variable between and in some cases within different tumor types, suggesting there may be methylation subtypes within tumors having similar histology (Costello, et al., Nat Genet. 24(2): 132-8 (2000)). Aberrant methylation of some of these genes correlates with loss of gene expression. Based on these observations, it should be feasible to use the methylation pattern of a sizable group of tumor suppressor genes or other cancer-related genes to classify and predict different kinds of cancer, or the same type of cancer in different stages. It promises to provide a useful tool for cancer diagnosis, or preferably, for detection of premalignant changes. When combined with the development of sensitive, non-invasive methods (e.g. a blood test; indeed, circulating tumor nucleic acids in blood have been demonstrated to reflect the biologic characteristics of tumors (Cui, et al., Science 299 (5613): 1753-5 (2003)), to detect such methylation signatures) this may provide a viable method to screen subjects at risk for cancer as well as to monitor cancer progression and response to treatment.
Because methylation detection interrogates genomic DNA, but not RNA or protein, it offers several technological advantages in a clinical diagnostic setting: (1) readily available source materials. This is particularly important for prognostic research, when only DNA can be reliably extracted from archived paraffin-embedded samples for study; (2) capability for multiplexing, allowing simultaneous measurement of multiple targets to improve assay specificity; (3) easy amplification of assay products to achieve high sensitivity; (4) robust measurement in tumors that arise from methylation inactivation of one allele of tumor suppressor genes—a process called “functional haploinsufficiency” (Balmain, et al., Nat Genet. 33 Suppl: 238-44 (2003)). It is much easier to detect a methylation change (from negative to positive) than to detect a two-fold gene expression change in these tumors. In summary, when combined with RNA-based gene expression profiling and/or protein-based immunoassays, DNA methylation profiling should provide a sensitive, accurate and robust tool for cancer diagnosis and prognosis (Wong, et al., Curr Oncol Rep. 4(6): 471-7 (2002)).
The present invention is directed to a method for the identification of differentially methylated CpG dinucleotides within genomic DNA that are particularly informative with respect to disease states. These may be used either alone or as components of a gene panel in diagnostic and/or prognostic assays.
In particular embodiments, the invention is directed to methods of prediction and diagnosis of conditions characterized by a pattern of one or more methylated genomic CpG dinucleotide sequences that is distinct from the pattern of methylated genomic CpG dinucleotide sequences exhibited in the absence of the particular condition, for example, cell proliferative disorders, such as cancer; dysfunctions, damages or diseases of the central nervous system (CNS), including aggressive symptoms or behavioral disorders; clinical, psychological and social consequences of brain injuries; psychotic disorders and disorders of the personality, dementia and/or associates syndromes; cardiovascular diseases, malfunctions or damages; diseases, malfunctions or damages of the gastrointestine diseases; malfunctions or damages of the respiratory system; injury, inflammation, infection, immunity and/or reconvalescence, diseases; malfunctions or damages as consequences of modifications in the developmental process; diseases, malfunctions or damages of the skin, muscles, connective tissue or bones; endocrine or metabolic diseases malfunctions or damages; headache; and sexual malfunctions; or combinations thereof.
It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.
EXAMPLE I
Design of Target Nucleic Acid Probes
This Example shows design of target nucleic acid probes for detection of genomic loci.
First, a human gene promoter database was prepared that includes all CpG regions of potential interest for methylation profiling. A fully automated SNP genotyping assay design program was adapted for methylation application and CpG islands of interest are selected and “converted by bisulfite” computationally. For each CpG locus, three probes are designed: two allele-specific oligonucleotides, one corresponding to the methylated, and the other to the unmethylated state of the CpG site and one locus-specific oligo (FIG. 4). If other CpG loci are present in the close vicinity of the chosen CpG site, a wobble base [A or G] is used for the corresponding probe position. Assays for more than 60 CpG sites from 20 different genes were designed, mostly selected from the methylation database on the world-wide-web at methdb.de. Approximately half of the sites were used in the assay development.
EXAMPLE II
Development of Internal Controls and Confirmation of Completeness of Bisulfite Conversion
This example shows the development of internal controls that allow optimization of protocols, determination of assay specificity, troubleshooting, and evaluation of overall assay performance.
Plasmids pUC19, pACYC184 and phage phiX174 were selected to serve as control DNAs. These DNAs can be spiked into the genomic DNA assays to provide internal controls, and would not interfere with human genomic DNA reactions. It is easy to prepare completely unmethylated plasmid DNAs and then methylate them in vitro using Sss I (CpG) methylase to produce substrates with known methylation status. Plasmids can be methylated virtually to completion. The quality of in vitro methylation was tested by restriction enzyme digestion of unmethylated and methylated DNAs using the methylation sensitive enzyme Hpa II and its isoschisomer Msp I, which is not sensitive to methylation. It was not possible to detect any bands resulting from restriction digest on the agarose gel after incubation of methylated pUC19, pACYC184 and phiX174 with Hpa II for two hours at 37° C., while the unmethylated DNAs were completely digested. Both methylated and unmethylated DNAs were completely digested by Msp I.
Plasmid controls (unmethylated, methylated or mixed at a 1:1 ratio) were spiked into human genomic DNA at a 1:1 molar ratio (at approximately 2-4 pg plasmid DNA/1 μg gDNA, depending on the plasmid size), and were used in every methylation experiment to monitor both bisulfite conversion efficiency and accuracy of methylation detection. As shown in FIG. 5, unmethylated, semi-methylated and fully-methylated loci can be easily distinguished by the assay.
The utility of bisulfite conversion of DNA for methylation detection is based on the different sensitivity of cytosine and 5-methylcytosine to deamination by bisulfite. Under acidic conditions, cytosine undergoes conversion to uracil, while methylated cytosine remains unreactive. An efficient bisulfite conversion protocol is a necessary prerequisite for a high-throughput methylation profiling assay. Incomplete conversion of cytosine to uracil by bisulfite can result in appearance of false-positive signals for 5-methylcytosine, and reduce the overall quality of the assay data. In order to monitor the effectiveness of bisulfite treatment, a set of oligonucleotides (a standard set of SNP genotyping probes) designed for unconverted genomic DNA sequences was included with the plasmid control oligos in the assay. As shown in FIG. 6, if bisulfite conversion is successful, the signal from oligonucleotides targeted to the unconverted DNA (the SNP set) will disappear, and signals from oligonucleotides targeted to the converted DNA will be present. Incomplete conversion will result in low and inconsistent signals across all targeted loci.
Data Processing
Development of a robust and high-throughput method for simultaneous measurement of methylation at many specific sites in many samples requires a highly efficient analysis and data export process. Each data point in the methylation assay can be represented as a ratio of the fluorescent signals from M (methylated) and U (unmethylated) specific PCR products after array hybridization. This value indicates the methylation status of the CpG locus and may range from 0 in the case of completely unmethylated sites to 1 in completely methylated sites. The value also can be visually presented as a red/green/yellow plot (FIG. 7). In addition, each locus is characterized by a locus intensity value, which allows filtering of failed loci. This combination of numerical and color outputs allows for quick comparison of genes and samples of interest, and processing of thousands of loci across hundreds of samples.
Reference Samples for Genome-wide Methylation Profiling
To calibrate quantitative measurements of methylation, “fully methylated” and “unmethylated” genomic templates were developed (FIG. 8). The fully unmethylated templates were generated by genome-wide amplification of human genomic DNA. With more than 1000-fold amplification, any endogenous methylation is effectively “diluted/erased”, and the sample can be used as an “unmethylated” reference DNA. Methylated templates were generated by in vitro methylation of amplified gDNA using Sss I (CpG-methylase) enzyme. The reproducibility of the methylation detection method was confirmed by typing 27 human gene-specific CpG sites in these reference DNAs over 30 times. A high degree of reproducibility was obtained (FIG. 8).
Methylation Profiles of Randomly Selected Human Genomic DNAs
We have also monitored a set of CpG sites in randomly selected human genomic DNAs. The DNA samples were obtained from the Human Genetic Cell Repository, Coriell Institute for Medical Research, NJ. In this experiment, we measured 7 females, 5 males and 3 of unknown gender specificity; each was done in duplicate and the results are shown in columns next to each other in FIG. 9. Distinguishable methylation patterns were obtained with DNAs isolated from male and female cell lines, especially in genes that are located on the X-chromosome (FIG. 9). The overall methylation profiles are quite reproducible among the duplicates.
Assay Reproducibility
One of the most important assay characteristics is its reproducibility. This was addressed by monitoring several plasmid loci with known methylation status across multiple replicates in several independent experiments. The experimental setup also allowed for estimation of the assay accuracy. A mixture of unmethylated, methylated and 1:1 mixed plasmid DNAs was prepared, and spiked into 1 μg of human genomic DNA in a 1:1 molar ratio. Each experiment included three plasmid mixtures:


















1
pUC19 U
pACYC 184 M
phiX174 U:M



2
pUC19 U:M
pACYC 184 U
phiX174 M



3
pUC19 M
pACYC 184 U:M
phiX174 U










A typical experiment included 32 replicates of each set of the three plasmid mixtures assayed on a 96 fiber bundle assay matrix. Results of the reproducibility study are summarized in Table 2, which involves (79+96+95+95) replicates×14 CpG sites=5,110 measurements. It is noticeable that some loci (e.g. phi—4972) tend to perform better than others (e.g. pACYC—360). There are also some performance variations from experiment to experiment. The overall call accuracy is averaged at  ̃97% with a high call rate of 99.6%. The accuracy was calculated using our existing SNP genotyping software, which uses a clustering algorithm to determine if a locus methylated, unmethylated or semi-methylated (FIG. 10).







TABLE 5







Methylation measurement with 14 plasmid CpG loci.













Experiment 1,
Experiment 2,
Experiment 3,
Experiment 4,
Summary
















79 replicates
96 replicates
95 replicates
95 replicates



Cor-
























No


No


No


No


No


rect,



call,
Correct,
Wrong
call,
Correct,
Wrong
call,
Correct,
Wrong
call,
Correct,
Wrong
call,
Wrong
Correct,
CV


Locus
%
%
call, %
%
%
call, %
%
%
call, %
%
%
call, %
%
call, %
%
(%)


























pUC—
0
100
0
0
93.75
6.25
0
93.68
6.32
0
100
0
0
3.14
96.86
3.75


 229


pUC—
0
100
0
0
89.58
10.42
0
94.74
5.26
0
100
0
0
3.92
96.08
5.20


 964


pUC—
1.27
100
0
0
96.88
3.13
0
91.58
8.42
0
100
0
0.32
2.89
97.11
4.09


1488


pUC—
0
100
0
8.33
88.64
11.36
6.32
91.01
8.99
0
96.84
3.16
3.66
5.88
94.12
5.55


2077


pUC—
0
100
0
0
88.54
11.46
0
90.53
9.47
0
100
0
0
5.23
94.77
6.43


2575


pACYC—
0
100
0
0
88.54
11.46
0
97.89
2.11
0
100
0
0
3.39
96.61
5.66


 167


pACYC—
0
98.73
1.27
1.04
74.74
25.26
0
91.58
8.42
0
97.89
2.11
0.26
9.26
90.73
12.27


 360


pACYC—
0
100
0
1.04
89.47
10.53
0
95.79
4.21
0
97.89
2.11
0.26
4.21
95.79
4.75


1289


pACYC—
0
100
0
1.04
92.63
7.37
0
95.79
4.21
0
100
0
0.26
2.89
97.11
3.69


1481


phi—
0
100
0
0
98.96
1.04
0
95.79
4.21
0
100
0
0
1.31
98.69
2.02


2191


phi—
0
100
0
0
100
0
0
98.95
1.05
0
98.95
1.05
0
0.53
99.47
0.61


3050


phi—
0
97.47
2.53
2.08
94.68
5.32
3.16
92.39
7.61
0
97.89
2.11
1.31
4.39
95.61
2.69


3687


phi—
0
100
0
0
100
0
0
98.95
1.05
0
98.95
1.05
0
0.53
99.47
0.61


4128


phi—
0
100
0
0
100
0
0
100
0
0
100
0
0
0
100
0


4972


Total:
0.09
99.73
0.27
0.97
92.60
7.40
0.68
94.90
5.10
0
99.17
0.83
0.43
3.40
96.60
4.09









Note: Experiment 1 included 80 replicates of bisulfite converted DNA and 16 replicates of unconverted samples for background control. The other 3 experiments included only bisulfite converted DNA.
As demonstrated above, assay sensitivity and specificity were shown to be sufficient to detect changes in methylation status at more than 50 loci simultaneously in 1 microgram of human genomic DNA. A minimum of three levels of methylation was clearly distinguished: fully methylated, hemi-methylated, and unmethylated. The ability to distinguish three levels of methylation was confirmed by using plasmid control DNAs with known methylation status, spiked into human genomic DNA in a 1:1 molar ratio. Furthermore, reproducibility of methylation determination was shown to be 96.6% (which is a more stringent measurement than reproducibility), at a call rate exceeding 90% (Table 2). A set of three reference samples for 14 CpG sites was analyzed in four independent experiments. The number of measurements in each experiment was 1106, 1344, 1330 and 1330 respectively.
Overall, this example demonstrates the development of a microtiter plate based, high throughput bisulfite conversion, which as described in the following Example, can be fully integrated into the SNP genotyping system for high-throughput methylation profiling. The methylation assays can be enlarged in both the scope and capacity of methylation detection with as many as 1500 methylation sites in each assay, while using reduced amounts of genomic DNA. Since the data collection and processing are largely automated, it is possible to do at least one, two, five or ten array matrix runs per day per system, with each run providing data from 96 samples at a time, creating a highly scalable system where multiple instruments can be run in parallel if needed.
EXAMPLE III
Integration of Microtiter Plate Based, High-throughput Bisulfite Conversion into Genotyping System for High Troughput Methylation Profiling
This example demonstrates the integration of a microtiter plate based, high throughput bisulfite conversion as described in Example II, into the SNP genotyping system for high-throughput methylation profiling.
The assay optimization process includes measuring the array-to-array experimental variability, both within a matrix and between matrices, and dissect out contributions to variability from samples, sample processing (bisulfite conversion, allele-specific extension, ligation, and PCR amplification), and array hybridization, using carefully designed controls. The resulting data also is useful in determining thresholds of significance for analyzing and interpreting results.
Improve Assay Performance with Fully “Methylated” and “Un-methylated” Genomic Templates
Currently, all the methods used to validate methylation status of any methylation site in any given sample, such as bisulfite sequencing and methylation-specific PCR, are either laborious and time consuming, or inaccurate and expensive. Indeed, a high performance methylation quantitation (or calibration) system is needed for large-scale genome-wide methylation assay development and validation. As described in Example II, a system has been developed that uses fully “methylated” and “un-methylated” genomic templates.
The un-methylated templates can be generated by genome-wide amplification of any genomic DNA, using random primed DNA amplification with enzymes such as Phi-29, Taq DNA polymerase or Klenow Fragment (3′->5′-exo-). After this amplification, the endogenous DNA methylation is diluted at least 100 to 1000-fold, effectively rendering the amplified genome DNA “un-methylated”.
The methylated templates can be generated by in vitro methylation using the SssI CpG-methylase. However, not all the CpG sites can be fully methylated in vitro. Some of these can result from base substitution at the CpG sites in the DNA tested, in particular, these sites become “methylation-resistant”. It is well known that CpG sites are mutation hot spots. In order to achieve higher levels of genomic DNA methylation, different experimental conditions are tested, for example, varying the concentration of magnesium in the methylation reaction and using multiple methylases.
The above-described templates are used for assay development and calibration. The fully methylated and unmethylated genomic DNA templates can be mixed at different ratio, for example, 0%, 25%, 50%, 75%, and 100% of methylated template. Methylation assays on these mixed templates generate a calibration curve for quantitative methylation measurement in unknown samples for any CpG site in the genome. The mixed templates can also be used to determine the sensitivity of methylation detection, for example, what percentage of the methylated template can be detected in the presence of un-methylated template.
Since this approach can be used to evaluate the methylation assay designed for any specific CpG site across the entire genome, it greatly aids methylation assay development such as assay specificity. For example, if an assay gives the same methylation “report” for both the unmethylated and the methylated DNA templates, it confirms that the assay is not working properly.
Finally, protocols for measuring DNA methylation in formalin-fixed, paraffin-embedded samples are created. Robust large-scale DNA methylation detection on these samples opens up a huge sample resource, for which clinical history is already available.
Improvement of Assay Sensitivity by Genomic DNA Amplification After Bisulfite Conversion
In order to improve assay sensitivity, DNA is amplified after bisulfite conversion, using a random priming approach. But, instead of using all possible random primers, advantage is taken of the unique sequence feature of genomic DNAs after bisulfite treatment, i.e. that un-methylated cytosines are converted to uracil. Therefore, these DNA templates contain mostly three bases, A, G and T (and U). The genomic amplification is carried out using (i) through (iii) as set forth in the following paragraphs.
(i) A mixture of two sets of primers that contain all possible combinations of three nucleotides: (i.e. A, T and C for one set, and A, T and G for the other set). Primers from the first set have higher affinity to the original bisulfite converted DNA strand, while primers from the second set preferentially anneal to the newly synthesized complementary strand. Using this scheme, having G and C in the same primer is avoided, thus preventing the primers from crossing over any CpG sites to be interrogated. Bias that may be introduced by the un-balanced annealing efficiency of primers corresponding to the two alleles (C or T) also is avoided. Lastly, since each primer set contains all possible combinations of three, but not four nucleotides, effective primer concentration is increased.
(ii) Simple primer sequences that contain only Adenines (A). The homopoly-A primers (for example, 6-mer, 9-mer, or longer) is used for the first strand synthesis. After that, a homopoly-T tail is added to the 3′-ends of the first strand products, using terminal deoxyribonucleotide transferase (TdT). A standard PCR is then be carried out to amplify the DNAs using a poly-A primer. Human chromosome 1 sequence was used to calculate the poly-T frequencies in the genome after bisulfite conversion: on average, the physical distance between any two poly-(T)n sequences (n>=9) is 330 bp, a perfect amplicon size range for robust amplifications.
(iii) Similar to approach (i), except that oligo-A primer is used for the first strand synthesis and primers containing combinations of A, T and G for the complementary strand synthesis.
Probe design is one of the critical components for a successful methylation assay. The fully automated SNP genotyping assay design program described herein can be used for methylation assay development. As shown above, for each CpG locus, three probes are designed: two allele-specific oligonucleotides, one corresponding to the methylated and the other to the unmethylated state of the CpG site, and one locus-specific oligo. If other CpG loci are present close to the chosen CpG site, a wobble base [A or G] is used in the corresponding position of the probes. Based on the observation that adjacent CpG sites tend to be co-methylated or co-de-methylated, a simpler design scheme is applied in which a “CG” sequence is used for all the CpG sites within the vicinity of the design, in particular for the landing sites for both ASO and LSO, to target any methylated CpG site; while a “TG” sequence is used for all the CpG sites within the vicinity of the design, to target any un-methylated CpG site. This approach requires two LSO oligos for some loci, but adds better discrimination between the methylated and unmethylated alleles.
A human gene promoter database, which includes all CpG regions of potential interest for methylation profiling was constructed by combining NCBI's RefSeq annotation, existing knowledge of some well-studied promoters, gene and promoter prediction algorithms, as well as observations of certain cancer-related genes. This database is continuously expanded by integrating more public information from literature and databases, and experimental observations. For the methylation study, a new database searching strategy is integrated into the primer design software. A modified genome database is generated in which all “C”s (except those located within a CpG dinucleotide sequence) are converted to “T”s in silico. The probe design program searches against this converted database to find unique sequences and compute melting temperature (Tm), self-complementarity and length for an optimal probe. An optimization program is applied to match address sequences with locus-specific oligos to minimize self-complementarities of combined address and probe sequences. A locus filtering program is used to filter out sequences predicted to be unsuitable on the basis of data from SNP genotyping experiments already carried out. Some sequence features have been shown to be troublesome, e.g. runs of six or more consecutive bases of a single type, extreme GC or AT content, inverted repeats (mostly due to secondary structure), and high numbers of hits in the human genome sequence based on similarity searches by BLAST. All of these parameters can be computed in advance. These parameters are stored in a relational database for further data analysis. In addition, the program computes the sequence complementarity between the probes designed for a given set of methylation sites, especially the sequence complementary at their 3′ ends. This calculation allows assessment of the compatibility of the assays, which in turn provides guidance regarding grouping of the assays properly for multiplexing.
The main problem for the methylation assay primer design lies in the reduced complexity of the genome after bisulfite conversion of the genomic DNA. Analysis of 5′-regulatory sequences from 1200 human genes was performed and preliminary computer simulation analysis indicates that the length of the primers designed for the bisulfite-converted DNAs will have to be increased by several bases as compared to the un-converted ones to achieve the same primer specificity and melting temperature. If necessary, longer primers along with increased assay stringency is used.
In another in-silico experiment, three BLAST searchable human genomic sequence databases were created. The first database mimics methylated condition (after bisulfite treatment), where all the C residues in CpG dinucleotide sequence remains as C; the second mimics un-methylated conditions, where all the C residues in CpG dinucleotide sequence are converted into T; both of these databases have C residues from non-CpG sites converted into T. The third database has normal genomic sequences. BLAST searching against these databases using designed probes as queries was performed and, as predicted, the probes had much larger number of hits to the database that have “C” converted into “T” (the first two databases), and less number of hits to the normal database (the third database). Subsequent empirical experiments with probes that have either large or small number of BLAST hits suggested that the probes with small number of hits usually generate good assay results, while the probes with large number of hits do not. In the future, this BLAST search process is automated and integrated into the probe design software. Furthermore, probes is designed for all the CpG sites in the promoter regions. After a subsequent BLAST filtering process, only three probes are synthesized for each gene. For a small number of genes that can't have three qualified probes designed due to limited number of CpG sites in the promoter region or CpG sites too close to each other, or/and severe sequence similarity to other genomic regions.
In order to search for specific methylation patterns in different cancer types or cancer stages, we first develop methylation assays for at least 1000 human genes. These genes are selected based on the following criteria:
(i) Biological relevance. Methylation patterns in previously characterized tumor suppressor genes and oncogenes are an initial focus (Esteller, Oncogene 21(35): 5427-40 (2002), Adorjan, et al., Nucleic Acids Res. 30(5): p. e21 (2002)). Then, the target group is enlarged to include genes that are indirectly involved in cancer development, for example, DNA repair genes; metastasis-inhibitor genes, genes regulated by various signaling pathways, and/or responsible for altered cell growth and differentiation; or genes considered to be targets for oncogenic transformation.
(ii) Previous knowledge, e.g. genes located within published recurrent loss of hetrozygosity (LOH) regions or amplified genomic regions (Pollack, et al., Proc Natl Acad Sci USA. 99(20): 12963-8 (2002)).
(iii) Gene expression profiling information, for example, genes differentially expressed in cancer and normal tissues. In the past few years, due to the rapid development of microarray technology, many specific gene-expression signatures have been identified for different cancer types (Golub, et al., Science 286(5439): 531-7 (1999), Ramaswamy, et al., Proc Natl Acad Sci USA. 98(26): 15149-54 (2001), Perou, et al., Nature 406(6797): 747-52 (2000), Bhattacharjee, et al., Proc Natl Acad Sci USA 98(24): 13790-5 (2001), Chen, et al., Mol Biol Cell 13(6): 1929-39 (2002), Welsh, et al., Proc Natl Acad Sci USA. 98(3): 1176-81 (2001)), cancer stages (Dhanasekaran, et al., Nature 412(6849): 822-6 (2001), Ramaswamy, et al., Nat Genet. 33(1): 49-54 (2003)), and cancer therapeutic outcomes (Shipp, et al., Nat Med. 8(1): 68-74 (2002)). Some of these differential expressions are regulated by imprinted or somatic methylation.
The methylation targets are grouped into functionally relevant sets that are useful for focused research (e.g. based on association with a particular pathway or disease; or expressed in particular tissues of interest; or representing a particular genomic region), as well as for more global studies. For example, genes can be grouped according to their biochemical properties, such as, oncogenes/tumor suppressor genes, kinases, phosphatases, and cell surface receptors. Genes can be also grouped based on their involvement in different biological pathways/functions, for example, tumor antigen, signal transduction, apoptosis, angiogenesis, cell cycle control, cell differentiation, DNA repair, cancer metastasis/invasion, drug resistance and detoxification, and transcriptional regulation, etc. The assays are then optimized to achieve a high degree of reliability and specificity within each set.
Selection of CpG Sites to be Interrogated
Several CpG sites within each 5′-regulatory region, including CpGs over the transcriptional start site is targeted, since redundant information from multiple CpG sites can provide a better measurement of the overall methylation status in the interrogated gene. While there are many CpG sites within each CpG island, only those for which robust assays can be designed is used. Each potential CpG site is BLAST searched against human dbSNP databases to avoid any potential “polymorphic” CpG site (i.e. the “methylation-resistant” site), to ensure clean data interpretation. If desired, the consequence of the polymorphic CpG sites, for example, their effect on methylation of adjacent CpG sites, and subsequently on gene expression level can be determined.
It has been estimated that the human genome has 26,000 to 45,000 CpG islands (Antequera, et al., Proc Natl Acad Sci USA. 90(24): 11995-9 (1993), Ewing, et al., Nat Genet. 25(2): 232-4 (2000)). Among them, those found in gene 5′-regulatory regions are the most biologically significant ones. The work does not only utilize CpG rich promoters, but also investigates less CpG rich promoters since they might also be subjected to aberrant methylation and silencing.
In order to correctly identify genes and their upstream regulatory regions, public databases such as NCBI RefSeq, UCSC Human Genome Project (HGP) Working Draft, ENSEMBL and Unigene are utilized. These databases annotate known or verified genes according to sequence similarity to mRNA, EST and protein sequences, and novel genes predicted using gene prediction programs. They also contain reference links to genetic and physical mapping data, as well as other features such as CpG islands. The accuracy of these annotated features is confirmed by re-running gene prediction programs and searching for sequence similarity to mRNA, EST, and known protein sequences. Existing promoter databases such as Eukaryotic Promoter Database (EPD) (Praz, V., et al., Nucleic Acids Res. 30(1): 322-4 (2002)), are used to identify the 5′-regulatory sequences. Meantime, promoter and first exon prediction algorithms such as FirstEF (Davuluri, et al., Nat Genet. 29(4): 412-7 (2001)) are used to identify potential regulatory sequences. This approach provides a well-annotated collection of regulatory sequences of human genes and allow for identification of the CpG sites within these regulatory regions and design assay probes as described above.
A CpG site within a CpG island doesn't automatically qualify it as a biologically significant methylation target and art knowledge is applied in order to design most valuable methylation assays. In the past few years, tremendous progress has been made in the epigenetics field, which uncovered many epigenetic regulation mechanisms in various biological pathways (Strichman-Almashanu, et al., Genome Res. 12(4): 543-54 (2002)), and cancers (Widschwendter, et al., Oncogene 21(35): 5462-82 (2002), Tsou, et al., 21(35): p. 5450-61 (2002)). Moreover, large methylation projects such as bisulfite sequencing of CpG island-enriched libraries (Cross, et al., Nat Genet. 6(3): 236-44 (1994)), or entire human chromosomes or even the entire genome are likely to be developed. These efforts should produce tremendous amounts of methylation data and reveal hundreds of thousands of new methylation sites in many different tissue types.
Assay Development, and Array Data Extraction and Analysis
Once the assays are designed, they are tested with publicly available genomic DNAs isolated from various cancerous or normal human tissues or cell lines of different tissue origins, and obtain tissue-specific methylation profiles for individual genes (CpG sites). These methylation profiles serve as references for analyzing unknown samples. Building on SNP genotyping technology, a quantitative metric to guide the methylation assay development is formulated and provide a quality assurance to data generated in a production setting. The metric takes into consideration all aspects of assay performance and data quality (e.g. assay specificity and quantitation), including efficiency of bisulfite conversion, overall signal intensity of all targeted CpG sites, concordance among the measurements of the three CpG sites within each gene, specificity of detection in control samples (e.g. plasmids, reference samples as well mixtures of the reference samples), and measurement variations in replicated samples, etc.
In addition the quantitative performance of methylation detection is tested at various multiplexing levels, for example, high (>1000-plex), medium ( ̃300-plex), and low (<100-plex), and validate the specificity and sensitivity of the assays at high multiplexing levels. Meanwhile, as a measurement of the assay specificity, concordance of methylation profiles generated from a given sample at different multiplexing levels are compared. Finally, methylation-specific PCR is used to validate some of the array results (Herman, et al., Proc Natl Acad Sci USA. 93(18): 9821-6 (1996)). All qualified assays are re-pooled and used for large-scale DNA methylation profiling.
Since the sequence complexity is significantly reduced following bisulfite conversion of the genomic DNA, the assay oligo/genomic DNA annealing protocol is optimized to minimize cross-hybridization. Tetramethylammonium (TMACl) (De Murcia, et al., Biophys Chem. 8(4): 377-83 (1978), Sorg, et al., Nucleic Acids Res. 19(17): 4782 (1991)), and/or Betaine (Rees, et al., Biochemistry, 32(1): 137-44 (1993)) is used to normalize the base composition dependence of DNA/oligo hybridization. Nevertheless, high locus specificity should be achieved by the requirement that both ASO and LSO oligos need to hybridize to the same genomic target site and then get extended and ligated (FIG. 3).
The existing SNP genotyping software is modified and adapted for the methylation data analysis. The current software takes the raw intensity data and transforms them into a genotype call using a clustering algorithm (FIG. 10). However, for most of the methylation data analysis cluster analysis cannot be utilized due to the need not only to distinguish three methylation states of each locus (unmethylated, methylated and semi-methylated), but do it in a more quantitative manner, for example, estimate percentage of methylation of certain loci in a given sample. The methylated and unmethylated reference samples and assay controls are used in every experiment for software calibration. Assay intensity data of unknown sample is compared to those obtained with the reference samples, and used to calculate the methylation level of the locus of interest. Software is developed for comparison of various samples and detection of differential methylation profiles to allow for identification of differences between normal tissues and tumors, and/or create tissue-specific methylation profiles for genes and loci of interest.
Search for Specific Methylation Patterns in Different Cancer Types or Cancer Stages
Once the gene-specific methylation assays are developed as described above, a large-scale DNA methylation survey is carried out in a large number of samples. The experiment is designed to compare methylation patterns in (1) normal and cancerous tissues; (2) different cancer types or cancer stages; (3) or responsive to (or associated with) treatment with certain growth factors or drugs, activation of oncogenes or inactivation of tumor suppressor genes, changes in a developmental program, etc. The main objective is to find unique methylation patterns for specific cancer types/stages and develop molecular markers for classification and diagnosis of cancers, which can be used to complement existing morphological and clinical parameters. This can be particularly useful for cancer types which appear similar by histological assessments, but follow different clinical courses (e.g. different therapeutic responses). The results also provide important clues to the mechanisms of specific cellular responses; and this information can prove critical for devising strategies for cancer prevention and treatment.
Malignant tissues obtained by laser capture microdissection (LCM) are used to identify specific cell and tissue types for methylation profiling. In these cases, a more sensitive strategy is employed, which involves DNA amplification after bisulfite conversion. If an assay can be established to detect tumor-specific methylation patterns in a very small amount of diseased tissue in the presence of a large amount of normal tissue, it may find wide application in clinical cancer diagnosis.
As a pilot study, DNA samples isolated from 50 or more lung tissues, for example, 98 lung tissues and 50 or more breast tissues, for example, 101 breast tissues, including both normal and cancerous tissues, are used. Among these tissues, a portion, for example, 169 (98 lung and 71 breast) are frozen and the remainder, for example, 30 (breast) are paraffin-fixed. These tumor tissues are classified upon resection, and basic (anonymous) data about each tumor is kept in the tumor bank database. The tissues were resected, sent to the Pathology Department for pathological examination, and then sent to the tumor bank with the initial pathology report. The tissue was quickly frozen and stored at −80° C. The tissue procurement, storage, and documentation of clinical specimens are very well documented, in accordance with guidelines for human subject research. Thirty of the 101 breast tumor tissues are formalin-fixed, paraffin-embedded tissues. Slides were made from the fixed tissue block and stored at room temperature. Each tumor sample was examined by a pathologist to confirm the clinical diagnosis. Basic (anonymous) data about each tumor, such as the information on the gender, age, and ethnic background of the patient as well as diagnosis are available to us for data analysis.
Initially, DNA methylation profiles are generated for these samples for a list of 141 genes, selected fully based on their biological functions (see Appendix I). If both DNA and RNA samples are available for a subject in the study, both DNA methylation and gene expression is measured, including allele-specific expression, using a sensitive RNA profiling method (Fan, et al., Genome Res. 14: 878-885 (2003), Yeakley, et al., Nat Biotechnol. 20(4): 353-8 (2002)). Gene-specific as well as allele-specific probes are designed to measure expression levels of specific transcripts and their isoforms. Cross-referencing gene expression results to DNA methylation data confirms not only the gene silencing caused by DNA methylation, but also helps interpret the association study results. Once specific methylation patterns are derived from this preliminary study, they are validated in (larger) independent sample sets.
Finally, comparing results obtained from human and animal studies sheds light on the underlying molecular mechanisms of tumorigenesis. For example, there is a well-characterized rat mammary tumor model involving mammary glands from virgin and parlous animals exposed to MNU (Sivaraman, et al., Carcinogenesis 19(9): 1573-81 (1998)). It provides an excellent biological model for studying molecular events in human breast cancer, especially those occurred in early cancer developments leading to mammary tumorigenesis (Russo, et al., Br J Cancer 64(3): 481-4 (1991)).
Bioinformatics, Array Information Management, and Statistical Data Analysis
Software development focuses on algorithms and software tools to process and analyze the large amount of methylation assay data as efficiently as possible. A database developer/administrator organizes, track and maintain all the methylation site information, primer design, sample information, the day-to-day experimental data, as well as design and implement web browser interfaces to provide search, query and report functions.
To develop a robust and high-throughput technology for simultaneous measurement of methylation at many specific sites in many samples, a highly efficient analysis and data export process is needed. Algorithms for analyzing data to determine methylation status automatically are developed. Again, this is based on experience with analysis of SNP genotyping data. There are some important differences in how the data is analyzed, such as the requirement for more quantitative analysis and output as described above. Experiments is designed and analysis procedures developed to determine the quantitative limits of our system, such as the limit of detection of methylated DNA in a mixed sample, and linearity of signal as a function of amount of methylated DNA.
Once tools are developed to extract information about methylation status from raw data, and to determine the significance of the measurements and assign confidence indices, the focus is on analyses of patterns of methylation and their correlations with different phenotypes. For example, analyses are carried out to detect and verify any correlations between specific methylation patterns and particular cancer types. Techniques (methods) to perform this type of analysis are the subject of intensive research in the microarray field. Many powerful algorithms/tools have been developed, such as supervised or unsupervised hierarchical clustering analysis (Dhanasekaran, et al., Nature 412(6849): 822-6 (2001), Eisen, et al., Proc Natl Acad Sci USA. 1998. 95(25): 14863-8 (1991), Khan, et al., Nat Med. 7(6): 673-9 (2001)), K-means clustering, bootstrapping or jackknife (Kerr, M. K. et al. Proc Natl Acad Sci USA. 98(16): 8961-5 (2001)), principal component analysis, etc. However, each of these methods has its own advantages and limitations; no clear advantage exists for any given algorithm in application to our study. Therefore, multiple algorithms are tested and the most suitable ones are selected to carry out the analyses.
The technology is upscaled to meet commercial requirements by implementing the entire process, including sample preparation, bisulfite treatment, genotyping-based assay and PCR amplification on a robotic platform; increasing the level of multiplexing to at least 96-plex, and as high as 1,500-plex; and reducing the amount of genomic DNA required such that, on average, <1 ng of genomic DNA is required per methylation site analyzed.
The assay described herein allows measurement of the methylation status in at least 10, 100, or 1,000 human genes' 5′-regulatory regions, and validate the sensitivity and specificity of the assays at low or high multiplexing levels. The assay further allows for a systematic search for specific methylation patterns in different cancer types and cancer stages.
Overall, this Example describes a system for methylation detection by leveraging various technologies for high-throughput array-based assays and SNP genotyping, and to validate the technology in real-world applications. The technology is highly scalable, both in terms of the number of assays carried out on a single sample, and the number of samples that can be processed in parallel. Furthermore, it can be used has the potential for broad application in many areas of cancer and fundamental biomedical research. The assays and assay protocols, and the specific methylation patterns (in various cancers) to be developed in this study can generally be useful to the research community.
EXAMPLE IV
DNA Methylation Profiling in Lung Cancer
This Example demonstrates the identification of adenocarcinoma markers by virtue of differential methylation patterns and use of the identified markers to distinguish adenocarcinoma from normal tissue with high specificity and sensitivity.
As described in detail above, the SNP genotyping system was adapted for DNA methylation detection based on “SNP” genotyping of bisulfite-converted genomic DNA. In this assay, non-methylated cytosines (C) are converted to uracil (U) when treated with bisulfite, while methylated cytosines remain unchanged. Hybridization behavior of uracil is similar to that of thymine (T). The detection of the methylation status of a particular cytosine can thus be carried out using a genotyping assay for a C/T polymorphism.
Assay Probe Design
A 1.5 kb sequence from the 5′-regulatory region (in some cases, including the first exon) is extracted for each target gene based on human genome public databases NCBI RefSeq build 33 released on Jun. 6, 2003. CpG islands of interest from this 1.5 kb region were selected and “converted by bisulfite” computationally. As described in Example 1, an automated SNP genotyping assay design program was adapted for methylation application. For each CpG locus, four probes were designed: two allele-specific oligos and two locus-specific oligos, each corresponding to the methylated or unmethylated state of the CpG site respectively (FIG. 20). If other CpG loci were present in the close vicinity of the chosen CpG site, an assumption is made that it has the same methylation status as the site of interest. While there are many CpG sites within each CpG island, only those for which robust assays could be designed were used. Assays were designed for 389 CpG sites from 141 genes: 114 genes with three CpG assays, 20 genes with two and 7 genes with only one assay. The designed site sequence information is included in Table 1.
DNA Samples for Methylation Analysis
Lung tissue specimens were collected from subjects with lung cancer. Under an Institutional Review Board approved protocol, the samples of lung tissue classified as cancerous and samples adjacent to the cancerous tissue but classified as normal were utilized in this study. After pathological classification upon resection, the tissues were frozen and stored at −80° C. Forty-six samples were obtained from Philipps-University of Marburg, Germany, and 25 samples were from The Pennsylvania State University College of Medicine Tumor Bank. Specifically, a total of 38 lung cancer and 33 normal tissues were used, including 22 matched pairs (Table 4). The cancer samples included 14 squamous cell carcinoma and 24 adenocarcinoma cancer tissues. The samples were pulverized under liquid nitrogen. DNA was extracted from the tissue powder by QIAamp® DNA Mini Kit (Qiagen Inc. Valencia, Calif.) according to manufacturer's instruction. The DNA was eluted from the column with dH2O, and stored at −80° C. until use.
Plasmid DNA Controls.
Plasmids pUC19, pACYC184 and phage phiX174 served as control DNAs as described above in Example II.
Bisulfite Conversion of DNA and Methylation Assay
The EZ DNA Methylation KitTM (Zymo Research, Orange Calif.) was used for bisulfite conversion of DNA samples, according to manufacturer's recommendations. One and a half microgram of genomic DNA was used for each conversion. Bisulfite-converted genomic DNA from one conversion was then used for up to 8 array experiments. After bisulfite conversion of genomic DNA, the remaining assay steps were identical to the GoldenGateTM assay (Fan et al., Cold Spring Harbor Symposia on Quantitative Biology 68: 69-78 (2003)), using manufacturer's reagents and conditions (BeadLab User Manual, Illumina).
Briefly, bisulfite-converted, biotinylated genomic DNA was immobilized on paramagnetic beads and washed. Pooled query oligos were annealed to the gDNA under a controlled hybridization program, and then washed to remove excess or mishybridized oligos. Hybridized oligos were then extended and ligated to generate amplifiable templates. A PCR reaction was performed with fluorescently labeled universal PCR primers. Single stranded PCR products were prepared by denaturation, then hybridized to a Sentrix® Array Matrix (Fan et al., Cold Spring Harbor Symposia on Quantitative Biology 68: 69-78 (2003)). The array hybridization was conducted under a temperature gradient program, and arrays were imaged using a BeadArray Reader 1000 scanner (Barker et al. Proc. SPIE 4966: 1-11 (2003)). Image processing and intensity data extraction software were as describe previously (Galinsky, Bioinformatics 19, 1832-6 (2003)).
Microarrays were assembled by loading pools of glass beads (3 μm in diameter) derivatized with oligos onto the etched ends of fiber-optic bundles (Barker et al., supra, 2003). About 50,000 optical fibers are hexagonally packed to form an approximately 1.4 mm diameter bundle. The fiber optic bundles are assembled into a 96-array matrix, Sentrix® Array Matrix, which matches the dimensions of standard microtiter plates. This arrangement allows simultaneous processing of 96 samples using standard robotics (Fan et al. supra, 2003). Because the beads are positioned randomly, a decoding process is carried out to determine the location and identity of each bead in every array location (Gunderson, Genome Res 14, 870-7 (2004)). Decoding is an automated part of array manufacture.
Methylation Data Analysis
Each methylation data point is represented by fluorescent signals from the M (methylated) and U (unmethylated) alleles. M and U signals were initially computed and normalized by requiring each sample to have the same average signal in each of the two fluorescent channels. Background intensity computed from a set of negative controls was subtracted from each of the analytical data point prior to normalization. The ratio of fluorescent signals from the two alleles




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The β value reflects the methylation status of each CpG locus, ranging from 0 in the cases of completely unmethylated sites to 1 in completely methylated sites. A constant offset of 100 was added to the denominator of the formula, as a compensation for any “negative signals” which may arise from global background subtraction (i.e. over-subtraction). Hierarchical clustering was performed using average linkage method and Pierson's correlation coefficient (r) as a similarity measure. K-nearest neighbor class prediction was done using 1−r for distance calculation and k=1 (nearest neighbor).

To choose optimal number of neighbor k, classification of the training set was performed first with different values of k. Leave-one-out cross validation gave misclassification rate of 0.03 (one out of 32 samples) using k=1. This result did not improve with a larger k.
According to the above-described protocols, for each CpG locus, four probes were designed: two allele-specific oligos (ASOs) and two locus-specific oligos (LSOs), each corresponding to the methylated or unmethylated state of the CpG site respectively (FIG. 1). Each ASO consists of a 3′ portion that hybridizes to the bisulfite-converted genomic DNA, with the 3′ base complementary to either the “C” or “T” allele of the targeted CpG locus, and a 5′ portion that incorporates a universal PCR primer sequence (P1 or P2, each associated with an allele respectively). The LSOs consists of three parts: At the 5′ end is a CpG locus-specific sequence; in the middle is an address sequence, complementary to a corresponding capture sequence on the array; and at the 3′ end is a universal PCR priming site (P3). The flexibility in primer design (the gap size between the ASO and LSO oligos varies from 1 base to 20 bases) is particularly important for methylation studies, since it can be implemented in a way that will avoid difficult sequences or ambiguous bases in the CpG islands of interest. In this study, assay oligos were designed for 389 CpG sites from the 5′-regulatory region of 141 genes.
Typically, 144-160 CpG sites were assayed together. Pooled assay oligos were first annealed to bisulfite-converted genomic DNA. An allele-specific primer extension step was then carried out; ASOs got extended if their 3′ base is complementary to their cognate CpG site in the gDNA template. Allele-specific extension was followed by ligation of the extended ASOs to their corresponding LSOs, to create PCR templates. Requiring the joining of two fragments to create a PCR template provides an additional level of genomic specificity. Any incorrectly hybridized ASOs and LSOs are unlikely to be adjacent, and therefore should not be able to ligate.
The ligated products were then amplified by PCR using common primers P1, P2, and P3, and hybridized to a microarray bearing the complementary address sequences. P1 and P2 are fluorescently labeled, each with a different dye, and associated with the “T” (unmethylated) allele or the “C” (methylated) allele respectively. As described in this example, methylation status of an interrogated locus is determined by quantifying β, which is defined as the ratio of the fluorescent signal from the methylated allele to the sum of the fluorescent signals of both methylated and unmethylated alleles.
Development of reliable internal controls is a prerequisite for development of a robust methylation detection method. Plasmids pUC19, pACYC184 and phagephiX174 as internal control DNAs. These DNAs (unmethylated, methylated or mixed at a 1:1 ratio) were spiked into 200 ng human genomic DNA at a 1:1 molar ratio (at approximately 2-4 pg plasmid DNA/1 μg gDNA, depending on the plasmid size), and were used in every methylation experiment to monitor both bisulfite conversion efficiency and accuracy of methylation detection. As shown in FIG. 2, unmethylated, semi-methylated and fully-methylated plasmid loci can be easily distinguished by the assay.
“Methylated” and “fully unmethylated” genomic templates were developed to calibrate quantitative measurements of methylation. The fully unmethylated templates were generated by genome-wide amplification of human genomic DNA using REPLI-g® DNA amplification kit (Molecular Staging). After this amplification, the endogenous DNA methylation is diluted at least 100 to 1000-fold, effectively rendering the amplified genomic DNA “unmethylated”. Methylated templates were generated by in vitro methylation using Sss I (CpG-methylase) enzyme (New England BioLabs). Among the 389 CpG sites targeted, 70 of them can only be methylated to less than 50% completion. Some of these sites may be less accessible to methylase because of the DNA secondary structure; some may result from base substitution at the CpG sites in the DNA tested, i.e. these sites become “methylation-resistant” (it is well known that CpG sites are mutation hot spots).
The above two reference templates were used for assay development and 20 CpG assays were disqualified due to similar β values for both unmethylated and methylated templates (a threshold of Δβ>0.1 was applied). Therefore, the data analyses is based on the remaining 369 functional CpG assays.
As described above, the use of bisulfite conversion of DNA for methylation detection is based on the different sensitivity of cytosine and 5-methylcytosine to deamination by bisulfite. Under acidic conditions, cytosine undergoes conversion to uracil, while methylated cytosine remains unreactive. Thus, an effective bisulfite conversion protocol is a necessary prerequisite for a robust methylation profiling assay. Incomplete conversion of cytosine to uracil by bisulfite can result in appearance of false-positive signals for 5-methylcytosine, and reduce the overall quality of the assay data. In order to monitor the effectiveness of bisulfite treatment, a set of oligos (a standard set of SNP genotyping probes) designed for unconverted genomic DNA sequences was used in the assay. If bisulfite conversion is successful, the signal from assays designed against the unconverted DNA (i.e. the SNP set) will disappear, and only signals from assays designed against the converted DNA are present. Incomplete conversion results in low and inconsistent signals across all loci targeted to both unconverted and converted DNA.
Each gDNA sample was assayed in duplicate (Table 4). According to the methods described herein, highly reproducible DNA methylation profiles within replicates were obtained with 200 ng genomic DNA (average r2 was 0.97) (FIG. 21, left and center panels). As a result, differential methylation between different samples was readily detected (FIG. 3, right panel).
As described above, DNA methylation is involved in transcriptional inactivation of genes on one of the two X-chromosomes in female somatic cells (i.e. X-chromosome silencing), compensating the dosage of functional X-linked genes between male and female. The methylation status of six X-linked housekeeping genes—BCAP31, GPC3, FMR, ELK1, G6PD and GLA, was analyzed to test if the assay could detect the methylation difference between males and females. As shown in FIGS. 16 and 22, methylation levels of these genes correlated well with the gender of the sample source.
There are four major histologic types of lung carcinomas: squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma. The methylation status of 369 CpG sites located in the 5′-regulatory regions of 141 genes in 14 squamous cell carcinoma, 24 adenocarcinoma and 33 normal tissue samples was measured. The genes were selected based on their biological relevance. These include tumor suppressor genes and oncogenes, genes that are indirectly involved in cancer development, for example, DNA repair genes; metastasis-inhibitor genes, genes regulated by various signaling pathways, and/or responsible for altered cell growth, differentiation and apoptosis; genes considered to be targets for oncogenic transformation; imprinting genes; previously reported differentially methylated genes (Esteller, Oncogene 21, 5427-40 (2002), Tsou et al., Oncogene 21, 5450-61 (2002); Adorjan et al., Nucleic Acids Res 30, e21 (2002)).
A data matrix of β values was used to identify CpG loci that show differential methylation in cancer. Using a Mann-Whitney test, 21 normal samples were first compared to 11 adenocarcinoma samples referred thereafter the “training set”. With a p-value cutoff of 1/369, 79 differentially methylated loci were identified. By choice of this p-value threshold, only one false positive marker was expected and this prediction was verified by randomly permuting columns in the data matrix and repeating Mann-Whitney test. Since the permutation process is independent of disease status, it provides an accurate estimate of actual false positive rate. On average, 0.63 differentially methylated markers were observed, indicating a lower than expected actual false positive rate, which is not surprising since not all CpG loci can be considered as independent variables. To select markers which have the largest difference between cancer and normal tissues (assuming this would cause the largest biological changes), an additional filter requiring |Δβ|>0.2 was required. Ultimately, a list of 14 differentially methylated markers was obtained: ADCYAP1—2, CDH13—3, GDF10—2, GDF10—3, HOXA5—2, MAGEA1—3, RUNX3—1, SCGB3A1—3, SERPINB5—1, SFN—2, SFTPA1—2, TERT—1, TERT—2, and TNF—2 (FIG. 23A).
The same differential methylation analysis was subsequently performed for the same 21 normal samples and 14 squamous cell carcinoma samples. When the same p-value threshold and |Δβ|>0.2 filtering was used, the following list of 21 differentially methylated markers was obtained: ADCYAP1—1, ADCYAP1—2, ADCYAP1—3, BCR—2, CALCA—1, GDF10—3, HOXA5—1, HOXA5—2, HOXA5—3, MAGEA1—1, MAGEA1—2, PGR—2, PRKCDBP—3, SCGB3A1—3, SERPINB5—1, SFN—2, SFTPC—3, TERT_I, TERT—2, TERT—3, TNF—2 (FIG. 23B).
In addition, methylation profiles of the cancer and normal samples were clustered using the selected markers. Normal samples formed a tight cluster (i.e. small cluster distances) in both adenocarcinoma and squamous cell carcinoma groups (FIGS. 24A & 24B). Two early stage cancer samples, G12029 (FIG. 24A) and G12003 (FIG. 24B), were co-clustered with normal samples. Quite large cluster distances existed among cancer samples which may reflect different disease stages.
An independent set of test samples was used to assess the specificity and sensitivity of the 14 adenocarcinoma markers. This test set contained 11 normal and 14 adenocarcinoma samples. We classified these samples using a k-nearest neighbor algorithm. The misclassification rate in the test set was 0.04, with one cancer sample (D12190) misclassified as normal. The 14 markers also were evaluated by measuring their p-values from Mann-Whitney test with the test sample set. The p-values ranged between 0.00017 and 0.029. Finally, the classification accuracy derived from these 14 specific markers and randomly selected 1,000 sets of 14 CpG loci was measured. Only 2.2% of the 1,000 random sets produced same or lower misclassification rates as compared to the 14 markers, with an average misclassification rate being 28%. The above-described analyses indicate that the differential methylation pattern for the identified markers is preserved in the two completely unrelated sample sets and the identified markers can be used to distinguish adenocarcinoma from normal tissue with high specificity and sensitivity.
These results are further demonstrated by cluster analysis of the methylation patterns of the 14 identified markers in the test set samples. As shown in FIG. 24C, the cancer and normal samples are clearly separated. The array data for some of the markers which show increased methylation in cancer samples was plotted to illustrate their performance in both training set and test set of samples (FIG. 25).
Throughout this application various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
The term “comprising” is intended herein to be open-ended, including not only the cited elements, but further encompassing any additional elements.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific examples and studies detailed above are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
















“GenBank®”

PROBE SEQUENCE (ASO1)
PROBE SEQUENCE (LSO1)
PROBE SEQUENCE (ASO2)


TARGET CPG SITE
ACCESSION #
TARGET SEQUENCE
SEQ ID NOS: 378-754
SEQ ID NOS: 755-1131
SEQ ID NOS: 1132-1508







GI29736559_ESR2_1R
NT_026437
GGATGCTCCTCAGCTCTGGGGACGCGGTGC
GCCCTCACACTTCTACACCG
GTCCCCAAAACTAAAAAACATC
AACACCCTCACACTTCTACACCA


(SEQ ID 1)

AGAAGTGTGAGGGCGCC





 


GI29736559_ESR2_2R
NT_026437
CTTCCGACTTTGTCACACACCTGCGCCGCC
CCGACTTTATCACACACCTACG
CGCCAAACTAAAATCGAACC
CTTCCAACTTTATCACACACCTACA


(SEQ ID 2)

AGACTGGGGTCGGGCCCC





 


GI29736559_ESR1_3R
NT_026437
CCCGGCTTCCAGGCAGTAATGGGCGGGTCC
CGACTTCCAAACAATAATAAAG
ATCCCTACGCGAAAACGTAAC
CCCAACTTCCAAACAATAATAAACA


(SEQ ID 3)

CTGCGCGGGAGCGTGGCGGGCG





 


GI29791372_TGFBR2_1R
NT_022517
AATCTGAAGAAAGCTGAGGGGAGGCGGCAG
CCCTAATAAATCAAAACATCCG
CTCCCCTCAACTTTCTTCAAAT
TCCCTAATAAATCAAAACATCTACCA


(SEQ ID 4)

ATGTTCTGATCTACTAGGGA





 


GI2979l372_TGFBR2_2R
NT_022517
AACTCCTGAGTGGTGTGGGAGGGCGGTGAG
ACTTTCAACTACCCCTCACCG
CCTCCCACACCACTCAAAAATT
AACTTTCAACTACCCCTCACCA


(SEQ ID 5)

GGGCAGCTGAAAGTC





 


GI29791372_TGFBR2_3R
NT_022517
TGGTCTAGGAAACGATGATTGGCAGCTACG
GTCCAACCCCTAACTCTCTCG
AACTACCAATCATATTTCCTAAACCA
ACATCCAACCCCTAACTCTCTCA


(SEQ ID 6)

AGAGAGCTAGGGGCTGGACGT





 


GI29791384_TP73_1R
NT_004321
CCGAGGAGCCCAGCGCTACTGGCGGCGGCC
ACCCGAATCTCTCCTAACCG
CGCCACTAACGCTAAACTCCT
TAACACCCAAATCTCTCCTAACCA


(SEQ ID 7)

AGGAGAGACCCGGGTGTCA





 


GI29791384_TP73_2R
NT_004321
GACAGCAGGGAGTCCGGGGGAAACGCAGGC
ACTCTATACCCGACGCCTACG
TTCCCCCGAACTCCCTACTAT
CCAACCTATACCCAACACCTACA


(SEQ ID 8)

GTCGGGCACAGAGTCGG





 


GI29791384_TP73_3R
NT_004321
GGCCCTGGCGCCGGACCTGCTTCGGCCCTG
CTAACGCCGAACCTACTTCG
CCCTACGTAAACGACCTCGC
AACCCTAACACCAAACCTACTTCA


(SEQ ID 9)

CGTGGGCGGCCTCGCCGG





 


GI29791621_PTGS2_1R
NT_004487
TCGGAAGCGCTCGGGCAAAGACTGCGAAGA
CGCCAAATATCTTTTCTTCTTCG
AATCTTTACCCGAACGCTTCC
CCACCAAATATCTTTTCTTCTTCA


(SEQ ID 10)

AGAAAAGACATCTGGCGG





 


GI29791621_PTGS2_3R
NT_004487
AGTCTGTCCCGACGTGACTTCCTCGTACCC
TATCCCGACGTAACTTCCTCG
CCCTCTAAAAACGTACAAACCAA
AATCTATCCCAACATAACTTCCTCA


(SEQ ID 11)

TCTAAAGACGTACAGACCAGA





 


GI29793179_DAPK1_1R
NT_023935
GCGCCGGCCTGGCAGGGCAGCTCGGAGGTG
CCGACCTAACAAAACAACTCG
AAATAAATAAACCGCGCCG
ACACCAACCTAACAAAACAACTCA


(SEQ ID 12)

GGTGGGCCGCGCCGCCAGC





 


GI29793179_DAPK1_3R
NT_023935
AAACCTTCTTGCCTTCAAGCCTCGGCTCCA
AACCTTCTTACCTTCAAACCTCG
CTCCAACACCAATCCGACA
AAACCTTCTTACCTTCAAACCTCA


(SEQ ID 13)

ACACCAGTCCGGCAGA





 


GI29794150_CASP8_1R
NT_005403
GGAAGTGAGAAACAACTGTGTGATAAACGG
AAATACTCCCATTCTCCACCG
TTATCACACACTTATTTCTCACTTCC
AAAAATACTCCCATTCTCCACCA


(SEQ ID 14)

TGGAGAATGGGAGCACTCTC





 


GI29794150_CASP8_2R
NT_005403
GCGGGAGGAGACGAGGAGGGCGTTCCCTGG
ACTACCACTCCCCAAAAAACG
CCCCTCGTCTCCTCCC
CACTACCACTCCCCAAAAAACA


(SEQ ID 15)

GGAGTGGCAGTG





 


GI029794150_STAT1_1R
NT_005403
AAGCCGGCGGAAATACCCCAGCGCGTGGGC
CGACGAAAATACCCCAACG
GTAAACGAAACAACGACCCG
AAACCAACAAAAATACCCCAACA


(SEQ ID 16)

GGAGCAGCGGCCCGCAGA





 


GI29794150_STAT1_2R
NT_005403
GGCGGTGGCGCCCACGGAACAGCCGCGTCT
GCGCTCAACCAATTAAACG
GACTATTCCGTAAACGCCACC
ACTCCACACTCAACCAATTAAACA


(SEQ ID 17)

AATTGGCTGAGCGCGGAGC





 


GI29794150_STAT1_3R
NT_005403
CCAGTTCCCAGCCTGGCAACACGGGACTGG
AATTCCCAACGTAACAACACG
AACTAAACTACAACTCACCCAACC
CCAATTCCCAACATAACAACACA


(SEQ ID 18)

GCTGCAGCTCACCCAGCCG





 


GI29794559_CDKN2A_1R
NT_008413
GACTCACCCCTCCTTTCTGCCGCTCCTTCC
ACTCACCCCTCCTTTCTACCG
TCCTTCCTTTCCTTACCCTACTTT
AACTCAGCCCTCCTTTCTACCA


(SEQ ID 19)

TTTCCTTGCCCTGCTTT





 


GI29794559_CDKN2A_2R
NT_008413
GCCCGCGTCCGAGTTCCTGGACGAGAGCCG
GCGTCCGAATTCCTAAACG
AAACCGAACCTCGCTTAAACC
ACCCACATCCAAATTCCTAAACA


(SEQ ID 20)

AGCCTCGCTTAGACCGC





 


GI29794559_CDKN2A_3R
NT_008413
TCCAGCCCGCGAGGTTTAGGACGGATCCAG
AACCCGCGAAAMAAAACG
ATCCAAACAAACCGCAAACT
TCCAACCCACAAAATTTAAAACA


(SEQ ID 21)

GCAGACCGCAGGCTCC





 


GI29794559_CDKN2B_1R
NT_008413
AGCAGTGCAGCCAGCATTCCTGGCGGCTCC
CAATACAACCAACATTCCTAACG
CTCCCTAACCCAATCTCTAACG
AACAATACAACCAACATTCCTAACA


(SEQ ID 22)

CTGGCCCAGTCTCTGGCGCA





 


GI29794559_CDKN2B_2R
NT_008413
GGCTGAAGGMCAGAAATCCTCTGCTCCGCC
CAACTCCTAATCCCCAATAAACG
AACAAAAAATTTCTATTCCTTCAACC
CAACTCCTAATCCCCAATAAACA


(SEQ ID 23)

TACTGGGGATTAGGAGCTG





 


GI29794559_CDKN2B_3R
NT_008413
AGCATCTTTGGGCAGGCTTCCCCGCCCTCG
CATCTTTAAACAAACTTCCCCG
CCTCGTAACGCGTCGAC
AACATCAAAAACAAACTTCCCCA


(SEQ ID 24)

TGACGCGTCGGCCCGGGC





 


GI29796774_MYC_1R
NT_008046
AGAGCAGGCAACCTCCCTCTCGCCCTAGCC
AAACAAACAACCTCCCTCTCG
CCTAACCCAACTCTAAAACAAACAA
AAAACAAACAACCTCCCTCTCA


(SEQ ID 25)

CAGCTCTGGAACAGGCAG





 


GI29796774_MYC_2R
NT_008046
ACCMGATTTCTCCCAAACCCGGCAGCCCGA
CTTTAATITCTCCCAAACCCG
CAACCCGAAACTATTACAAACCG
ACCAAAATTTCTCCCAAACCCA


(SEQ ID 26)

GACTGTTGCAAACCGG





 


GI29796774_MYC_3R
NT_008046
CAAGGGTCTCTGCTGACTCCCCCGGCTCGG
AAAAATCTCTACTAACTCCCCCG
CTCGATCCACAAACTCTCCACT
CAAAAATCTCTACTAACTCCCCCA


(SEQ ID 27)

TCCACAAGCTCTCCACTT





 


GI29797939_APC_1R
NT_034772
CAGCAACACCTCTCACGCATGCGCATTGTA
AACAACACCTCTCACGCATACG
ATTATAATCTTCCCACCTCCCAC
CAACAACACCTCTCACACATACA


(SEQ ID 28)

GTCTTCCCACCTCCCAC





 


GI29797939_APC_2R
NT_034772
GACAGAACAGCGAAGCAGTGCCCGGCAAGC
CAAAACAACGAAACAATACCCG
CAAACGAAACGCAACACC
AACAAAACAACAAAACAATACCCA


(SEQ ID 29)

GGAGCGCAGCACCCATTG





 


GI29797939_APC_3R
NT_034772
CGCCGGAAGCCTAGCCGCTGCTCGGGGGGG
CGAAAACCTAACCGCTACTCG
AAAAAACCTACGAACTCAAACCC
CACCAAAAACCTAACCACTACTCA


(SEQ ID 30)

ACCTGCGGGCTCAGGCCCG





 


GI29798364_HIC1_1R
NT_010718
TGCCCAGGCCGCAGGGCTGATGCCCCCGCT
CCCCTTCCCTCAACTAAACG
AAACATCAACCCTACGACCTAAA
CTTCCCCTTCCCTCAACTAAACA


(SEQ ID 31)

CAGCTGAGGGAAGGGGAAG





 


GI29798364_HIC1_2R
NT_010718
CTCCTGCTCCTTCTCCTGGTCCGGGCGGGC
CTCCTACTCCTTCTCCTAATCCG
ACGAACCGACCTAAACTCCC
CTCCTACTCCTTCTCCTAATCCA


(SEQ ID 32)

CGGCCTGGGCTCCCACT





 


GI29798364_HIC1_3R
NT_010718
AACTGGGGCAACTTCTCCCGAGGCGGGAGG
TAAAACAACTTCTCCCGAAACG
AAAACGCTAATTCCTCGACTCC
AACTAAAACAACTTCTCCCAAAACA


(SEQ ID 33)

CGCTGGTTCCTCGGCTCCC





 


GI29798364_TP53_1R
NT_010718
GTTAGTATCTACGGCACCAGGTCGGCGAGA
TATCTACGACACCAAATCGACG
AAATCCTAACTCTACACCCTCCTC
ATTAATATCTACAACACCAAATCAACA


(SEQ ID 34)

ATCCTGACTCTGCACCCTCCTC





 


GI29798364_TP53_2R
NT_010718
CTCCATTTCCMGCTTCCTCCGGCAGGCGGA
CCATTTCCMACTTCCTCCG
CAAACGAATTACTTACCCTTACTTATCA
CTCCATTTCCTTTACTTCCTCCA


(SEQ ID 35)

TTACTTGCCC1TACTIGTCA





 


GI29798364_TP53_3R
NT_010718
CCCCGCCGCCTGCAGAGGGCGCAGCAGGTC
CGCCGCCTACAAAAAACG
AACAAATCTTACACCTCTTCTACATCTC
CCCCACCACCTACAAAAAACA


(SEQ ID 36)

TTGCACCTCTTCTGCATCTC





 


GI29798595_BRCA1_1R
NT_010755
TTTCGTATTCTGAGAGGCTGCTGCTTAGCG 
CACGAAAACCAAAAAACTACCG
TAAACAACAACCTCTCAAAATACG
CCACAAAAACCAAAAAACTACCA


(SEQ ID 37)

GTAGCCCCTTGGTTTCCGTGG





 


GI29798595_BRCA1_2R
NT_010755
TGGGTGGCCAATCCAGAGCCCCGAGAGACG
AATAACCAATCCAAAACCCCG
AAAACGCTTAACTCTTTCTATCCC
TAAATAACCAATCCAAAACCCCA


(SEQ ID 38)

CTIGGCTCTTTCTGTCCCTCC





 


GI29798595_BRCA1_3R
NT_010755
CTCAGGTAGAATTCTTCCTCTTCCGTCTCT
CAAATAAAATTCTTCCTCTTCCG
CTCMCCUTTACGTCATCCG
CTCAAATAAAATTCTTCCTCTTCCA


(SEQ ID 39)

TTCCTTTTACGTCATCCGGGG





 


GI29800185_PTEN_1R
NT_030059
AGACTCGAGTCAGTGACACTGCTCAACGCA
CTCGAATCAATAACACTACTCAACG
ACCCATCTCAACTTTCATCATCA
AAACTCAAATCAATAACACTACTCAACA


(SEQ ID 40)

CCCATCTCAGCTTTCATCATCA





 


GI29800185_PTEN_2R
NT_030059
AGGGTATTCCCCTTGCAGGGACCGTCCCTG
AAATATTCCCCTTACAAAAACCG
CCCTACATTTCCCTCTACACTAAA
AAAATATTCCCCTTACAAAAACCA


(SEQ ID 41)

CATTTTTCCTCTACACTGAG





 


GI29800185_PTEN_3R
NT_030059
AAGGGAGCCGGATGAGGTGATACACGCTGG 
CAACCTACTATTATATCGCCAACG
ATATCACCTCATCCGACTCCCTT
AAAACAACCTACTATTATATCACCAACA


(SEQ ID 42)

CGACACAATAGCAGGTTGCTCT





 


GI29800407_REL1 R
NT_033985
CGGCTCTGCGTAGGTGCGCGGACCCGGGCT
TACGTAAATACGCGAACCCG
ACTCCTAAATTCCATCCCCG
CAACTCTACATAAATACACAAACCCA


(SEQ ID 43)

CCTGGGTTCCATCCCCGC





 


GI29800407_RET_2R
NT_033985
GTCGGCCAGACCTGCATCCCGCGTAGCATC
CAAACCTACATCCCGCG
AACATCCCTACCCTCTCTATACAA
ATCAACCAAACCTACATCCCACA


(SEQ ID 44)

CCTGCCCTCTCTGTGCAGCG





 


GI29800407_RET_3R
NT_033985
CCGCGGAGAGCCTGGAGGCGGGGCGCCCTT
CGAAAAACCTAAAAACGAAACG
CCTTCTCTAAATCCGCGAAAT
CCACAAAAAACCTAAAAACAAAACA


(SEQ ID 45)

CTCTGAGTCCGCGGGGTCGC





 


GI29800594_TGF131_1R
NT_011109
CCCGTGCGCTTCCTGGGTGGGGCCGGGGGC
CGTACGCTTCCTAAATAAAACCG
AAACGACTTCAAAACCCCCTAC
CCCATACACTTCCTAAATAAAACCA


(SEQ ID 46)

GGCTTCAAAACCCCCTGCC





 


GI29800594_TGFB1_2R
NT_011109
CTCTTTCTCTGGTGACCCACACCGCCCGCA
TCTTTCTCTAATAACCCACACCG
CCGCAAAACCACAACGC
CTCTTTCTCTAATAACCCACACCA


(SEQ ID 47)

AAGCCACAGCGCATCTGG





 


GI29800594_TGFB1_3R
NT_011109
CCCAGCCTGACTCTCCTTCCGTTCTGGGTC
CCAACCTAACTCTCCTTCCG
TCTAAATCCCCCTCCTCTAATCG
CCCAACCTAACTCTCCTTCCA


(SEQ ID 48)

CCCCTCCTCTGGTCG





 


GI29802188_SNRPN_1R
NT_026446
TCTCTTGAATCTTGACAATCCCCGAACACT
TCTTAAATCTTAACAATCCCCG
ACACTACTCCCCGCTTTATTTATT
TCTCTTAAATCTTAACAATCCCCA


(SEQ ID 49)

GCTCCCCGCTTTATTTGTTTAG





 


GI29802188_SNRPN_2R
NT_026446
TCCCTGGGTGCCTCCTGCGCAAGCGCAGTT
CCTAAATACCTCCTACGCAAACG
AATTATCCTCCTACGCCGACCT
TCCCTAAATACCTCCTACACAAACA


(SEQ ID 50)

GTCCTCCTGCGCCGACCTCG





 


GI29802395_GLA_1R
NT_011651
GAAAGGAAGAGGGTGATTGGTTAGCGGAAC
ATCAATCACGTAATTTCGTTCCG
TAACCAATCACCCTCTTCCTTTC
CAATAATCAATCACATAAAACATTCCA


(SEQ ID 51)

GTTTTACGTGACTGATTATTG





 


GI29802395_GLA_2R
NT_011651
ACACACCAACCTCTAACGATACCGGGTAAT
ACACCAACCTCTAACGATACCG
ATAATTTTCCTCCITCTTCCCTC
ACACACCAACCTCTAACAATACCA


(SEQ ID 52)

TTTCCTCCTTCTTCCCTC





 


GI29802395_GLA_3R
NT_011651
AACTCAGGGCCGTGGTTTTCAAACGTTTTT
CTCAAAACCGTAATTTTCAAACG
TTTTCGCCTTACGATCACC
AACTCAAAACCATAATTTTCAAACA


(SEQ ID 53)

CGCCTTACGGTCACCCTTAG





 


GI29802670_AR_1R
NT_011669
TAGAGCTAGCCTCTCCTGCCCTCGCCCACG
AAAACTAACCTCTCCTACCCTCG
CCACGCTACGCCAACACTTAT
TAAAACTAACCTCTCCTACCCTCA


(SEQ ID 54)

CTGCGCCAGCACTAGTTTCT





 


GI29802670_AR_2R
NT_011669
TCCTAGAGCAAATGGCACAATGCCACGAGG
CCTAAAACAAATAACACAATACCACG
AACCCGATCTATCCCTATAACGA
TCCTAAAACAAATAACACAATACCACA


(SEQ ID 55)

CCCGATCTATCCCTATGACGGA





 


GI29802670_AR_3R
NT_011669
CCGGTGGGGGCGGGACCCGACTCGCAAACT
ATAAAAACGAAACCCGACTCG
AAACTATTACATTTACTCACCACCTCC
CCAATAAAAACAAAACCCAACTCA


(SEQ ID 56)

GTTGCATTTGCTCTCCACCTCC





 


GI29802882_MAGEA1_1R
NT_011726
TCCCGCCAGGAAACATCCGGGTGCCCGGAT
CAAATCAATAACGTCACATCCG
ACACCCGAATATTTCCTAACGTTT
CACAAATCAATAACATCACATCCA


(SEQ ID 57)

GTGACGCCACTGACTTGCG





 


GI29802882_MAGEA1_2R
NT_011726
CTCAACCCTGATGCCCATCCGCCCAGCCAT
CTCAACCCTAATACCCATCCG
CCAACCATTCCACCCTCA
CTCAACCCTAATACCCATCCA


(SEQ ID 58)

TCCACCCTCA





 


GI29802882_MAGEA1_3R
NT_011726
CCACTCCAATGCTCACTCCCGTGACCCAAC
CCACTCCAATACTCACTCCCG
AACCCAACCCCCTCTTCATTAT
CCACTCCAATACTCACTCCCA


(SEQ ID 59)

CCCCTCTTCATTGT





 


GI29804485_CTAG2_1R
NT_025965
GACGGACAGGGCAGGCAGGGTCCGGGACGA
CGAACAAAACAAACAAAATCCG
AACGATAACCGCACAATCC
AACAAACAAAACAAACAAAATCCA


(SEQ ID 60)

TGGCCGCACAGTCCCGGC





 


GI29804485_CTAG2_2R
NT_025965
GAGACCTTGGCTGGCGCGAGGCCACGCCCA
CTTAACTAACGCGAAACCACG
CCACCAAACATACAATTCCAACT
AAAACCTTAACTAACACAAAACCACA


(SEQ ID 61)

CCAGACATGCAGTTCCAGCT





 


GI29804485_CTAG2_3R
NT_025965
GGGCGACTAGGACAGGGACAGAACCCGTTG
GAATCTCACTCCTAAATTCAACG
ATTCTATCCCTATCCTAATCGCC
CCAAATCTCACTCCTAAATTCAACA


(SEQ ID 62)

AACCCAGGAGTGAGATCCGG





 


GI29804185_G6PD_1R
NT_025965
TCCCGCTCCGGAGAGAAGTCTGAGTCCGCC
GCGAACCTACAAAACCTAACG
ACTCAAACTTCTCTCCGAAACG
TCCACAAACCTACAAAACCTAACA


(SEQ ID 63)

AGGCTCTGCAGGCCCGCGGA





 


GI29804485_G6PD_2R
NT_025965
CGGAAGCTCGGTAATGATAAGCACGCCGGC
ACTCGATAATAATAAACACGCCG
CCACTTTACAAAACGTCACCG
CAAAAACTCAATAATAATAAACACACCA


(SEQ ID 64)

CACTTTGCAGGGCGTCACCGCC





 


GI29804485_G6PD_3R
NT_025965
CCCAATAGGGCCGGCTTGACCCGCGAACAG
CCAATAAAACCGACTTAACCCG
GAACAAACGAAAATTCCCGA
CCCAATAAAACCAACTTAACCCA


(SEQ ID 65)

GCGAGGGTTCCCGGGGG





 


GI29804485_SLC6A8_1R
NT_025965
GGGAAGCTGTAGCCCCCAGTGGGCAGCGGT
CTTCCTTAAACTCTCTCCACCG
TACCCACTAAAAACTACAACTTCCC
CCTTCCTTAAACTCTCTCCACCA


(SEQ ID 66)

GGAGAGAGCTCAAGGAAGG





 


GI29804485_SLC6A8_2R
NT_025965
GGTAGCAACCATCCTGCCTCCCGCTGGAGC
ATAACAACCATCCTACCTCCCG
TAAAACGACGTCTCCTCCCC
AATAACAACCATCCTACCTCCCA


(SEQ ID 67)

GGCGTCTCCTCCCCGG





 


GI29804485_SLC6A8_3R
NT_025965
CCGGCCGGCCGTGGGGGTGGGGCGATAGTG
GACCGACCGTAAAAATAAAACG
TAATAACATCACCCCGAAATCG
CCAACCAACCATAAAAATAAAACA


(SEQ ID 68)

ACATCACCCCGGAGTCGG





 


GI29805823_GSTP1_2R
NT_033903
CCTCTCCCCTGCCCTGTGAAGCGGGTGTGC
CCTCTCCCCTACCCTATAAAACG
ATATACAAACTCCGAAATCGCAA
CCTCTCCCCTACCCTATAAAACA


(SEQ ID 69)

AAGCTCCGGGATCGCAGCGG





 


GI29805823_GSTP1_3R
NT_033903
CGGCCAGCTGCGCGGCGACTCCGGGGACTC
AACTACGCGACGACTCCG
AAACTCCAAAACGCCCCTCTAC
CAACCAACTACACAACAACTCCA


(SEQ ID 70)

CAGGGCGCCCCTCTGCG





 


GI29807292_BCR_1 R
NT 011520
GCTGTGAGGTGTGAGGAACTTACCTGCGTC
GAAAAACACCTTCCATAAAAACG
AAATAAATTCCTCACACCTCACAAC
CAAAAAACACCTTCCATAAAAACA


(SEQ ID 71)

TCCATGGAAGGTGCCCTCCG





 


GI29807292_BCR_2R
NT_011520
CTCTGACACGACGACTGGGCAGTGCCGGTG
GCAATACCATAAACGTCACCG
CACTACCCAATCGTCGTATCAAA
ACCACAATACCATAAACATCACCA


(SEQ ID 72)

ACGCTTATGGCACTGCGGC





 


GI29807292_BCR_3R
NT_011520
GGGCTGCGAGTTGCACAGTCCAATTCGCTG
ACGAATTACACAATCCAATTCG
TATTATTAAAACCTCAATTTCCCAAA
AAACTACAAATTACACAATCCAATTCA


(SEQ ID 73)

TTGTTAGGGCCTCAGTTTCCCAAA





 


GI29807292_TIMP3_1R
NT_011520
AAGGAGCAGTGGAAAGGGGTGACGAGTTCC
AATTAATAACCAACCAAAAACTCG
CACCCCTTTCCACTACTCCTT
AATAATTAATAACCAACCAAAAACTCA


(SEQ ID 74)

TGGCTGGCCACCAATCATC





 


GI29807292_TIMP3_2R
NT_011520
AACTTGTGGCATTTCTAACAGGATGAAGCG
TCTCTTCCCTTCTCTTCCG
TTCATCCTATTAAAAATACCACAAATT
TCTCTTCCCTTCTCTTCCA


(SEQ ID 75)

GAAGAGAAAGGGAAAGAGA





 


GI29807454_CALCA_1R
NT_009237
GGGGCGAGGGTTCAAAACCAGGCCGGACTG
AATAAATTTCACCTCTCAATCCG
CCTAATTTTAAACCCTCGCCC
CATAATAAATTTCACCTCTCAATCCA


(SEQ ID 76)

AGAGGTGAAATTCACCATG





 


GI29807454_CALCA_2R
NT_009237
CAGGTTCTGGAAGCATGAGGGTGACGCAAC
AAAATCCTTTACCCCTAAATTACG
CACCCTCATACTTCCAAAACCTA
AAAAATCCTTTACCCCTAAATTACA


(SEQ ID 77)

CCAGGGGCAAAGGACCCCT





 


GI29807454_KAI1_1R
NT_009237
CAGGGCAGGGCAGGATTAGGAAGGCGCTGA
GCACCAACCTAAACTCAACG
CTTCCTAATCCTACCCTACCCTA
CCACACCAACCTAAACTCAACA


(SEQ ID 78)

GCCCAGGCTGGTGCGG





 


GI29807454_KAI1_2R
NT_009237
GATAGAGGAGAGACTCCGTAGCGGGGCGGG
CCCAACTAAAACTAAAACCCCG
CCCGCTACGAAATCTCTCCT
ACCCAACTAAAACTAAAACCCCA


(SEQ ID 79)

GCCTCAGCTCCAGCTGGGC





 


GI29807454_KAI1_3R
NT_009237
GCCGCCTCCTGATAGAGGCCCCGACTTAGG
GCCTCCTAATAAAAACCCCG
CTTAAAACACAAACCGCTCCC
ACCACCTCCTAATAAAAACCCCA


(SEQ ID 80)

ACACAAACCGCTCCCAC





 


GI29807454_MYOD1_1R
NT_009237
TTGCCTCTCTCCAAATCTCTCACGACCTGA
TACCTCTCTCCAAATCTCTCACG
CCTAATTTCTACAACCGCTCTACC
TTACCTCTCTCCAAATCTCTCACA


(SEQ ID 81)

TTTCTACAGCCGCTCTACCC





 


GI29807454_MYOD1_2R
NT_009237
CTTTCTGCGTGTCTCTCAGCCTCTTTCGGT
TACGTATCTCTCAACCTCTTTCG
TCCCTCTTTCACGATCTCACTC
CTTTCTACATATCTCTCAACCTCTTTCA


(SEQ ID 82)

CCCTCTTTCACGGTCTCACTCC





 


GI29807454_MYOD1_3R
NT_009237
GGAATATCAGAGCCTCTACGACCCGTTTGT
AATATCAAAACCTCTACGACCCG
TTATCTCGAACTCGCCCACTT
AAAATATCAAAACCTCTACAACCCA


(SEQ ID 83)

CTCGGGCTCGCCCACTTCAA





 


GI29808625_CDH1_2R
NT_010498
CGAACCCAGTGGAATCAGAACCGTGCAGGT
GAACCCAATAAAATCAAAACCG
ACAAATCCCATAACCCACCTAAA
CAAACCCAATAAAATCAAAACCA


(SEQ ID 84)

CCCATAACCCACCTAGA





 


GI29808625_CDH1_3R
NT_010498
ACCCTCAGCCAATCAGCGGTACGGGGGGCG
CCTCAACCAATCAACGATACG
AAAACGATACCTCCGAAACTCAC
ACCCTCAACCAATCAACAATACA


(SEQ ID 85)

GTGCCTCCGGGGCTCACC





 


GI29823164_TYMS_1R
NT_010859
CTGTGCTGCTGGCTTAGAGAAGGCGCGGTC
TTAAAAACCGTCTAATCGACCG
GCCTTCTCTAAACCAACAACACA
CCTTTAAAAACCATCTAATCAACCA


(SEQ ID 86)

GACCAGACGGTTCCCAAAGG





 


GI29823164_TYMS_2R
NT_010859
GGCTCCGTTCTGTGCCACACCCGTGGCTCC
TCCGTTCTATACCACACCCG
AACTCCTACGTTTCCCCCTAAC
AACTCCATTCTATACCACACCCA


(SEQ ID 87)

TGCGTTTCCCCCTGGCG





 


GI29823171_BCL2_1R
NT_025028
CGTGTGACGTTACGCACAGGAAACCGGTCG
TACTTCATTCTCTACACAACCCG
CCGATTTCCTATACGTAACGTCA
CTTACTTCATTCTCTACACAACCCA


(SEQ ID 88)

GGCTGTGCAGAGAATGAAGTAAG





 


GI29823171_BCL2_2R
NT_025028
CGGGAGGCGGCCGTAGCCAGCGCCGCCGCG
CCTCCTAATCCTACGCGACG
CGCTAACTACGACCGCCTC
CCTCCTCCTAATCCTACACAACA


(SEQ ID 89)

CAGGACCAGGAGGAGG





 


GI29823171_BCL2_3R
NT_025028
CCGTCAGCGCTCGGAGCGGGCTGCGCGGCG
AACGCTCGAAACGAACTACG
GACGAAAACTCCGAAAAACGAC
CCATCAACACTCAAAACAAACTACA


(SEQ ID 90)

GGAGCTCCGGGAGGCGGCC





 


GI29734309_SYK_1R
NT_008476
GCCCAGCTCCGGGCTCATGGGCGCGGTCAG
CCAACTCCGAACTCATAAACG
GATCAACAAAACGAACCAAAACG
ACCCAACTCCAAACTCATAAACA


(SEQ ID 91)

CAGGGCGGGCCAGGGCGG





 


GI29734309_SYK_2R
NT_008476
GGGCTCCTCTACACCTGCCGCCGCCTGGGC
ACTCCTCTACACCTACCGCCG
CTAAACCGATTCCGCGAAC
AAACTCCTCTACACCTACCACCA


(SEQ ID 92)

CGATTCCGCGGGCCTCG





 


GI29734309_SYK_3R
NT_008476
GGACCGATGCGGTCCATGTCCCGGGCAGCC
ACCGATACGATCCATATCCCG
ACAACCCCACCTTCTCTACCTAC
AAACCAATACAATCCATATCCCA


(SEQ ID 93)

CCACCTTCTCTGCCTGC





 


GI29740881_HTR1B_1R
NT_007299
GGGAATGCAAGATCTCGGGACCTCTCGCTG
AAACCAAAACTTACAAACCAACG
AAAATCCCGAAATCTTACATTCC
AAAACCAAAACTTACAAACCAACA


(SEQ ID 94)

GCCTGCAAGCTTTGGTCTC





 


GI29740881_HTR1B_2R
NT_007299
GGCGGAGGAATAATTGAGGAACTCACGGAA
TTTATCCCCAATTAATAATTCCG
AAATTCCTCAATTATTCCTCCGC
AATTTATCCCCAATTAATAATTCCA


(SEQ ID 95)

CTATCAACTGGGGACAAACC





 


GI29740881_HTR1B_3R
NT_007299
CCCTTTTATGGCTCCGTCTCCGCGGGGCAG
TTTATAACTCCGTCTCCGCG
AACAACTCGTCCGAATAACCAA
CCCTTTTATAACTCCATCTCCACA


(SEQ ID 96)

CTCGTCCGAGTGGCCAGA





 


GI29741420_MGMT_1R
NT_008818
GGCTTGTACCGGCCGAAGGGCCATCCGGGT
CTACCCTATACGCCTAACCCG
ATAACCCTTCGACCGATACAAAC
CCACTACCCTATACACCTAACCCA


(SEQ ID 97)

CAGGCGCACAGGGCAGCGG





 


GI29741420_MGMT_2R
NT_008818
GGCCGGCGTGCCGGCGTCCAGCGAGGATGC
ACGTACCGACGTCCAACG
AAATACGCAAACTACCTCAAACC
AACCAACATACCAACATCCAACA


(SEQ ID 98)

GCAGACTGCCTCAGGCCCG





 


GI29741420_MGMT_3R
NT_008818
GACCACTCGGGCACGTGGCAGGTCGCTTGC
CACTCGAACACGTAACAAATCG
TTACACGCCCGCGAACTAT
AACCACTCAAACACATAACAAATCA


(SEQ ID 99)

ACGCCCGCGGACTATCCCT





 


GI29791697_CDC25A_1R
NT_005825
GAAGTTGCTTACTGATTGGTGGATTCCGTT
CTTTCCTAATTAACGCCAAACG
AATCCACCAATCAATAAACAACTTC
CCCTTTCCTAATTAACACCAAACA


(SEQ ID 100)

TGGCGCCAACTAGGAAAGGG





 


GI29791697_CDC25A_2R
NT_005825
GAGGCCGAGCCAAGGCTGGATCCGGCCAGA
AACCGAACCAAAACTAAATCCG
CCAAACCTCCACAAATCTTCCTT
AAAACCAAACCAAAACTAAATCCA


(SEQ ID 101)

CCTCCACAGGTCTTCCTT





 


GI29791697_CDC25A_3R
NT_005825
CTCCAGAGTTGGGCCCTGGTGGTCGAGTCC
AATAACCCCCAAAACTAAACTCG
CCACCAAAACCCAACTCTAAAA
CAATAACCCCCAAAACTAAACTCA


(SEQ ID 102)

AGTCCTGGGGGTCATTG





 


GI29791697_PTHR1_1R
NT_005825
GGGCCCAGCCCTGGGCATCTGAACACCGGC
CAAAAACAAATCCAAATATACCG
TATTCAAATACCCAAAACTAAACCC
AACAAAAACAAATCCAAATATACCA


(SEQ ID 103)

ACACTTGGATCTGCCTCTGTT





 


GI29791697_PTHR1_2R
NT_005825
CAGCCCTGGGCATCTGAACACCGGCACACT
CAACCCTAAACATCTAAACACCG
CACACTTAAATCTACCTCTATTACCTCC
CAACCCTAAACATCTAAACACCA


(SEQ ID 104)

TGGATCTGCCTCTGTTGCCTCC





 


GI29791697_PTHR1_3R
NT_005825
GGGGCTGTAGGGGTCAGGACACGGGCTGGA
TCATTCCACTCCAACCCG
ATCCTAACCCCTACAACCCC
TCATTCCACTCCAACCCA


(SEQ ID 105)

GTGGAATGA





 


GI29792366_SOD3_1R
NT_006316
TCTGGGAAGTCTCCCTCTTATCTCGCAGAA
CTAAAAAATCTCCCTCTTATCTCG
AAAATACCTATCCCTAAATAAAAATCATT
TCTAAAAAATCTCCCTCTTATCTCA


(SEQ ID 106)

TGCCTGTCCCTGGATAAAGATCATT





 


GI29792366_SOD3_2R
NT_006316
TTCTCAGCGGGCCAGAAGGAAGGACGGTAG
AACCCTTCCACCCTACCG
CCTTCCTTCTAACCCGCTAAA
CAACCCTTCCACCCTACCA


(SEQ ID 107)

GGTGGAAGGGCTG





 


GI29792366_SOD3_3R
NT_006316
CACAAGTCCTAGAATACCAGAACGGAGACG
CACAAATCCTAAAATACCAAAACG
AAACGTACMCTTAAACCTTAAACG
CACAAATCCTAAAATACCAAAACA


(SEQ ID 108)

TGCTTTCTTGGACCTTAAACGAAA





 


GI29792503_VHL_1R
NT_005927
CAAGGCTGCAGTGAGCCAAGCTCGCGCCAC
AACTACAATAAACCAAACTCGCG
CACTACACTCCAACCCGAAC
CAAAACTACAATAAACCAAACTCACA


(SEQ ID 109)

TGCACTCCAGCCCGGGCGAC





 


GI29792503_VHL_3R
NT_005927
CCCGTGTGAGATGCGCCACCCTCGAACCTT
GTATAAAATACGCCACCCTCG
ACCTTATTACGACGTCGACACAT
CCCATATAAAATACACCACCCTCA


(SEQ ID 110)

GTTACGACGTCGGCACATTGCG





 


GI29793120_CD1A_1R
NT_004668
GGGGAATTAAGACCCAATGGGCAATCCGTG
CCCATCCCCTAATAATCACG
ATTACCCATTAAATCTTAATTCCCC
TCCCATCCCCTAATAATCACA


(SEQ ID 111)

ACTATTAGGGGATGGGA





 


GI29793120_CD1A_2R
NT_004668
CATAGCAGTAGCAGACATCTCTTGTACCGT
AACAATAACAAACATCTCTTATACCG
ACCTTACACCTCAACCCATTT
CATAACAATAACAAACATCTCTTATACCA


(SEQ ID 112)

GCCTTACACCTCAGCCCATTT





 


GI29793120_S100A2_1R
NT_004668
AGACTGTGGCCCAGCCCAACTGCGGCTGTG
ACTATAACCCAACCCAACTACG
CTATATATAAAACAACCCCATTTCTCA
AAACTATAACCCAACCCAACTACA


(SEQ ID 113)

TGTAGAGCAACCCCATTTCTCA





 


GI29793120_S100A2_2R
NT_004668
GAGCACTGGACCATGAAGTCTCAGCGTGTG
AACACTAAACCATAAAATCTCAACG
ATACTCACAACCTCTCACACAAAA
AAACACTAAACCATAAAATCTCAACA


(SEQ ID 114)

CTCACAGCCTCTCACACAGGA





 


GI29793234_ABCC5_1R
NT_005962
GGGCAGTTGGTGTTAAAGTGAGAAAGGCGG
CCTACAATCTACGCAAATACCG
CTTTCTCACTTTAACACCAACTACCC
AACCCTACAATCTACACAAATACCA


(SEQ ID 115)

TATCTGCGCAGACTGCAGGGTC





 


GI29793234_ABCC5_2R
NT_005962
CGCAGGGTGGGACTGCTCTGCCTACGAACA
CCTATATTCGCACACAATACTATTCG
AAACAAAACAATCCCACCCTAC
CCCTATATTCACACACAATACTATTCA


(SEQ ID 116)

GCACTGTGTGCGAACACAGGG





 


GI29793234_ABCC5_3R
NT_005962
CTGGCCGGCCTGACGACTCTCAACGGGTTA
ACCGACCTAACGACTCTCAACG
ATTAAACGCGAACTACGATAACC
CTAACCAACCTAACAACTCTCAACA


(SEQ ID 117)

GACGCGGGCTACGATGACCTTCACG





 


GI29793234_HRASLS_2R
NT_005962
TGGAGGTCTCTGCTGGCTATCTGGCGTGTG
ACACCACACACACACACACG
CAAATAACCAACAAAAACCTCCA
CACACCACACACACACACACA


(SEQ ID 118)

TGTGTGTGTGGTGTG





 


GI29793234_HRASLS_3R
NT_005962
CAAAACCGATTATCTTTATAACCGCGGCGC
AACCGATTATCTTTATAACCGCG
CGCCTAACACAACGCCTAATAC
CAAAACCAATTATCTTTATAACCACA


(SEQ ID 119)

CTAGCACAGCGCCTGGTGCCCT





 


GI29793705_CD2_1R
NT_004754
GGAGCACATCAGAAGGGCTGGCTTGTGCGC
CACAAAAAACAAAAACGCG
ACAAACCAACCCTTCTAATATACTCC
CACATACACAAAAAACAAAAACACA


(SEQ ID 120)

GCTCTTGCTCTCTGTGTATGTG





 


GI29793872_RASSF1_1R
NT_006014
TCCTCACCCCAAGTGAAGGCTCGAGACTTC
CCTCACCCCAAATAAAAACTCG
AACTTCCTACCCCACCCAATAAA
TCCTCACCCCAAATAAAAACTCA


(SEQ ID 121)

CTGCCCCACCCAGTGGG





 


GI29793872_RASSF1_2R
NT_006014
CCTGCACCCAGGTTTCCATTGCGCGGCTCT
CTACACCCAAATTTCCATTACG
GACTCTCCTCAACTCCTTCCC
CCTACACCCAAATTTCCATTACA


(SEQ ID 122)

CCTCAGCTCCTTCCCGC





 


GI29793872_RASSF1_3R
NT_006014
AGATCACGGTCCAGCCTCTGCCGGAGCCCC
AATCACGATCCAACCTCTACCG
AACCCCAATCTCCGCAATAAA
AAATCACAATCCAACCTCTACCA


(SEQ ID 123)

AGTCTCCGCAGTGGAG





 


GI29794089_PRDM2_1R
NT_004873
CTGGAACATCGCGGGCACAGGGCAGCGTTG
ATACCCAAAAACAATAACCAACG
TACCCTATACCCGCGATATTCC
AATACCCAAAAACAATAACCAACA


(SEQ ID 124)

GTCACTGCTCCTGGGTACT





 


GI29794089_PRDM2_2R
NT_004873
GGGGGTCCCGGAGCCCCCCAGCCCCGCAGG
GAACCACGAAACAATAACCTACG
AACTAAAAAACTCCGAAACCCC
TCAAACAACAAAACAATAACCTACA


(SEQ ID 125)

CCACTGCCTCGCCGCCCGA





 


GI29794089_PRDM2_3R
NT_004873
TCTTCATCAGAACATCTATGAGTCGTCGTC
TCATCAAAACATCTATAAATCGTCG
CTCTACAATCCATTTATTTACCCG
TCTTCATCAAAACATCTATAAATCATCA


(SEQ ID 126)

TCTGCAGTCCATTTGTTTGCCCGC





 


GI29794150_NCL_1R
NT_005403
ACTCCAAGGCTGCCCAAGCCTACGGACCCA
TCCAAAACTACCCAAACCTACG
ACCCAACCACATTAACGAACC
ACTCCAAAACTACCCAAACCTACA


(SEQ ID 127)

GCCACATTGGCGAACCG





 


GI29794150_NCL_2R
NT_005403
ACTCCGCGGTCCCTGAACTTCCGGTGCTGG
GCCATCCCTAAACTTCCG
TACTAACGAACTCCTCGCTCCAA
ACTCCACAATCCCTAAACTTCCA


(SEQ ID 128)

CGGACTCCTCGCTCCAGG





 


GI29794150_NCL_3R
NT_005403
GGGGTGCCTCCGGCCCCATGCTCGCGGGCA
CACAACTTATCCCTACTTACCCG
GAACATAAAACCGAAAACACCC
AACACAACTTATCCCTACTTACCCA


(SEQ ID 129)

AGCAGGGATAAGCTGTGCC





 


GI29784150_TMEFF2_1R
NT_005403
GGCCATCCGGCAAAGACCCGAGTAAGGAAC
TTAACCCAAACAATAACCCTACG
TCCTTACTCCAATCTTTACCCAA
ATTAACCCAAACAATAACCCTACA


(SEQ ID 130)

GCAGGGTCACTGCCTGGGCCAAC





 


GI29794150_TMEFF2_2R
NT_005403
TTTCTCTCAGACCACTTGTCCCGACCAATC
TTCTCTCAAACCACTTATCCCG
CCAATCTAACCTTCCAAACACAT
TTTCTCTCAAACCACTTATCCCA


(SEQ ID 131)

TGACCTTCCAAACACAT





 


GI29794150_TMEFF2_3R
NT_005403
CGCAGAGCCAGAGACTCCTGCCCAGTTAGA
GCAAAACCAAAAACTCCTACCG
ATTAAACCTTCTCTCGTCGCC
CACAAAACCAAAAACTCCTACCA


(SEQ ID 132)

CCTTCTCTCGTCGCCCC





 


GI29795229_DBCCR1_1R
NT_008470
GCGCGGTCCCTTTGGATGCTCGTGCGCATA
CGATCCCTTTAAATACTCGTACG
ATAAACACAACACCCTACACGC
ACACAATCCCTTTAAATACTCATACA


(SEQ ID 133)

GACACAACACCCTACACGCC





 


GI29795229_DBCCR1_2R
NT_008470
CCCCAGAAACACTCAGGTACTCGCGACACA
CCCAAAAACACTCAAATACTCG
GACACACACAATACAATCACGCT
CCCCAAAAACACTCAAATACTCA


(SEQ ID 134)

CACAGTACAGTCACGCTTAA





 


GI29795229_DBCCR1_3R
NT_008470
GCAGACACAAACGGACCCACACGGGCAACT
ACACAAACCAACCCACACG
ACAACTCCCGAAACAAAACC
ACAAACACAAACAAACCCACACA


(SEQ ID 135)

CCCGAGACAAAACCC





 


GI29795229_TMEFF1_1R
NT 008470
GGACACAAAGGGAAGGCGAGGAGGCGAGCA
AACAAACCCAACCTCTTACTCG
CTCCTCGCCTTCCCTTTATATC
AAACAAACCCAACCTCTTACTCA


(SEQ ID 136)

AGAGGCTGGGCCTGCCT





 


GI29795229_TMEFF1_2R
NT_008470
CGGGCGGGCCGGGGTCTTTGTGACGCGGTG
GAACCGAAAGGTTTATAACGCG
TAACAACGACCACGAACACAA
CAAACAAACCAAAATCTTTATAACACA


(SEQ ID 137)

GCAACGGCCACGGACACAAAG





 


GI29795229_TMEFF1_3R
NT_008470
GACAAGTCACCTTTACCTCTTCCGTGACTC
CAAATCACCTTTACCTCTTCCG
AACTCAATTTCTICCACCTAAAAAC
AACAAATCACCTTTACCTCTTCCA


(SEQ ID 138)

AGTTTCTTCCACCTAAAAAC





 


GI29796755_HOXA5_1R
NT_007819
TGCAGCCCCCGGTCGGAAGCTGGGCGATGA
CCGCTAAAAACAAAACTCATCG
CCAACTTCCGACCGAAAACTAC
ACCCACTAAAAACAAAACTCATCA


(SEQ ID 139)

GCCCTGCCTCCAGCGGGT





 


GI29796755_HOXA6_2R
NT_007819
GGGCGATGAGCCCTGCCTCCAGCGGGTGGC
CGATAAACCCTACCTCCAACG
ATAACGCTCGAATCCGACTAAAC
AAACAATAAACCCTACCTCCAACA


(SEQ ID 140)

GCTCGAGTCCGGCTGAACGGCG





 


GI29796755_HOXA5_3R
NT_007819
ATCCTCTGCATCCTCGCCGGGCGCGCGATC
CTACATCCTCGCCGAACG
GCGATCGACAACTAACGACCTAA
ATCCTCTACATCCTCACCAAACA


(SEQ ID 141)

GGCAGCTGACGGCCTAACAA





 


GI29796755_IGFBP1_2R
NT_007819
GGCGCGGCCTGTGCCCTTTATAAGGTGCGC
ATACTCGCTAAACACAACGCG
ACCTTATAAAAAACACAAACCGC
AACCAATACTCACTAAACACAACACA


(SEQ ID 142)

GCTGTGTCCAGCGAGCATCGGCC





 


GI29796755_IGFBP1_3R
NT_007819
CGCGGCCTGTGCCCTTTATAAGGTGCGCGC
GACCTATACCCTTTATAAAATACGCG
TATATCCAACGAACATCGACCA
CACAACCTATACCCTTTATAAAATACACA


(SEQ ID 143)

TGTGTCCAGCGAGCATCGGCCACC





 


GI29796755_IL6_1R
NT_007819
TCTGCTTCTTAGCGCTAGCCTCAATGACGA
TCTTAACGCTAACCTCAATAACG
CCTAAACTACACTTTTCCCCCT
TCTACTTCTTAACACTAACCTCAATAACA


(SEQ ID 144)

CCTAAGCTGCACTTTTCCCCCT





 


GI29796755_IL6_2R
NT_007819
CCTGGAGACGCCTTGAAGTAACTGCACGAA
TACCTAACCATCCTCAAATTTCG
ACAATTACTTCAAAACGTCTCCAA
CTACCTAACCATCCTCAAATTTCA


(SEQ ID 145)

ATTTGAGGATGGCCAGGCAG





 


GI29796755_TWIST1_1R
NT_007819
GTTTTTGAATGGTTTGGGAGGACGAATTGT
CTTCCTCGAAATCTAACAATTCG
CCTCCCAAACCATTCAAAAAC
CCTTCCTCAAAATCTAACAATTCA


(SEQ ID 146)

TAGACCCCGAGGAAGG





 


GI29796755_TWIST1_2R
NT_007819
CCACTCCGGATGGGGCTGCCACCGCGGCCA
ACTCCGAATAAAACTACCACCG
GACCAAAACAATCTCCTCCGAC
CCACTCCAAATAAAACTACCACCA


(SEQ ID 147)

GGACAGTCTCCTCCGAC





 


GI29796755_TWIST1_3R
NT_007819
GGCCTTTGGAACTCCAAGGGGTTCGTCTAC
CCCACCCAATAATCAAATAAACG
ACCCCTTAAAATTCCAAAAACC
CCCACCCAATAATCAAATAAACA


(SEQ ID 148)

CTGACCATTGGGTGGG





 


GI29796774_TRC8_1R
NT_008046
GAAGCTAGAGTAAGCTGAGGAGGTGGGCGG
ACCCATAATTACCATAATTTCCG
CCACCTCCTCAACTTACTCTAACTTC
CACCCATAATTACCATAATTTCCA


(SEQ ID 149)

AAACCATGGCAACCATGGGTG





 


GI29796774_TRC8_2R
NT_008046
ACCACACCCAGCCTAGTGCCACGCACCGCA
CACACCCAACCTAATACCACG
ACCGCAAACGCTCCATAA
ACCACACCCAACCTAATACCACA


(SEQ ID 150)

AGCGCTCCATAAACGCA





 


GI29796774_TRC8_3R
NT_008046
CGCAGGCAGTGGGCGCGGACTCTGCGGTTC
AAACAATAAACGCGAACTCTACG
TTCGCTTAACTAACGACGCAAC
CACAAACAATAAACACAAACTCTACA


(SEQ ID 151)

GCTTGACTGACGGCGCAGCCTCC





 


GI29804415_C4B_1R
NT_007592
TTGTGAGGGCCTTTAAATATCCTGTACTCG
AAACCCAACATAACCCACG
ATACAAAATATTTAAAAACCCTCACAA
AAAACCCAACATAACCCACA


(SEQ ID 152)

TGGGCCATGTTGGGCCCT





 


GI29804415_C4B_2R
NT_007592
TCAGGCACTGGAATGAGAGGAGTTAACGGG
AACCCTATCCTTCCCCG
TAACTCCTCTCATTCCAATACCTAA
AAAATAACCCTATCCTTCCCCA


(SEQ ID 153)

GAAGGACAGGGTTATTTC





 


GI29804415_C4B_3R
NT_007592
CCCATGGACACCCAGGTGTCCGGGGTGCCC
CCATAAACACCCAAATATCCG
AATACCCCCACAACTCTAAACCT
CCCATAAACACCCAAATATCCA


(SEQ ID 154)

CCACAACTCTGGGCCT





 


GI29804415_CDKN1A_1R
NT_007592
TGGCTCTGATTGGCTTTCTGGCCGTCAGGA
TCAACATATTAAAACATATTCC
CCAAAAAACCAATCAAAACCA
ACTCAACATATTAAAACATATTCCTAACA


(SEQ ID 155)

ACATGTCCCAACATGTTGAGC
TAACG




 


GI29804415_CSNK2B_1R
NT_007592
CCCACATTACTTGAGGGCTCGGGCGTGCGC
CCACATTACTTAAAAACTCGAACG
ACGCAAAACTCCGAATTCAAT
CCCACATTACTTAAAAACTCAAACA


(SEQ ID 156)

AAAGCTCCGGGTTCAGTTTC





 


GI29804415_CSNK2B_2R
NT_007592
ACAATCAAATAGCCACACGGCACGAAGACG
AATCAAATAACCACACGACACG
AAACGCATACGTAACGACAACA
ACAATCAAATAACCACACAACACA


(SEQ ID 157)

CATGCGTGGCGACAACAACAAC





 


GI29804415_CSNK2B_3R
NT_007592
CAGCCTGGCCCTTTAAGTCTTCCGCGATCC
AACCTAACCCTTTAAATCTTCCG
GATCCCATTTCGAAATTTCCTCT
CAACCTAACCCTTTAAATCTTCCA


(SEQ ID 158)

CATTTCGGAGTTTCCTCT





 


GI29804415_EDN1_1R
NT_007592
TCTTTTTCTTAGCCCTGCCCCCGAATTGTC
CTTTTTCTTAACCCTACCCCCG
ATTATCAAACGACGAACGTCTACC
TCTTTTTCTTAACCCTACCCCCA


(SEQ ID 159)

AGACGGCGGGCGTCTGCCTCT





 


GI29804415_EDN1_2R
NT_007592
GGCTGGCAGCTTGCAAAGGGGAAGCGGACT
TACCCGTACAATACTAAAATCCG
TTCCCCTTTACAAACTACCAACC
CCTACCCATACAATACTAAAATCCA


(SEQ ID 160)

CCAGCACTGCACGGGCAGG





 


GI29804415_EDN1_3R
NT_007592
GGCCTGGCCTTATCTCCGGCTGCACGTTGC
ATTATTAATCACCAACAAACAACG
ACAACCGAAAATAAAACCAAACC
TTATATTATTAATCACCAACAAACAACA


(SEQ ID 161)

CTGTTGGTGACTAATAACACAA





 


GI29804415_HLA-F_1R
NT_007592
AAGTTCAATCAAGGGACTGGGATTTCGGAA
TCTCCCTTCATTATTCATTCCG
AATCCCAATCCCTTAATTAAACTT
CATCTCCCTTCATTATTCATTCCA


(SEQ ID 162)

TGAATAATGAAGGGAGATG





 


GI29804415_HLA-F_2R
NT_007592
TCCTTCTTCCTGGATACTCATAACGCGGCC
TCTTCCTAAATACTCATAACGCG
CCCCATTTCTCACTCCCATTAA
TCCTTCTTCCTAAATACTCATAACACA


(SEQ ID 163)

CCATTTCTCACTCCCATTGG





 


GI29804415_LY6G6E_1R
NT_007592
GGGGCAGGCTGGGGGCCCCCGCTGCCTGCT
AACCTAACCCAACAAACAACG
AAACCCCCAACCTACCCC
CAACCTAACCCAACAAACAACA


(SEQ ID 164)

GGGTCAGGCTG





 


GI29804415_LY6G6E_2R
NT_007592
AACTCTACAAATCCCGGGATCTCGGGGTGC
TCTACAAATCCCGAAATCTCG
AATACAAATCACCTCTCCCAAA
AACTCTACAAATCCCAAAATCTCA


(SEQ ID 165)

AGATCACCTCTCCCAGA





 


GI29804415_LY6G6E_3R
NT_007592
TCTGAAGCTGGGTGGGGTCCGCCCTTACAC
CACAAAACCCAACTATATAAAAACG
ACCCCACCCAACTTCAAA
ACACAAAACCCAACTATATAAAAACA


(SEQ ID 166)

AGCTGGGCTTTGTGT





 


GI29804415_NEU1_1R
NT_007592
GCTGAGGCAGGAGAATCGCTTGAACCCGGG
TCACTACAACCTCTACCTCCCG
ATTCAAACGATTCTCCTACCTCA
ACTCACTACAACCTCTACCTCCCA


(SEQ ID 167)

AGGCAGAGGTTGCAGTGAGC





 


GI29804415_NEU1_2R
NT_007592
GAGGGCCAATCGGAAGGGCAAGCTTCGAGA
ACGTAATCACGCAACATCTCG
AACTTACCCTTCCGATTAACCCT
CCACATAATCACACAACATCTCA


(SEQ ID 168)

TGCTGCGTGATCACGTGG





 


GI29804415_NEU1_3R
NT_007592
GGGCGGATCACCTGAGTCAGGAGTTCGAGA
TCACCATATTAACCAAACTAATCTCG
ACTCCTAACTCAAATAATCCGCC
TTCACCATATTAACCAAACTAATCTCA


(SEQ ID 169)

CCAGCCTGGCCAACATGGTGAA





 


GI29804415_RDBP_1R
NT_007592
GGTGAAGGAGGTTTGGACTCAATGCGGGTC
TTTTAACCCTAACCTTTAACCCG
ATTAAATCCAAACCTCCTTCACC
CTTTTAACCCTAACCTTTAACCCA


(SEQ ID 170)

AAAGGTTAGGGTCAAAAG





 


GI29804415_RDBP_2R
NT_007592
GGGATCTGGAGGGGGTAGCACTACGGGGAA
CCTAACTTTTAACCTTTCCCCG
AATACTACCCCCTCCAAATCCC
CCCTAACTTTTAACCTTTCCCCA


(SEQ ID 171)

AGGTCAAAAGTCAGGG





 


GI29804415_RDBP_3R
NT_007592
TGGCAGCCCGGAAGTGCGGCAAGTAGTCGC
AAACGAAACTTACTTCGCAACG
CTACTTACCGCACTTCCCAACTA
CCAAAACAAAACTTACTTCACAACA


(SEQ ID 172)

TGCGAAGTAAGCCCCGCCCCGG





 


GI29804415_TNF_1R
NT_007592
ATAGGTTTTGAGGGGCATGGGGACGGGGTT
AAACCCTAAAAACTAAACCCCG
CCCCATACCCCTCAAAACCTAT
AAAACCCTAAAAACTAAACCCCA


(SEQ ID 173)

CAGCCTCCAGGGTCCT





 


GI29804415_TNF_2R
NT_007592
CCCGCCCCCGCGATGGAGAAGAAACCGAGA
AATAAACCCTACACCTTCTATCTCG
TTTCTTCTCCATCGCGAAA
ATAATAAACCCTACACCTTCTATCTCA


(SEQ ID 174)

CAGAAGGTGCAGGGCCCACTAC





 


GI29804415_TNF_3R
NT_007592
CAGAAGGTGCAGGGCCCACTACCGCTTCCT
AAAAAATACAAAACCCACTACCG
TTCCTCCAAATAAACTCATAAATTTC
CAAAAAATACAAAACCCACTACCA


(SEQ ID 175)

CCAGATGAGCTCATGGGTTTC





 


GI29804415_VEGF_1R
NT_007592
GTCTCTGGACAGAGTTTCCGGGGGCGGATG
ACAACCTAAAAATTACCCATCCG
CCCCGAAAACTCTATCCAAAA
CACAACCTAAAAATTACCCATCCA


(SEQ ID 176)

GGTAATTTTCAGGCTGTG





 


GI29804415_VEGF_2R
NT_007592
TCCCCTTCATTGCGGCGGGCTGCGGGCCAG
CCCTTCATTACGACCAACTACG
ACCAAACTTCACTAAACGTCCG
TCCCCTTCATTACAACAAACTACA


(SEQ ID 177)

GCTTCACTGAGCGTCCGCA





 


GI29804415_VEGF_3R
NT_007592
GAACGGCTCTCAGGCCCTGTCCGCACGTAA
AACGACTCTCAAACCCTATCCG
ACGTAACCTCACTTTCCTACTCC
AAACAACTCTCAAACCCTATCCA


(SEQ ID 178)

CCTCACTTTCCTGCTCCCT





 


GI29807454_WT1_1R
NT_009237
CATCTCTACTCCCACCGCATTCGACCCTGC
CTACTCCCACCGCATTCG
CCCTACCCGAACTCACTACTTAC
CATCTCTACTCCCACCACATTCA


(SEQ ID 179)

CCGGACTCACTGCTTACC





 


GI29824571_MOS_1R
NT_008183
TCAGTCATGTTTCCAAAGTCCCGCGGTTTC
CAATCATATTTCCAAAATCCCG
GATTTCCCCTAATCTCTTCATTCA
TCAATCATATTTCCAAAATCCCA


(SEQ ID 180)

CCCTAGTCTCTTCATTCA





 


GI29789877_CSF1_2R
NT_019273
GTTTGCTGAAGGCTTGGAAGTGCAGCGCAG
TCCTAATCACCCTCTATCTTCTACG
TACACTTCCAAACCTTCAACAAAC
TTCCTAATCACCCTCTATCTTCTACA


(SEQ ID 181)

AAGACAGAGGGTGACTAGGAA





 


GI29789877_CSF1_3R
NT_019273
TGTGTGTGTGTATGTGTGTGTCTGGCGCCT
AAAAAATCACCCTAACCAAACG
CAAACACACACATACACACACACA
ATAAAAAATCACCCTAACCAAACA


(SEQ ID 182)

GGCCAGGGTGATTTCCCAT





 


GI29789881_MTHFR_1R
NT_021937
GAGCATCTCTGGTTGGAATCATAATGCTTC
AAATAACCTAATCACTTCAAACCG
AACATTATAATTCCAACCAAAAATACTC
AATAAATAACCTAATCACTTCAAACCA


(SEQ ID 183)

GGCCTGAAGTGACCAGGCCACTCACT





 


GI29791372_MLH1_1R
NT_022517
CGGGAGGCCACAAGAGCAGGGCCAACGTTA
CCCCTTACGACCTTTCTAACG
TAACCCTACTCTTATAACCTCCCG
TCTCCCCTTACAACCTTTCTAACA


(SEQ ID 184)

GAAAGGCCGCAAGGGGAGA





 


GI29791372_MLH1_2R
NT_022517
TGGCGTAAGCTACAGCTGAAGGAAGAACGT
CACCTCAATACCTCGTACTCACG
TCTTCCTTCAACTATAACTTACGCC
TCACCTCAATACCTCATACTCACA


(SEQ ID 185)

GAGCACGAGGCACTGAGGTGA





 


GI29791372_MLH1_3R
NT_022517
CCGCCACATACCGCTCGTAGTATTCGTGCT
CACATACCGCTCGTAATATTCG
ACTCAACCTCGTAATAACGCCTA
CCACCACATACCACTCATAATATTCA


(SEQ ID 186)

CAGCCTCGTAGTGGCGCCTGACG





 


GI29791392_EGR4_1R
NT_022184
CCACAGGAAATGCACAGGTGAGAAACTGAC
TAACACTCAATCCCCCTTAACG
CAATTTCTCACCTATACATTTCCTATAA
TTAACACTCAATCCCCCTTACA


(SEQ ID 187)

GTTAAGGGGGACTGAGTGTCAA





 


GI29791392_EGR4_2R
NT_022184
AGGGCAGATTCAAACCCAACACGGTCCTCC
AAACAAATTCAAACCCAACACG
TCCTCCCCTACTACCCCTCG
AAAACAAATTCAAACCCAACACA


(SEQ ID 188)

CCTGCTGCCCCTCGGC





 


GI29791392_EGR4_3R
NT_022184
CGGTCGGCCCGGTGAGGCGCAGCGCCCCAG
ACCCGATAAAACGCAACG
CCCAAACTAACGCATCCG
CAATCAACCCAATAAAACACAACA


(SEQ ID 189)

ACTGGCGCATCCGCGGC





 


GI29791392_POMC_1R
NT_022184
GGCGAGCGGCCAGGTGCGCCTTCGGCAGGA
AACGACCAAATACGCCTTCG
CAAAACAATACTAATTCCAACCCC
AACAAACAACCAAATACACCTTCA


(SEQ ID 190)

CAGTGCTAATTCCAGCCCC





 


GI29791392_POMC_2R
NT_022184
CACACGGGGGTGCTAAGCCTCCCGCCCGTT
CACGAAAATACTAAACCTCCCG
CCGTTCTAAACGAAAACCCAAC
CACACAAAAATACTAAACCTCCCA


(SEQ ID 191)

CTAAGCGGAGACCCAACG





 


GI29791392_POMC_3R
NT_022184
CGCACGCAGGTAACTTCACCCTCGCCTCAA
ACGCAAATAACTTCACCCTCG
CTCAACGACCTCAAAAACTACCC
CACACACAAATAACTTCACCCTCA


(SEQ ID 192)

CGACCTCAGAGGCTGCCC





 


GI29791392_SFTPB_1R
NT_022184
GAGGTCGCTGCCACTCCTACAGAGCCCCCA
CCCCTTATAACTAAACGAAACG
AAAAACTCTATAAAAATAACAACGACCT
CATAACCCCTTATAACTAAACAAAACA


(SEQ ID 193)

CGCCCCGCCCAGCTATAAGGGGCCATG





 


GI29791392_SFTPB_2R
NT_022184
ACTCCTACAGAGCCCCCACGCCCCGCCCAG
TCCTACAAAACCCCCACG
CCCGCCCAACTATAAAAAA
ACTCCTACAAAACCCCCACA


(SEQ ID 194)

CTATAAGGGGCCA





 


GI29791392_TGFA_1R
NT_022184
ACGGTAGCCGCCTTCCTATTTCCGCCCGGC
ATAACCGCCTTCCTATTTCCG
CCGACGAACAACGCTACG
ACAATAACCACCTTCCTATTTCCA


(SEQ ID 195)

GGGCAGCGCTGCGGGGCGA





 


GI29791392_TGFA_2R
NT_022184
CGCCGCCTAGAGCCTGGAAGCCGCCACTGC
CCGCCTAAAACCTAAAAACCG
CACTACGACCCAAAACAATCC
CACCACCTAAAACCTAAAAACCA


(SEQ ID 196)

GGCCCAGGACAATCCGG





 


GI29791392_TGFA_3R
NT_022184
CCAAGTCTTGGCAAGCGGCCGGCGAAACTC
AAATCTTAACAAACGACCGACG
AACTCACAAATCCCTTTCCTAAC
CCAAATCTTAACAAACAACCAACA


(SEQ ID 197)

ACAGGTCCCTTTCCTGGC





 


GI29794065_N33_1R
NT_015280
CTCCTCTCCTCAGCGCTGGTCCGGGAAAGG
CTCTCCTCAACGCTAATCCG
AAAAAACAAACTCCGAACGAAA
CTCCTCTCCTCAACACTAATCCA


(SEQ ID 198)

CAAGCTCCGGGCGGGAGCG





 


GI29794065_N33_2R
NT_015280
GGGAATAGAGATTGCTGGGGACCGCAGGGG
AAAATAACAACCTAACCCCTACG
TCCCCAACAATCTCTATTCCC
AAAAATAACAACCTAACCCCTACA


(SEQ ID 199)

CCAGGTTGTCATTCCC





 


GI29794065_N33_3R
NT_015280
CCCGAAGCCTGGCTCCCTCGCCACGCCCAC
CCGAAACCTAACTCCCTCG
CACGCCCACTTCCTACCC
CCCAAAACCTAACTCCCTCA


(SEQ ID 200)

TTCCTGCCCC





 


GI29798364_UBB_1R
NT_010718
CACCAATCAGCGCCGACCTCGCCTTCGCAG
AATCAACGCCGACCTCG
CTTCGCAAACCTAACCAATCAAT
CACCAATCAACACCAACCTCA


(SEQ ID 201)

GCCTAACCAATCAGT





 


GI29798364_UBB_2R
NT_010718
CGCGCTCAGTTACTTAGCAACCTCGGCGCT
CGCTCAATTACTTAACAACCTCG
CGCTAAACCACCCCAAATAAAAC
CACACTCAATTACTTAACAACCTCA


(SEQ ID 202)

AAGCCACCCCAGGTGGAGC





 


GI29798364_UBB_3R
NT_010718
GGAGAATCGCTTGAACCCGGGAGGCGGAGG
GACTCATCGCAACCTCCG
CTCCCGAATTCAAACGATTCT
ATCTCAACTCATCACAACCTCCA


(SEQ ID 203)

TTGCGATGAGCCGAGAT





 


GI29798595_ERBB2_1R
NT_010755
GGCAGCCTAGGGAATTTATCCCGGACTCCG
CTCTACCCCCTCCCCCG
AATCCGAAATAAATTCCCTAAACT
ATAACTCTACCCCCTCCCCCA


(SEQ ID 204)

GGGGAGGGGGCAGAGTCAC





 


GI29798595_ERBB2_2R
NT_010755
GATTCTCCGAGGAAAAGTGTGAGAACGGCT
AACGCCTAAATTACCTACAACCG
TCTCACACTTTTCCTCGAAAAAT
CAAAACACCTAAATTACCTACAACCA


(SEQ ID 205)

GCAGGCAACCCAGGCGTCCCG





 


GI29798595_ERBB2_3R
NT_010755
AGGAGGGACGCACCCAGGCCTGCGCGAAGA
ACTTCACTTTCTCCCTCTCTTCG
GCAAACCTAAATACGTCCCTCCT
AACTTCACTTTCTCCCTCTCTTCA


(SEQ ID 206)

GAGGGAGAAAGTGAAGCT





 


GI29799031_ATP5G1_1R
NT_010783
GTGGCCCAAGCGCCCGAATGAAGGCTCGGG
TCAAACACATAACTTTAACCCCG
ACCTTCATTCGAACGCTTAAAC
AATCAAACACATAACTTTAACCCCA


(SEQ ID 207)

GCCAAAGTCATGTGTCTGACT





 


GI29799031_ATP5G1_2R
NT_010783
CTGTGGAGGGAGAGGAAGCAGCTGCGGAAA
TCCCCACTCTTATTAACTTTCCG
AACTACTTCCTCTCCCTCCACAA
TTCCCCACTCTTATTAACTTTCCA


(SEQ ID 208)

GCCAATAAGAGTGGGGAA





 


GI29799031_ATP5G1_3R
NT_010783
TCCCGCCAACTCTATGGTCGAGCGTTTCAG
CGCCAACTCTATAATCGAACG
TTCAAAAATCGACCAATCGTAAT
TCCCACCAACTCTATAATCAAACA


(SEQ ID 209)

GGATCGGCCAATCGTAATCC





 


GI29799031_NME1_1R
NT_010783
CTCGGGAAGCCAATTTGCTCGCGAACGAAG
GAATTCTCTAACTCACTTCCTTCG
TCGCGAACAAATTAACTTCCC
CAAATTCTCTAACTCACTTCCTTCA


(SEQ ID 210)

GAAGTGAGTCAGAGAACCCG





 


GI29799031_NME1_2R
NT_010783
GGGTGGAGAGAAGAAAGCAAGCAGCTAACC
GCTAACTTTTTCAAACCTTTCCG
TTAACTACTTACTTTCTTCTCTCCACC
CACTAACTTTTTCAAACCTTTCCA


(SEQ ID 211)

GGAAAGGTCTGAAAAAGCTAGCG





 


GI29799031_NME1_3R
NT_010783
CGCAACGTGTGAGCGCCACCTCTCGGGAAG
AACGTATAAACGCCACCTCTCG
AAAACCAATTTACTCGCGAACG
CACAACATATAAACACCACCTCTCA


(SEQ ID 212)

CCAATTTGCTCGCGAACGAAG





 


GI29799354_NF1_1R
NT_010799
CTCCCGGGTCAGCTCTGGCACTCGCCAGCT
CCCGAATCAACTCTAACACTCG
CAACTAAACCCAACGCCAATCTA
CTCCCAAATCAACTCTAACACTCA


(SEQ ID 213)

GAGCCCAGCGCCAGTCTAG





 


GI29799354_NF1_2R
NT_010799
GGGCGCCCTAACTTCCAACTCCGGGAGCAA
GCCCTAACTTCCAACTCCG
AAACAATCCAAACCCGAAAAC
AAACACCCTAACTTCCAACTCCA


(SEQ ID 214)

TCCAAACCCGGAGGCC





 


GI29799354_NF1_3R
NT_010799
TTTTCATTAATGAAACCGGCCGGCGCGGGC
TCATTAATAAAACCGACCGACG
GAACGCATACGCGACAAAC
TTTTCATTAATAAAACCAACCAACA


(SEQ ID 215)

GCATGCGCGGCAGGCCGCC





 


GI29800594_APOC2_1R
NT_011109
TTCAGGAGGGTGAGGGCAGGAGCGTGGGTG
AAACCTACTAACTCCACCCACG
TCCTACCCTCACCCTCCTAAA
AAAAACCTACTAACTCCACCCACA


(SEQ ID 216)

GAGTCAGCAGGTCCCC





 


GI29800594_APOC2_2R
NT_011109
CAACCCAGCCTCTGTCGGAGGCGAATTCTC
AACCCAACCTCTATCGAAAACG
ATTCTCAAAATAAAAATTCCCTATCA
CAACCCAACCTCTATCAAAAACA


(SEQ ID 217)

AGAGTGAGGGTTCCCTGTCA





 


GI29800594_APOC2_3R
NT_011109
TTCCCTGTGACGTGACCTTGGGGGACGTCA
TCCCTATAACGTAACCTTAAAAAACG
CATTACCCTTTCTATCCCCACC
TTCCCTATAACATAACCTTAAAAAACA


(SEQ ID 218)

TTGCCCTTTCTGTCCCCACC





 


GI29800594_KLK10_3R
NT_011109
GGTTTCACTATGTTGGCCAGGCTGGTCTCG
GAATCACGAAATCAAAAATTCG
AACCAACCTAACCAACATAATAAAACC
AACAAATCACAAAATCAAAAATTCA


(SEQ ID 219)

AACTCCTGACCTCGTGATCCGCC





 


GI29800594_TSLL2_2R
NT_011109
CGGTGGCTCAGCTCACAGCCTCTCGCTAGA
ATAACTCAACTCACAACCTCTCG
TAAACTCCAAACACCCACCTACC
CAATAACTCAACTCACAACCTCTCA


(SEQ ID 220)

CTCCAGACACCCACCTGCC





 


GI29800594_TSLL2_3R
NT_011109
ATCCTTGACCTGCCTCGACCTCCGGACCCT
CTTAACCTACCTCGACCTCCG
ACCCTCCGAACCTCCAACTAC
ATCCTTAACCTACCTCAACCTCCA


(SEQ ID 221)

CCGGACCTCCAGCTGC





 


GI29801019_STK11_1R
NT_011255
AGCCAGCCTGGGGACTGGAGGGTGGCGGCT
ACCAACCTAAAAACTAAAAAATAACG
CTACATCTACTCATTTCCTCCCA
AACCAACCTAAAAACTAAAAAATAACA


(SEQ ID 222)

GCATCTGCTCATTTCCTCCCA





 


GI29801019_STK11_2R
NT_011255
GGAGTCTTGCTGTGTCTCCCAGGCCGGAGT
AAATCTTACTATATCTCCCAAACCG
AATACAATAACATAACCTCTACCTCCC
AAAATCTTACTATATCTCCCAAACCA


(SEQ ID 223)

GCAATGGCATGACCTCTGCCTCCC





 


GI29801019_STK11_3R
NT_011255
TGGAGTCTTGCTGTGTCTCCCAGGCCGGAG
CAAAAATCATACCATTACACTCCG
CCTAAAAAACACAACAAAACTCCA
CAAAAATCATACCATTACACTCCA


(SEQ ID 224)

TGCAATGGCATGACCTCTG





 


GI29801560_CDKN2D_1R
NT_011295
GCCATTGCCGGCGGTCCACCGGTTGCCACA
ATTACCGACGATCCACCG
TTACCACACTCTAACCAATCAAAA
ACCATTACCAACAATCCACCA


(SEQ ID 225)

CTCTGACCAATCAGGA





 


GI29801560_CDKN2D_2R
NT_011295
AGTTAAACCAGCCTTCTTTCCCGCCTGCCG
ATTAAACCAACCTTCTTTCCCG
CTACCGAATTCATTTAAAAACCG
AATTAAACCAACCTTCTTTCCCA


(SEQ ID 226)

GGTTCATTTGAAAACCGAAA





 


GI29801560_CDKN2D_3R
NT_011295
CTCATCCACTCCGTCTCTCCGTTTCCCTTT
CATCCACTCCGTCTCTCCG
TTCCCTTTCTTCACGATACTTAACA
CTCATCCACTCCATCTCTCCA


(SEQ ID 227)

CTTCACGGTGCTTGACA





 


GI29801560_ICAM1_1R
NT_011295
AGCTTGGAAATTCCGGAGCTGAAGCGGCCA
ATCCTCCCTCGCTAACCG
TTCAACTCCGAAATTTCCAAACT
AAATCATCCTCCCTCACTAACCA


(SEQ ID 228)

GCGAGGGAGGATGACCC





 


GI29801560_ICAM1_2R
NT_011295
TCCCCGGACGTGGTGAGACCGCGCTTCGTC
CGAACGTAATAAAACCGCG
TTCGTCACTCCCACGATTAAC
TCCCCAAACATAATAAAACCACA


(SEQ ID 229)

ACTCCCACGGTTAGCGG





 


GI29801560_ICAM1_3R
NT_011295
CCTGGCTCTGCTCTGGCCGCTTCTCGAGAA
CCTAACTAACACGAACATTTCTCG
AAAACGACCAAAACAAAACCAA
CACCTAACTAACACAAACATTTCTCA


(SEQ ID 230)

ATGCCCGTGTCAGCTAGGTG





 


GI29801767_THBS1_1R
NT_010194
CCAGTCTCTAGTATCCACCTCTCGCCATCA
CCAATCTCTAATATCCACCTCTCG
CATCAACCAAACATTCCGAAA
CCAATCTCTAATATCCACCTCTCA


(SEQ ID 231)

ACCAGGCATTCCGGGAG





 


GI29801767_THBS1_2R
NT_010194
GGGCCAGCTCAGGACAGGCGCTCGGGGGAC
CAACTCAAAACAAACGCTCG
AAAACGCGTATCCTCACCC
AAACCAACTCAAAACAAACACTCA


(SEQ ID 232)

GCGTGTCCTCACCCCAC





 


GI29801767_THBS1_3R
NT_010194
GTGGAGGAGAGTCAGCGAGGGCCCGAGGGG
TTCGTTAAAATACCTACCCCTCG
ACCCTCGCTAACTCTCCTCC
CATTCATTAAAATACCTACCCCTCA


(SEQ ID 233)

CAGGTACTTTAACGAATG





 


GI29801784_ELK1_1R
NT_011568
GCCTTTTCAGTTGCTCACAGTCCGTCAGTC
CCTTTTCAATTACTCACAATCCG
CAATCCTTAAACCAATCGACGTA
ACCTTTTCAATTACTCACAATCCA


(SEQ ID 234)

CTTGAGCCAATCGGCGTGGA





 


GI29801784_ELK1_2R
NT_011568
AGCGCTTTGGCCAATCAGCGAGCGGCGGGA
GCTTTAACCAATCAACGAACG
CGAAACATTAAACTCCTCCTCCT
AACACTTTAACCAATCAACAAACA


(SEQ ID 235)

CATTGGGCTCCTCCTCCTC





 


GI29801784_ELK1_3R
NT_011568
CCATTTCTATACAAGCCCTGCTTCCGCTGA
CATTTCTATACAAACCCTACTTCCG
TAAACAACATAACGTACGACACCG
CCATTTCTATACAAACCCTACTTCCA


(SEQ ID 236)

GCAGCATGGCGTGCGACACCGCC





 


GI29802832_FMR1_1R
NT_011681
CGGGGGTTCGGCCTCAGTCAGGCGCTCAGC
AAAATTCGACCTCAATCAAACG
TCAACTCCGTTTCGATTTCACT
CAAAAATTCAACCTCAATCAAACA


(SEQ ID 237)

TCCGTTTCGGTTTCACTTC





 


GI29802832_FMR1_2R
NT_011681
CAGAAATGGGCGTTCTGGCCCTCGCGAGGC
AATAAACGTTCTAACCCTCGCG
AACAATACGACCTATCACCGC
CAAAAATAAACATTCTAACCCTCACA


(SEQ ID 238)

AGTGCGACCTGTCACCGCCCT





 


GI29802832_FMR1_3R
NT_011681
ACCTCCCGCTCAGTCAGACTGCGCTACTTT
TCCCGCTCAATCAAACTACG
TACTTTAAACCGAACCAAACCAA
ACCTCCCACTCAATCAAACTACA


(SEQ ID 239)

GAACCGGACCAAACCAAA





 


GI29802923_APAF1_1R
NT_019546
TCCACTGCGATATTGCTCCAAATCCGAGGA
CACTACGATATTACTCCAAATCCG
AAAAATTCAAACTCCCGAACG
TCCACTACAATATTACTCCAAATCCA


(SEQ ID 240)

AATTCAAACTCCCGGGCGCG





 


GI29802923_APAF1_2R
NT_019546
AGCAGGGGCTCCCTTGGGCCCCGACTTCTT
ACAAAAACTCCCTTAAACCCCG
CTTCTTCCGACTCTTCACCTCAA
AACAAAAACTCCCTTAAACCCCA


(SEQ ID 241)

CCGGCTCTTCACCTCAG





 


GI29802923_APAF1_3R
NT_019546
CCCGAGTCCGGCATTGGTGGGAACGCGGCG
CGAATCCGACATTAATAAAAACG
GACGCGTCCCTAAAACTTAACC
CCCAAATCCAACATTAATAAAAACA


(SEQ ID 242)

CGTCCCTGAGGCTTAGCCACG





 


GI29803889_GPC3_1R
NT_011786
GCTGAAAGAAGGCCTGTGGCGAAGCGGAGG
CCTAACTTAAATCCCCCTCCG
TTCGCCACAAACCTTCTTTC
CCTAACTTAAATCCCCCTCCA


(SEQ ID 243)

GGGACCTAAGTCAGG





 


GI29803889_GPC3_2R
NT_011786
CCAGAGCGCCCTGTGTAGAGCGGCTGCGAG
ACGCCCTATATAAAACGACTACG
ACGAACAACTAAACTCGACTACCG
CCAAAACACCCTATATAAAACAACTAA


(SEQ ID 244)

CGGGCAGCTGGGCTCGGCTGCCGGG





 


GI29803889_GPC3_3R
NT_011786
CCAGAGCCAGTCAGAGCGGACGGCTGCTGG
CAAAACCAATCAAAACGAACG
CTACTAAAAAACCAATCAACGCG
CCAAAACCAATCAAAACAAACA


(SEQ ID 245)

GAAGCCAATCAGCGCGCTCG





 


GI29805200_CCND2_1R
NT_009759
AAATACAAGGGCAGGAGGATTAGGATCCGT
CCAACTTTAACTTCTTCAAAACG
ATCCTAATCCTCCTACCCTTATATTT
CTCCAACTTTAACTTCTTCAAAACA


(SEQ ID 246)

TTTGAAGAAGCCAAAGTTGGAG





 


GI29805200_CCND2_3R
NT_009759
GGGAGGAGAGCTAACTGCCCAGCCAGCTTG
TCTCCGCTCTAAAACGATAACG
AAACTAACTAAACAATTAACTCTCCTC
CTCTTCTCCACTCTAAAACAATAACA


(SEQ ID 247)

CGTCACCGCTTCAGAGCGGAGAAGAG





 


GI29806267_SOD1_1R
NT_011512
AGAAGGTTGTTTTCTCCACATTTCGGGGTT
AAAAATTATTTTCTCCACATTTCG
AATTCTAAACGTTTCCCGACTAC
AAAAAATTATTTTCTCCACATTTCA


(SEQ ID 248)

CTGGACGTTTCCCGGCTGCGGG





 


GI29806267_SOD1_2R
NT_011512
TTTACATCATTTTGCCAATTTCGCGTACTG
ACATCATTTTACCAATTTCGCG
ACTACAACCGACGAACCACG
TTTACATCATTTTACCAATTTCACA


(SEQ ID 249)

CAACCGGCGGGCCACGCC





 


GI29806267_SOD1_3R
NT_011512
CAGTCATTCCCGGCCACTCGCGACCCGAGG
AATCATTCCCGACCACTCG
GACCCGAAACTACCGCAAA
CAATCATTCCCAACCACTCA


(SEQ ID 250)

CTGCCGCAGGGGGC





 


GI29806588_GP1BB_1R
NT_011519
CCCCAGCACCGCTGTGGTGTGCCGGGATCC
CCAACACCGCTATAATATACCG
AATCCTAAACCTAAACCTCCCGA
CCCCAACACCACTATAATATACCA


(SEQ ID 251)

TGAGCCTAGGCCTCCCGA





 


GI29806588_GP1BB_2R
NT_011519
GAGACCCCATTTTCTGTCGAGGCGGGCCGA
AACCCCATTTTCTATCGAAACG
ACCGAATCTTCCCTTATCCC
AAAACCCCATTTTCTATCAAAACA


(SEQ ID 252)

GTCTTCCCTTATCCCC





 


GI29806588_GP1BB_3R
NT_011519
AAGGAGGCCAGAGGCTGCAAGGAGCGGGGT
AATATAAACGATCACGACCCCG
TCCTTACAACCTCTAACCTCCTT
AAAATATAAACAATCACAACCCCA


(SEQ ID 253)

CGTGACCGCTTACACCCC





 


GI29807454_WT1_2R
NT_009237
TTTCTGCGCTTTCCTGAAGTTCCCGCCCTC
TACGCTTTCCTAAAATTCCCG
CCTCTTAAAACCTACCTACCCCTC
TTTCTACACTTTCCTAAAATTCCCA


(SEQ ID 254)

TTGGAGCCTACCTGCCCCTC





 


GI29807454_W71_3R
NT_009237
CGCCCGGCTTATAACTGGTGCAACTCCCGG
AACGTCCCTCAATTAAATAACCG
AAATTACACCAATTATAAACCGAACG
AAACATCCCTCAATTAAATAACCA


(SEQ ID 255)

CCACCCAACTGAGGGACGTTC





 


GI29808062_SOCS1_1R
NT_010393
CGAACAGGCGGGCAGAGGGCCCCGCGGGAG
CCTCTCTTCTAAACCCTCCCG
GAAACCCTCTACCCGCCTATT
CCTCTCTTCTAAACCCTCCCA


(SEQ ID 256)

GGTCCAGAAGAGAGG





 


GI29808062_SOCS1_2R
NT_010393
GGGTCCGAGAAGTGGCCGGAAGGCGCAGGG
CCGAAAAATAACCGAAAAACG
AAAATCGAAACCAAAACCCC
AAATCCAAAAAATAACCAAAAAACA


(SEQ ID 257)

TCGGGGCCAGAGCCCCTC





 


GI29808062_SOCS1_3R
NT_010393
CGGGCGCCGAACAGAGCGAGCTGCGGCCGT
GCCGAACAAAACGAACTACG
CCGTAACAACTACACGACTCCTAA
CAAACACCAAACAAAACAAACTACA


(SEQ ID 258)

GGCAGCTGCACGGCTCCTGGCC





 


GI29808625_CDH3_2R
NT_010498
GAGAATCGCTTCAGCCCGGGAGGTCGAGGC
GATCTCGACTCACTACAACCTCG
CCTCCCGAACTAAAACGATTCT
CAATCTCAACTCACTACAACCTCA


(SEQ ID 259)

TGTAGTGAGCCGAGATCG





 


GI29808625_CDH3_3R
NT_010498
CGAGATCGCGCTACTGCACTCCTGGGCGAC
CGCGCTACTACACTCCTAAACG
CAAAACGAAACCCTATCTCCAA
CAAAATCACACTACTACACTCCTAAAA


(SEQ ID 260)

AGAGCGAGACCCTGTCTCCAAA





 


GI29808952_TUBB4_1R
NT_010542
AAAGACAGGGAGCTGGGATGGTGCGGGTTG
CCGATTTAAAAACCAACCCG
ACCATCCCAACTCCCTATCTTT
ACACCAATTTAAAAACCAACCCA


(SEQ ID 261)

GTCTCTAAACCGGCGT





 


GI29808952_TUBB4_2R
NT_010542
CGCGCGGTGCGGAGCCTGCGGGCCGGGCGG
CGATACGAAACCTACGAACCG
ACGAAACTCTACGACGACGC
CACACAATACAAAACCTACAAACCA


(SEQ ID 262)

GGCTCTGCGGCGGCGCCTCC





 


GI29808952_TUBB4_3R
NT_010542
CCCGCGGTGACATCAGCCGATGCGAAGGGC
GCGATAACATCAACCGATACG
AAAACGAAACCGCGACTATAAA
CCCACAATAACATCAACCAATACA


(SEQ ID 263)

GGGGCCGCGGCTATAAGAGCG





 


GI29823164_ADCYAP1_1R
NT_010859
ATGCCTCTCGGGTGGTGACTCCAGCGCAGG
AACGCTTCTTCAAATTCCTACG
TAAAATCACCACCCGAAAAACAT
CAAAACACTTCTTCAAATTCCTACA


(SEQ ID 264)

AACTTGAAGAAGCGCAAG





 


GI29823164_ADCYAP1_2R
NT_010859
GGTAAGGGAGGGAAAATCTTACCAAAGCGA
CAACAATCGAATAAACCGATCG
TTTAATAAAATTTTCCCTCCCTTACC
AAATCAACAATCAAATAAACCAATCA


(SEQ ID 265)

CCGGCTCACTCGACTGCTGATTC





 


GI29823164_ADCYAP1_3R
NT_010859
TCCTGCTGCTCCCGCTGGTTCCTGCGGCTT
TACTACTCCCGCTAATTCCTACG
CTTCTACTCAAACACCAACGCC
TCCTACTACTCCCACTAATTCCTACA


(SEQ ID 266)

CTGCTCAGACACCAACGCCA





 


GI29823164_MC2R_1 R
NT_010859
CCTGAAGAATCAATCAAGTTTTCCGTGAAG
AAAAAATCAATCAAATTTTCCG
AAAATCAAATCCAAATAACATCCC
CCTAAAAAATCAATCAAATTTTCCA


(SEQ ID 267)

TCAAGTCCAAGTAACATCCC





 


GI29823167_ATP5A1_1R
NT_010966
TCTCCTCCTGCGGCTCCATAGCTGCGGCTC
CCTCCTACGACTCCATAACTACG
CTCCCGCCACTTTACTAAAAACT
TCTCCTCCTACAACTCCATAACTACA


(SEQ ID 268)

CCGCCACTTTACTAGGAACTCC





 


GI29823167_ATP5A1_2R
NT_010966
GGCTCTGGCATTGCAAGCCTCGCTTCGTTG
CTAACATTACAAACCTCGCTTG
TACCACTTCCCAACTCTTCCC
AACTCTAACATTACAAACCTCACTTCA


(SEQ ID 269)

CCACTTCCCAGCTCTTCCC





 


GI29823167_ATP5A1_3R
NT_010966
CTCCCAGTGTTGGGATGCAGGGCGCGGAAG
CTTCCACATCTAAACTACTTCCG
GCCCTACATCCCAACACTAAAA
CAACTTCCACATCTAAACTACTTCCA


(SEQ ID 270)

CAGTTCAGATGTGGAAGCCG





 


GI17458490_PLAGL2_1R
NT_028392
CGGGTCCCAGGAGCCCGCAGGCATCCCCGG
TACTACTACTTCTACCGCTCCCG
AAATACCTACGAACTCCTAAAACCC
TTCCTACTACTACTTCTACCACTCCCA


(SEQ ID 271)

GAGCGGCAGAAGCAGCAGCAGGAA





 


GI17458490_PLAGL2_2R
NT_028392
ACGCTCAGCGGCCGTGCCCACGCGGCGGCG
TCAACGACCGTACCCACG
GACGACGCCCATATCGAC
ACACTCAACAACCATACCCACA


(SEQ ID 272)

CCCATGTCGGCCCG





 


GI17458490_PLAGL2_3R
NT_028392
GGCTTAGACCTCAAGCAAGTCACGTAACCT
ACTTAAACCTCAAACAAATCACG
AACCTCTCCCAACCTCAATTTT
AACTTAAACCTCAAACAAATCACA


(SEQ ID 273)

CTCCCAGCCTCAGTTTT





 


GI29732427_ABL1_1R
NT_035014
AAAGCAGAGAAGCGAGAGCGGCCACTAGTT
TTCCAACAAATTTCCTACCG
ACTAATAACCGCTCTCGCTTCT
ATCTTCCAACAAATTTCCTACCA


(SEQ ID 274)

CGGCAGGAAATTTGTTGGAAGAT





 


GI29732427_ABL1_2R
NT_035014
CAATGCATTCCTTTTCGTCAGAGTCGAGGG
ACATTCCTTTTCGTCAAAATCG
AAACAAACTCGCTAAAATCTAAATAAC
CAATACATTCCTTTTCATCAAAATCA


(SEQ ID 275)

CAAACTCGCTGAAATCTGGGTGACCC





 


GI29732427_ABL1_3R
NT_035014
GGATTGGTTCCACAGGCCTTTTAAAAAGCG
CATTACCAACAACTTTTAAATCCG
TTTTTAAAAAACCTATAAAACCAATCC
CATTACCAACAACTTTTAAATCCA


(SEQ ID 276)

GACTTAAAAGTTGCTGGCAATG





 


GI29736559_CRIP1_1R
NT_026437
GGGACAGACTCCGCCTGGCACCGGGACCAT
ACAAACTCCGCCTAACACCG
AACCATCCTCCGCCTCA
AAAACAAACTCCACCTAACACCA


(SEQ ID 277)

CCTCCGCCTCAACTTTG





 


GI29736559_CRIP1_2R
NT_026437
GTGCCAGACGCCTGTGCCTTGGCCGTCCTA
CCAAACGCCTATACCTTAACCG
CCTACAACCCTACTACCCCTACC
ATACCAAACACCTATACCTTAACCA


(SEQ ID 278)

CAGCCCTGCTGCCCCTGCC





 


GI29736559_CRIP1_3R
NT_026437
CTGGAGTGTGGCCTCTGTGCCAGACGCCTG
TAAAACGACCAAAACACAAACG
CTAACACAAAAACCACACTCCAA
CTATAAAACAACCAAAACACAAACA


(SEQ ID 279)

TGCCTTGGCCGTCCTACAG





 


GI29736559_DAD1_1R
NT_026437
CCTAGAGGAAGTTTAGTCCTTGGGACGCTT
TCAAAAACTAACAAACCAAACG
CCCAAAAACTAAACTTCCTCTAAA
TTTCAAAAACTAACAAACCAAACA


(SEQ ID 280)

GGCCTGCCAGTCTCTGAAA





 


GI29736559_DAD1_2R
NT_026437
GATGGTGGTGGTGGTAGTGCACGTTGGGTT
ATCCCCTCAACCCAACG
ACACTACCACCACCACCATC
ATCCCCTCAACCCAACA


(SEQ ID 281)

GAGGGGAC





 


GI29736559_FOS_1R
NT_026437
TCTGAGACAGGAACTGCGAAATGCTCACGA
CCTTAACGCGTATCCTAATCTCG
AAACATTTCGCAATTCCTATCTC
CCACCTTAACACATATCCTAATCTCA


(SEQ ID 282)

GATTAGGACACGCGCCAAGGCGG





 


GI29736559_FOS_2R
NT_026437
CAAGGGTCCGCATTGAACCAGGTGCGAATG
AATCCGCATTAAACCAAATACG
ATATTCTCTCTCATTCTACGCCG
CAAAAATCCACATTAAACCAAATACA


(SEQ ID 283)

TTCTCTCTCATTTCTGCGCCGTTCC





 


GI29736559_FOS_3R
NT_026437
CGGGAGCCGAGGCTTAAGTCCTCGGGGTCC
GAAAACCGAAACTTAAATCCTCG
AATCCTATACTCGATACCGTTTCTC
CAAAAACCAAAACTTAAATCCTCA


(SEQ ID 284)

TGTACTCGATGCCGTTTCTCC





 


GI29736559_HSPA2_1R
NT_026437
AGACATAGGGTTTGGTGTGGCTGTGCGGGG
AAAACCCGAAAACCCCG
ACAACCACACCAAACCCTATATCT
ACAAAACCCAAAAACCCCA


(SEQ ID 285)

TTCCCGGGCCTTGC





 


GI29736559_HSPA2_2R
NT_026437
GCTGGCCTGTCGGCCTCTGATGCACTCGAA
CCTATCGACCTCTAATACACTCG
ACTTCCAACCCTTAAAACAAACA
ACTAACCTATCAACCTCTAATACACTA


(SEQ ID 286)

CTTCCAGCCCTTGGAGCAGACA





 


GI29738185_ESR1_1R
NT_023451
TCAGGGAAGCTGCTCTTTGGGATCGCTCCA
CAAACACAACTCGATTTAAAACG
TCCCAAAAAACAACTTCCCTAA
TCCAAACACAACTCAATTTAAAACA


(SEQ ID 287)

AATCGAGTTGTGCCTGGA





 


GI29738185_ESR1_2R
NT_023451
GGCCCCTGGATCCGTCTTTCGCGTTTATTT
CCTAAATCCGTCTTTCGCG
TTATTTTAAACCCAATCTTCCCT
AACCCCTAAATCCATCTTTCACA


(SEQ ID 288)

TAAGCCCAGTCTTCCCT





 


GI29738185_ESR1_3R
NT_023451
CCCCATTCTATCTGCCCTATCTCGGTTACA
CCCATTCTATCTACCCTATCTCG
TTACAATATAATCCTCCCCAAAATC
CCCCATTCTATCTACCCTATCTCA


(SEQ ID 289)

GTGTAGTCCTCCCCAGGGTC





 


GI29738645_RB1_1R
NT_024524
CCTGGAAGGCGCCTGGACCCACGCCAGGTT
TAAAAAACGCCTAAACCCACG
CAAATTTCCCAATTTAATTCCTCA
CCTAAAAAACACCTAAACCCACA


(SEQ ID 290)

TCCCAGTTTAATTCCTCA





 


GI29738645_RB1_3R
NT_024524
GCGGGCGGAAGTGACGTTTTCCCGCGGTTG
AACGAAAATAACGTTTTCCCG
GATTAAACGCGACGCTCAATTAC
ACAAACAAAAATAACATTTTCCCA


(SEQ ID 291)

GACGCGGCGCTCAGTTGCCGGG





 


GI29738863_GDF10_1R
NT_030772
TCGCAGCCAGGGCAGGCTGCGGCGCCGACA
GCAACCAAAACAAACTACGACG
CGACACACAAAAACCGACTAC
TCACAACCAAAACAAACTACAACA


(SEQ ID 292)

CACAGGAGCCGGCTGCGGG





 


GI29738863_GDF10_2R
NT_030772
TCATCTTGCAATCACATAGCCATCGCATGC
TCATCTTACAATCACATAACCATCG
ATACCATTACAAAAACAACCCAACA
TCATCTTACAATCACATAACCATCA


(SEQ ID 293)

CATTGCAGGGGCAACCCAGCA





 


GI29738863_GDF10_3R
NT_030772
CATACGCTGACTCCGAGGCAACTGGGCGCA
GCTAACTCCGAAACAACTAAACG
ACACATATCCCCTCTCTAATCCTC
CATACACTAACTCCAAAACAACTAAAA


(SEQ ID 294)

CACATATCCCCTCTCTGGTCCTC





 


GI29739550_OAT_1R
NT_035040
CAGGGTTCAAGGGCATGGGGCTCCCGCAGC
CTCTACCCCAACCACAACTACG
AAACCCCATACCCTTAAACCCTA
ACTCTACCCCAACCACAACTACA


(SEQ ID 295)

TGTGGCTGGGGCAGAGC





 


GI29739550_OAT_2R
NT_035040
GCCCAACAGACTTTTCCTTTTCGGGCCTCA
CCCAACAAACTTTTCCTTTTCG
ACCTCAATCTTCTCGTCAACAAA
ACCCAACAAACTTTTCCTTTTCA


(SEQ ID 296)

GTCTTCTCGTCAGCAAG





 


GI29739550_OAT_3R
NT_035040
CACGCGATTGGTATCCTGCCCTCCGCCCCA
GCGATTAATATCCTACCCTCCG
CCCAACCAATAAACGACGAAA
CACACAATTAATATCCTACCCTCCA


(SEQ ID 297)

GCCAATGAGCGGCGAGGGT





 


GI29789893_CTNNB1_1R
NT_037565
TCCCCGATGCAGACCACAGCGCCCTCACGG
CCCGATACAAACCACAACG
CCTCACGAACTACCCTCAAAC
TCCCCAATACAAACCACAACA


(SEQ ID 298)

GCTGCCCTCAGGCC





 


GI29791372_RARB_1R
NT_022517
AAAGGGAGAGAAGTTGGTGCTCAACGTGAG
GAAACGCTACTCCTAACTCACG
TAAACACCAACTTCTCTCCCTTT
AACCAAAACACTACTCCTAACTCACA


(SEQ ID 299)

CCAGGAGCAGCGTCCCGGCT





 


GI29791372_RARB_2R
NT_022517
CGTGAGCCAGGAGCAGCGTCCCGGCTCCTC
GTAAACCAAAAACAACGTCCCG
CTCCTCCCCTACTCATTTTAAAA
CATAAACCAAAAACAACATCCCA


(SEQ ID 300)

CCCTGCTCATTTTAAAA





 


GI29791372_RARB_3R
NT_022517
GAGCGGGCGCAGGCGGAACACCGTTTTCCA
AACGCAAACGAAACACCG
TTTCCAAACTAAACCGCCG
AAACAAACACAAACAAAACACCA


(SEQ ID 301)

AGCTAAGCCGCCGCAAA





 


GI29791375_ARHI_1R
NT_032977
GGCTGGGGCACACAAAACCAGGTGCTACCG
ACCCTATAAACCTAATCGTTAACG
TAACACCTAATTTTATATACCCCAACC
CAATACCCTATAAACCTAATCATTAAA


(SEQ ID 302)

TCAACGACTAGGCCCATAGGGTACTG





 


GI29791375_ARHI_2R
NT_032977
AAGGGAGAAAGAAGCCAGACGGAGCTCGGA
GTCTACCCTCCACATCTCCG
ACTCCGTCTAACTTCTTTCTCCC
TACATCTACCCTCCACATCTCCA


(SEQ ID 303)

GATGTGGAGGGCAGACGCA





 


GI29791375_ARHI_3R
NT_032977
CCCAAAACGCTCGGTAGGCGGTGGTGCGCA
AACGCTCGATAAACGATAATACG
AACTTTCAATACATCCGCCG
CCCAAAACACTCAATAAACAATAATAA


(SEQ ID 304)

GCTTTCAATGCATCCGCCGCCA





 


GI29791382_SFN_2R
NT_037485
CTCCTGAACCTTTATGGGCTCTGTCGAGGC
ACCCCTAACTACTTCAACCTCG
CAAAACCCATAAAAATTCAAAAA
AACCCCTAACTACTTCAACCTCA


(SEQ ID 305)

TGAAGCAGCCAGGGGCT





 


GI29791382_SFN_3R
NT_037485
GCAGGTCTTGGGCCCTGGTCCCGCCCCCTG
CAAATCTTAAACCCTAATCCCG
CCCCTACTCCTCCCCAC
ACAAATCTTAAACCCTAATCCCA


(SEQ ID 306)

CTCCTCCCCAC





 


GI29794267_SFTPC_1R
NT_023666
GAGGGGCTGGAGGAGGAAACGGGGAGGCCC
CCCTTCTATATAAACCTCCCCG
TTCCTCCTCCAACCCCTC
CCCTTCTATATAAACCTCCCCA


(SEQ ID 307)

ACACAGAAGGG





 


GI29794267_SFTPC_2R
NT_023666
GGGCAACTGTCCCCACCCGTCCCTGGCACG
AAACAACTATCCCCACCCG
CCCTAACACGACTCTACCCAA
AAACAACTATCCCCACCCA


(SEQ ID 308)

GCTCTGCCCAGT





 


GI29794267_SFTPC_3R
NT_023666
TCCTTGCTGCCACACATTTAGCCGCCCTCC
CCTTACTACCACACATTTAACCG
CCTCCCCATACCAACTTAAAA
TCCTTACTACCACACATTTAACCA


(SEQ ID 309)

CCATGCCAGCTTGGGG





 


GI29794674_EGFR_1R
NT_033968
GTTGGGTGCCCTCATTTCAGATGATTTCGA
TCAAATCTTATCAAACACCCTCG
AATCATCTAAAATAAAAACACCCAAC
TTCAAATCTTATCAAACACCCTCA


(SEQ ID 310)

GGGTGCTTGACAAGATCTGAA





 


GI29794674_EGFR_2R
NT_033968
GGGGACCCTGGCACAGATTTGGCTCGACCT
AAACCCAACCTATATCCAAATCG
ACCAAATCTATACCAAAATCCCC
CAAACCCAACCTATATCCAAATCA


(SEQ ID 311)

GGACATAGGCTGGGCCTG





 


GI29794674_EGFR_3R
NT_033968
GGCAGTGCTGGGAACGCCCCTCTCGGAAAT
AATACTAAAAACGCCCCTCTCG
AAATTAACTCCTCAAAACACCCG
AACAATACTAAAAACACCCCTCTCA


(SEQ ID 312)

TAACTCCTCAGGGCACCCGC





 


GI29796148_TERT_1R
NT_023089
GACCTGGAGGCAGCCCTGGGTCTCCGGATC
CTTTAACCGCTAACCTAATCCG
AAACCCAAAACTACCTCCAAATC
CCCTTTAACCACTAACCTAATCCA


(SEQ ID 313)

AGGCCAGCGGCCAAAGGG





 


GI29796148_TERT_2R
NT_023089
CCGCCCGGAGCAGCTGCGCTGTCGGGGCCA
CCCGAAACAACTACGCTATCG
AACCAAACCGAACTCCCAATAA
CCACCCAAAACAACTACACTATCA


(SEQ ID 314)

GGCCGGGCTCCCAGTGGA





 


GI29796148_TERT_3R
NT_023089
GGGTTACCCCACAGCCTAGGCCGATTCGAC
AATTACCCCACAACCTAAACCG
TTCGACCTCTCTCCGCTAAA
AAATTACCCCACAACCTAAACCA


(SEQ ID 315)

CTCTCTCCGCTGGGGCC





 


GI29797939_IL13_1R
NT_034772
GGCCGCGCAGATCCCGCTTATCGGGCCCAT
CGCAAATCCCGCTTATCG
ACCCATCTCCCGTTACATAAAAC
AACCACACAAATCCCACTTATCA


(SEQ ID 316)

CTCCCGTTACATAAGGC





 


GI29797939_IL13_2R
NT_034772
GAGCCGCTGGCCCGGGTGTCCAGCCGGCCC
GCCAAACAAAACAAAAACCG
CTAAACACCCGAACCAACGACT
AACACCAAACAAAACAAAAACCA


(SEQ ID 317)

TTGCCCTGCCTGGCGCT





 


GI29797939_IL13_3R
NT_034772
CACGCAGCGCATTGCAGGCAGGTGCGAGCC
GCAACGCATTACAAACAAATACG
ACCACATACGCCAAATACCC
CACACAACACATTACAAACAAATACA


(SEQ ID 318)

ACATGCGCCAAGTGCCCGGA





 


GI29797939_LOX_1R
NT_034772
ATCCTGGGGTTTATTTGCTGAGGGCGCTTC
CACTCTCTCGCTTTTATAAAAAACG
CCTCAACAAATAAACCCCAAAAT
CACACTCTCTCACTTTTATAAAAAACA


(SEQ ID 319)

CCATAAAAGCGAGAGAGTGTG





 


GI29797939_LOX_2R
NT_034772
GTGAACAAATAGCTGAGGGGCGGCCGGGCC
ATTACACAAACCGTTCTAACCCG
CCGCCCCTCAACTATTTATTC
AAATTACACAAACCATTCTAACCCA


(SEQ ID 320)

AGAACGGCTTGTGTAACTT





 


GI29797939_LOX_3R
NT_034772
AGGCGAGGAGCTGTCCGCCTTGCACGTTTC
GAAAAACTATCCGCCTTACACG
TTCCAATCGCATTACGTAAACA
AAACAAAAAACTATCCACCTTACACA


(SEQ ID 321)

CAATCGCATTACGTGAACAAATAG





 


GI29800185_SFTPD_1R
NT_030059
TTGTGAATATCAGTGGCAGGTTTCCAGAAC
TCACTCTTATCCCCACCTACG
TCTAAAAACCTACCACTAATATTCACA
ACTCACTCTTATCCCCACCTACA


(SEQ ID 322)

GCAGGTGGGGATAAGAGTGAGT





 


GI29800185_SNCG_1R
NT_030059
CGGTTTCATGGCAGCCCAGGGTCCAGCGGC
CCACCAACATCCTAAATACCG
TAAACCCTAAACTACCATAAAACCG
CCCACCAACATCCTAAATACCA


(SEQ ID 323)

ATCCAGGATGCTGGTGGG





 


GI29800185_SNCG_2R
NT_030059
AGCTGCACAGCCCAGGCCGCGGGAGGTTGG
ACACAACCCAAACCGCG
AAAATTAACTACTCTCACCTAACAAAC
AACTACACAACCCAAACCACA


(SEQ ID 324)

CTGCTCTCACCTAACAGGCC





 


GI29800185_SNCG_3R
NT_030059
ACCTCAGATCCTCCAACCGGTTTCATGGCA
ACCTCAAATCCTCCAACCG
TTTCATAACAACCCAAAATCCA
ACCTCAAATCCTCCAACCA


(SEQ ID 325)

GCCCAGGGTCCA





 


GI29801752_PLAGL1_1R
NT_025741
AGTGCCTGGCTCACGGTCAGTGCTCACGTT
CCCAAATACAACTACCCAAACG
AAACACTAACCGTAAACCAAACAC
ACCCAAATACAACTACCCAAACA


(SEQ ID 326)

TGGGCAGCTGCACTTGGGC





 


GI29801752_PLAGL1_2R
NT_025741
CCAGGCCGGCTCGGGTCTACCTGCGCCAGC
AACCGACTCGAATCTACCTACG
CAACGCTATACCTAAACGACCTT
CCAAACCAACTCAAATCTACCTACA


(SEQ ID 327)

GCTGTACCTGGGCGACCTTGGCTT





 


GI29801752_PLAGL1_3R
NT_025741
GGGCTCTGGCGGCCCATCCTGGCGGAGACT
ACTCTAACGACCCATCCTAACG
AAACTTCGACTAACAAACCCCG
AAACTCTAACAACCCATCCTAACA


(SEQ ID 328)

TCGGCTAGCAGGCCCCGC





 


GI29803948_CD63_1R
NT_029419
CTGGCTTTGTCTTCTGGGATAAGACGGGGA
CTTCATCCTCTAAATTTCCCCG
CTTATCCCAAAAAACAAAACCAA
ACTTACATCCTCTAAATTTCCCCA


(SEQ ID 329)

AATCCAGAGGATGTAAGC





 


GI29803948_CD63_2R
NT_029419
GGCAGCTTGATCCCCCTAGCCCGCTTCCAG
ACAACTTAATCCCCCTAACCCG
TTCCAACCCTCACCTAACTTTATC
AACAACTTAATCCCCCTAACCCA


(SEQ ID 330)

CCCTCACCTGGCTTTGTC





 


GI29803948_CD63_3R
NT_029419
GGTGTGACGGCCGGTCCTACTCTCCGGCCT
ATAACGACCGATCCTACTCTCCG
CCTATTCAAATCAACTCCTTCTAAA
AATATAACAACCAATCCTACTCTCCA


(SEQ ID 331)

GTTCAGATCAGCTCCTTCTGGG





 


GI29803948_CDK4_1R
NT_029419
TCCTGGGGACCCTTGACCCTCCGACACAAA
CTAAAAACCCTTAACCCTCCG
CACAAACAAACGCTCCAAAA
TCCTAAAAACCCTTAACCCTCCA


(SEQ ID 332)

CAAGCGCTCCAGGAGTC





 


GI29803948_CDK4_2R
NT_029419
CCCTTAGCTTACGGAGACCCTGGGGCGAGG
ACTATTCTCTCCTATTTCCCTCG
CCCAAAATCTCCGTAAACTAAAA
AACTACTATTCTCTCCTATTTCCCTCA


(SEQ ID 333)

GAAATAGGAGAGAACAGCAGTC





 


GI29803948_CDK4_3R
NT_029419
TGTTAATGGGTGAGAGGGATTTTGCGCTCT
TAAAACTTTCTCCCCGAAAAACG
AAAATCCCTCTCACCCATTAACA
ATAAAACTTTCTCCCCAAAAAACA


(SEQ ID 334)

CCGGGGAGAAAGCCCCAC





 


GI29804485_BCAP31_1R
NT_025965
GGCCCTCCAGGGTCGCGGCCTCTTCGGGCA
AATTCCTCCACTTAAACTACCCG
AAAAACCGCGACCCTAAAAA
AAATTCCTCCACTTAAACTACCCA


(SEQ ID 335)

GCCCAAGTGGAGGAACTT





 


GI29804485_BCAP31_2R
NT_025965
GGGCTCAGGGCCCTCCAGGGTCGCGGCCTC
AACTCAAAACCCTCCAAAATCG
GACCTCTTCGAACAACCCAAATA
AAACTCAAAACCCTCCAAAATCA


(SEQ ID 336)

TTCGGGCAGCCCAAGTG





 


GI29804485_CTAG1_1R
NT_025965
CCAGCTGGGCGACCAGGACAGGGACGGAGG
CTAACTAAACTCAACAACCTCCG
CCCTATCCTAATCGCCCAACTAA
CTCTAACTAAACTCAACAACCTCCA


(SEQ ID 337)

CTGCTGAGCCCAGTTAGAG





 


GI29804485_CTAG1_2R
NT_025965
GAGACCTTGGCTGGCGCGAGGCCACGCCCA
CTTAACTAACGCGAAACCACG
CCACCAAACATACAATTCCAACT
AAAACCTTAACTAACACAAAACCACA


(SEQ ID 338)

CCAGACATGCAGTTCCAGCT





 


GI29804485_CTAG1_3R
NT_025965
GGGCGACTAGGACAGGGACAGAACCCGTTG
GAATCTCACTCCTAAATTCAACG
ATTCTATCCCTATCCTAATCGCC
CCAAATCTCACTCCTAAATTCAACA


(SEQ ID 339)

AACCCAGGAGTGAGATCCGG





 


GI29804485_STK23_1R
NT_025965
GGGGGGCCTCTGCCCGGGGGCCGGGCTCTC
AAAACCTCTACCCGAAAACCG
ACTCTCTAAAACCGCCGTCG
AAAAAACCTCTACCCAAAAACCA


(SEQ ID 340)

TGGGGCCGCCGTCGCGCC





 


GI29804485_STK23_2R
NT_025965
GGGGTACGGGCTGCTTCACCCCCGCGTGGA
AATACGAACTACTTCACCCCCG
GTAAACCTCCCCCTCAACTAAAA
AAAATACAAACTACTTCACCCCCA


(SEQ ID 341)

CCTCCCCCTCAGCTGGGG





 


GI29804485_STK23_3R
NT_025965
CTGGGAGCCAGAGCGCCAGGCAGCGTCGCA
CAACCCAAAACCTACTCCTACG
CGCTACCTAACGCTCTAACTCC
CCAACCCAAAACCTACTCCTACA


(SEQ ID 342)

GGAGCAGGTTCTGGGCTGG





 


GI29804900_PRKCDBP_1R
NT_028310
TGAAACAGGCACACCAGGGAATTGGAGCGG
AAATTTAAATTACCCTCCTCCG
TCCAATTCCCTAATATACCTATTTCA
CTCTAAATTTAAATTACCCTCCTCCA


(SEQ ID 343)

AGGAGGGTAACTCAAACTCAGAG





 


GI29804900_PRKCDBP_2R
NT_028310
GGCCCCCTGGTTATCTCTTTGCCGTGCCAA
CCCCTCTAATTATCTCTTTACCG
ACCAACACAATCTCTACGCCC
AACCCCTCTAATTATCTCTTTACCA


(SEQ ID 344)

CACAGTCTCTGCGCCCAC





 


GI29804900_PRKCDBP_3R
NT_028310
GCAGCCTGTCCAAGTCACAAAGCGGGGCCT
CAACCTATCCAAATCACAAAACG
AACCTCGAACCTTAACAATTCG
ACAACCTATCCAAATCACAAAACA


(SEQ ID 345)

CGGGCCTTGACAGTTCGCG





 


GI29805597_APOA1_1R
NT_033899
AAGGGGATGAGTGCAGGGAACCCCGACCCC
CAAATCTCCCGAATAAAATCG
AATTCCCTACACTCATCCCCTT
CTTACAAATCTCCCAAATAAAATCA


(SEQ ID 346)

ACCCGGGAGACCTGCAAG





 


GI29805597_APOA1_2R
NT_033899
GCAGACACTCCCCTCCCGCCCCCACTGAAC
CAAACACTCCCCTCCCG
CCCCACTAAACCCTTAACCC
ACAAACACTCCCCTCCCA


(SEQ ID 347)

CCTTGACCC





 


GI29805597_APOA1_3R
NT_033899
CGAAACCTCAGTCTGGGAGCCACGGAGGGC
AAACCTCAATCTAAAAACCACG
AAAACTCTCCCCTCTCCCC
CAAAACCTCAATCTAAAAACCACA


(SEQ ID 348)

TCTCCCCTCTCCCC





 


GI29805597_PGR_1R
NT_033899
GCCCCTCCCCAGATGATCGAGGGGTCCAGG
CCCCTCCCCAAATAATCG
AAAATCCAAACTCCTTACCTCTAAT
ACCCCTCCCCAAATAATCA


(SEQ ID 349)

CTCCTTACCTCTAGT





 


GI29805597_PGR_2R
NT_033899
CATATTATATCCTGCCATTTGATGCCCGGTG
CATATTATATCCTACCATTTAATA
TAAACATTCTATACCTACTTCCCAAAA
CATATTATATCCTACCATTTAATACCA


(SEQ ID 350)

AACATTCTATACCTGCTTCCCAGAA
CCCG




 


GI29809602_CDH13_1R
NT_024797
GAAAAGTGGAATCAGCTGGCATTGCCCAGC
AAACTCAACCTCACAAATCACG
TAAACAATACCAACTAATTCCACTTTC
TTAAAACTCAACCTCACAAATCACA


(SEQ ID 351)

GTGATTTGTGAGGCTGAGCCCCAA





 


GI29809602_CDH13_2R
NT_024797
ACGAGGGAGCGTTAGGAAGGAATCCGTCTT
CCAAAACCAATAACTTTACAAAACG
ATTCCTTCCTAACGCTCCCTC
CCAAAACCAATAACTTTACAAAACA


(SEQ ID 352)

GTAAAGCCATTGGTCCTGG





 


GI29809602_CDH13_3R
NT_024797
AAGCAAATGGGATGCCACCTCCGCGGGGCT
AAATAAAATACCACCTCCGCG
AACTCGCTCCTCGCGAAATAC
AAACAAATAAAATACCACCTCCACA


(SEQ ID 353)

CGCTCCTCGCGAGGTGCTCAC





 


GI29809804_ASC _1R
NT_024812
GGTTCGGGGAACCGCGGAGGTTTCGGGGTT
AAAAACCGCGAAAATTTCG
AATTCTAAAAATCCGAAATTCTAAACC
AATTCAAAAAACCACAAAAATTTCA


(SEQ ID 354)

CTAGAAATCCGAGGTTCTAAGCC





 


GI29809804_ASC_2R
NT_024812
GGAGGTTTCGGGGTTCTAGAAATCCGAGGT
AAAACACCTAAACTTAAAACCTCG
ATTTCTAAAACCCCGAAACCTC
TTAAAACACCTAAACTTAAAACCTCA


(SEQ ID 355)

TCTAAGCCTAGGTGCTCCAA





 


GI29809804_ASC_3R
NT_024812
GTGAGAGCCAGCCCAGGTTTCCGGTCTGTA
AAACCAACCCAAATTTCCG
TCTATACCCGCTAATACAAACCC
ATAAAAACCAACCCAAATTTCCA


(SEQ ID 356)

CCCGCTGGTGCAAGCCCAGA





 


GI29809804_PRSS8_1R
NT_024812
CACCACATACACACTACACACCGGGACACA
ACCACATACACACTACACACCG
AACACACACACAAAAAACCTATACA
CACCACATACACACTACACACCA


(SEQ ID 357)

CACACAAAAGGCCTATGCA





 


GI29809804_PRSS8_2R
NT_024812
CACACACACAAACCACATGCACGTGCATGC
ACACACACAAACCACATACACG
ACATACACGCACACTCAAACA
CACACACACAAACCACATACACA


(SEQ ID 358)

ACGCACACTCAGACACAC





 


GI29809804_PRSS8_3R
NT_024812
TGAGCTCAGGGCTCAAGGGGGACGGGGCTG
AAACAAACACCAACCCCG
CCCCCTTAAACCCTAAACTCA
AAAACAAACACCAACCCCA


(SEQ ID 359)

GTGCCTGTTTT





 


GI29823171_SERPINB5_1R 
NT_025028
TTCACCTTCCGGTCCTGCGTGGGCCGAGAG
ACATACGTACGACAATCCTCTCG
CCCACGCAAAACCGAAAA
CAAACATACATACAACAATCCTCTCA


(SEQ ID 360)

GATTGCCGTACGCATGTCTG





 


GI29823171_SERPINB5_3R
NT_025028
CAAACTCCTGGGCTCAAGCAATCCGCTCAC
AACTCCTAAACTCAAACAATCCG
TCACGTCAACCTCCCCAAATA
CAAACTCCTAAACTCAAACAATCCA


(SEQ ID 361)

GTCAACCTCCCCAAATGCT





 


GI29789893_CTNNB1_2R
NT_037565
AGCGGTACTCGAAGGCCGGGGCCGAGATGC
GATACTCGAAAACCGAAACCG
AATACCACCTTCCGCAAACC
AACAATACTCAAAAACCAAAACCA


(SEQ ID 362)

CACCTTCCGCAGGCCGC





 


GI29789893_CTNNB1_3R
NT_037565
GGGTGCTGTGAGACTGGGCTGCGACCCAGG
CACACTCCCTACTAAACCTAAATCG
AACCCAATCTCACAACACCC
CACACTCCCTACTAAACCTAAATCA


(SEQ ID 363)

TCCAGCAGGGAGTGTG





 


GI29794147_RUNX3_1R
NT_077383
TGAGCCAAGGCCGCGAGCAGGCTTCTCGCA
CCAACCTCAACTCACAAAATACG
AAAACCTACTCGCGACCTTAACT
CCAACCTCAACTCACAAAATACA


(SEQ ID 364)

TCCTGTGAGCTGAGGTTGG





 


GI29794147_RUNX3_2R
NT_077383
GTGACATCACGGCCCAGGTGACCGCGGCCC
TAACATCACGACCCAAATAACCG
GACCCAACCAATAAACCAAAAC
ATAACATCACAACCCAAATAACCA


(SEQ ID 365)

AGCCAATGAGCCAAGGCC





 


GI29794147_RUNX3_3R
NT_077383
TGGTGGACAATGGCAGGGAGTCAGCCGCAG
ACCTCGCCCCTAAACTACG
CTAACTCCCTACCATTATCCACCA
ATACCACCTCACCCCTAAACTACA


(SEQ ID 366)

CCCAGGGGCGAGGTGGCAT





 


GI29794313_MYCL1_1R
NT_077386
CCCAGCTCGGAGTGGGCAAGGAGCACGGTT
TACCAACCGACTAAACTAAACCG
ACTCCTTACCCACTCCGAACTAA
CAATACCMCCAACTAAACTAAACCA


(SEQ ID 367)

TAGCTCAGCCGGCTGGCACCG





 


GI29794313_MYCL1_2R
NT_077386
CGACGGGTGACCAGACGGACGCCGGGACCA
AATAACCAAACGAACGCCG
AACCAACCCAAATTCAAAACTAA
CAACAAATAACCAAACAAACACCA


(SEQ ID 368)

GCCCAAGTTCAGGGCTGA





 


GI29799662_SCGB3A1_1R
NT_077451
GGCAGGGACCAGGGAGCCAGGAACTGCGCC
CAAAACAAAAACGAAAACGACG
AATTCCTAACTCCCTAATCCCTACC
ACCAAAACAAAAACAAAAACAACA


(SEQ ID 369)

GCCCCCGCCCCTGCCCTGGC





 


GI29799662_SCGB3A1_2R
NT_077451
GGCGAGGACCGGGTATAAGAAGCCTCGTGG
ACTACCCGAACAAAACCACG
AACTTCTTATACCCGATCCTCG
ACAACTACCCAAACAAAACCACA


(SEQ ID 370)

CCTTGCCCGGGCAGCCGC





 


GI29799662_SCGB3A1_3R
NT_077451
CGCGTGGGGTCAGACCGCAAAGCGAAGGTG
GTAAAATCAAACCGCAAAACG
AAATACGAACCGAAATAAACCTC
CACATAAAATCAAACCACAAAACA


(SEQ ID 371)

CGGGCCGGGGTGGGCCTCGCG





 


GI29801002_SFTPA1_1R
NT_077575
TGGGGCTCATGGCTGAGCCAGGTCGCAGGA
CCAAACCAACTTATCTATCCTACG
CCTAACTCAACCATAAACCCCA
TCCAAACCAACTTATCTATCCTACA


(SEQ ID 372)

CAGACAAGTTGGCCTGGA





 


GI29801002_SFTPA1_2R
NT_077575
TTACAGACCTGGAGTTCCTCTTTCGCAGGT
CAAACCTAAAATTCCTCTTTCG
AAATTCTATACTCCCCTCAAAAATC
TTACAAACCTAAAATTCCTCTTTCA


(SEQ ID 373)

TCTGTGCTCCCCTCAAGGGTC





 


GI29801002_SFTPA1_3R
NT_077575
AGACAGAAACTGCAGCTCTCCCGTGACCTT
AACAAAAACTACAACTCTCCCG
AACCTTCCAAATACTACCCTAACTC
AAACAAAAACTACAACTCTCCCA


(SEQ ID 374)

CCAGGTGCTGCCCTGACTC





 


GI29804083_UNG_1R
NT_078089
CCGAGAGACAGGGTCTCGCTCTGTCGCCCA
CCAATACACTCCAAACCTAAACG
CAAAACGAAACCCTATCTCTCG
ACCAATACACTCCAAACCTAAACA


(SEQ ID 375)

GGCCTGGAGTGCATTGGC





 


GI29804083_UNG_2R
NT_078089
CGCTAGGATTACAGGCGTGGGCCACCGCGC
AAAATTACAAACGTAAACCACCG
GCCTAACCAATCTTCTCTTCTTACA
CACTAAAATTACAAACATAAACCACCA


(SEQ ID 376)

CTGACCAGTCTTCTCTTCTTGCAG





 


GI29804083_UNG_3R
NT_078089
GCTCAGACCCTCTGGCCTCAAGCGATCCTC
TCAAACCCTCTAACCTCAAACG
TCCTCCAACCTAAACCTCCC
ACTCAAACCCTCTAACCTCAAACA


(SEQ ID 377)

CAGCCTGGGCCTCCC
















TABLE 2







Methylation Markers for Squamous Cell Carcinoma


METHYLATION MARKERS FOR SQUAMOUS CELL CARCINOMA










Panel_I
Panel_II
Panel_III
Panel_IV














Marker Name
p-value
Marker Name
p-value
Marker Name
p-value
Marker Name
p-value





HTR1B_2r
2.559E−06
MTHFR_1r
0.00004
HTR1B_2r
0.00043
HTR1B_2r
0.00043


GDF10_3r
1.065E−05
TSLL2_2r
0.00053
MTHFR_1r
0.00080
MTHFR_1r
0.00080


ARHI_2r
2.616E−05
HTR1B_2r
0.00056
MLH1_2r
0.00099
MLH1_2r
0.00099


SFN_1r
4.169E−05
ABCC5_2r
0.00206
GDF10_3r
0.00183
GDF10_3r
0.00183


CALCA1_1r
4.187E−05
GDF10_3r
0.00244
APC_1r
0.00228
APC_1r
0.00228


ADCYAP1_2r
4.533E−05
MLH1_2r
0.00290
TP53_3r
0.00279
TP53_3r
0.00279


TERT_1r
1.094E−04
VHL_1r
0.00350
ARHI_2r
0.00339
ARHI_2r
0.00339


RASSF1_3r
1.384E−04
UBB_2r
0.00400
BRCA1_3r
0.00397
BRCA1_3r
0.00397


MOS_1r
1.497E−04
CALCA_1r
0.00474
CALCA_1r
0.00468
CALCA_1r
0.00468


SFTPC_3r
4.626E−04
SNCG_3r
0.00522
ADCYAP1_2r
0.00505
ADCYAP1_2r
0.00505


TERT_3r
4.681E−04
S100A2_1r
0.00532
SFTPC_3r
0.00555
SFTPC_3r
0.00555


MYOD1_2r
6.924E−04
SFN_2r
0.00639
TSLL2_2r
0.00676
TSLL2_2r
0.00676


C4B_2r
7.712E−04
SFTPC_3r
0.00817
TERT_1r
0.00713
TERT_1r
0.00713


CALCA_2r
7.857E−04
ADCYAP1_2r
0.00910
MYC_2r
0.00745
MYC_2r
0.00745




SFN_3r
0.00923
RASSF1_3r
0.00825
RASSF1_3r
0.00825






MOS_1r
0.00872
MOS_1r
0.00872






SNCG_3r
0.00876
SNCG_3r
0.00876






TGFBR2_3r
0.00957
TGFBR2_3r
0.00957








APC_2r
0.01187








HOXA5_3r
0.01204








TYMS_2r
0.01442








CDKN2B_3r
0.01455








CALCA_2r
0.01579








MYOD1_2r
0.01594








TERT_3r
0.01734








PTEN_1r
0.01896








C4B_2r
0.01927








GSTP1_3r
0.01964
















TABLE 3







Methylation Markers for Adenocarcinoma


Methylation Markers for Adenocarcinoma










Panel_I

Panel_II











Marker Name
p-value
Marker Name
p-value





SFN_2r
0.00000000007
SFN_2r
0.000009


TWIST1_3r
0.00000093146
CD1A_1r
0.000511


SERPINB5_1r
0.00000098742
TNF_2r
0.000526


GDF10_3r
0.00000130650
SERPINB5_1r
0.000638


PGR_1r
0.00000188610
PGR_1r
0.001117


SFTPD_1r
0.00000420738
TWIST1_3r
0.001288


ARHI_3r
0.00000598158
GDF10_3r
0.001469


CALCA_1r
0.00000933174
SFTPD_1r
0.002031


SFTPB_1r
0.00004835043
ADCYAP1_2r
0.002176


CALCA_2r
0.00008410116
ARHI_3r
0.002211


WT1_1r
0.00013356747
IL13_3r
0.002844


PRDM2_3r
0.00014989843
HOXA5_2r
0.002966


TERT_1r
0.00019412964
CALCA_1r
0.003758


S100A2_2r
0.00031181656
HOXA5_1r
0.004410


ADCYAP1_3r
0.00033644602
SFTPB_1r
0.004675


RUNX3_2r
0.00045917044
MC2R_1r
0.005590


MYOD1_2r
0.00061315386
HOXA5_3r
0.005836


TWIST1_2r
0.00077702738
WT1_1r
0.005920




SERPINB5_3r
0.008011




CDH13_3r
0.008365




CALCA_2r
0.008434




RUNX3_1r
0.008640
















TABLE 4







Samples for Methylation Analysis














Sample
Diagnosis
Age
Sex
Norm Match
Staging
Grading
R2 correlation

















D12152
Adenocarcinoma
73
F

no data

0.958


D12155
Adenocarcinoma
60
M

no data

0.964


D12157
Normal
79
M



0.811


D12158
Adenocarcinoma
87
M

no data

0.963


D12160
Adenocarcinoma
79
M

no data

0.986


D12162
Adenocarcinoma
47
M

no data

0.970


D12163
Adenocarcinoma
54
M

no data

0.990


D12164
Normal
21
M



0.980


D12165
Adenocarcinoma
76
F

no data

0.964


D12170
Adenocarcinoma
62
M

no data

0.984


D12173
Normal
77
M



0.988


D12180
Normal
69
M



0.876


D12181
Adenocarcinoma
64
M

no data

0.962


D12182
Normal
64
M



0.981


D12184
Normal
75
F



0.984


D12188
Normal
66
M



0.940


D12190
Adenocarcinoma
49
M

no data

0.961


D12195
Normal
62
F



0.982


D12197
Adenocarcinoma
50
F

no data

0.982


D12198
Normal
74
F



0.976


D12202
Normal
71
M



0.988


D12203
Adenocarcinoma
71
M
D12202
no data

0.940


D12205
Normal
68
M



0.961


D12207
Adenocarcinoma
62
M

no data

0.978


D12209
Normal
6
M



0.990


G12001
Squamous cell
74
M
B12001
pT1 N0
1
0.995



carcinoma



Mx


G12002
Squamous cell
63
M
B12002
pT4 N2
3
0.988



carcinoma



Mx


G12003
Squamous cell
67
M
B12003
pT1 N3
2
0.981



carcinoma



Mx


G12004
Squamous cell
69
M
B12004
pT2 N1
3
0.983



carcinoma



Mx


G12005
Squamous cell
59
M
B12005
pT2 N0
2
0.994



carcinoma



M0


G12006
Squamous cell
49
M
B12006
pT2 N1
2
0.989



carcinoma



Mx


G12007
Squamous cell
71
M

pT2 N1
2
0.988



carcinoma



Mx


G12008
Squamous cell
57
F
B12008
pT2 N0
3
0.964



carcinoma



Mx


G12009
Squamous cell
62
M
B12009
pT2 N1
3
0.986



carcinoma



Mx


G12010
Squamous cell
67
M
B12010
pT2 N1
2
0.921



carcinoma



Mx


G12011
Squamous cell
73
M

pT2 N0
2
0.977



carcinoma



Mx


G12012
Squamous cell
71
M
B12012
pT2 N0
2
0.994



carcinoma



Mx


G12013
Squamous cell
73
M

pT2 N0
1
0.938



carcinoma



Mx


G12014
Squamous cell
64
M

pT2 N2
3
0.933



carcinoma



Mx


G12018
Adenocarcinoma
60
F
B12018
pT1 N0
2
0.996







Mx


G12019
Adenocarcinoma
78
M
B12019
pT2 N1
3
0.985







M0


G12020
Adenocarcinoma
64
M
B12020
pT2 N0
3
0.990







Mx


G12022
Adenocarcinoma
76
M
B12022
pT2 N0
3
0.989







Mx


G12023
Adenocarcinoma
55
M
B12023
pT2 N0
2
0.832







Mx


G12024
Adenocarcinoma
54
M
B12024
pT2 N3
3
0.971







Mx


G12025
Adenocarcinoma
52
F
B12025
pT2 N2
2
0.987







Mx


G12026
Adenocarcinoma
57
F
B12026
pT2 N2
3
0.992







Mx


G12027
Adenocarcinoma
65
M
B12027
pT2 N0
1
0.990







Mx


G12028
Adenocarcinoma
75
M
B12028
pT3 N3
3
0.980







Mx


G12029
Adenocarcinoma
71
M
B12029
pT2 N0
3
0.992







M0


B12001
Normal
74
M



0.994


B12002
Normal
63
M



0.977


B12003
Normal
67
M



0.994


B12004
Normal
69
M



0.982


B12005
Normal
59
M



0.997


B12006
Normal
49
M



0.985


B12008
Normal
57
F



0.987


B12009
Normal
62
M



0.994


B12010
Normal
67
M



0.991


B12012
Normal
71
M



0.995


B12018
Normal
60
F



0.992


B12019
Normal
78
M



0.978


B12020
Normal
64
M



0.978


B12022
Normal
76
M



0.993


B12023
Normal
55
M



0.979


B12024
Normal
54
M



0.964


B12025
Normal
52
F



0.973


B12026
Normal
57
F



0.931


B12027
Normal
65
M



0.984


B12028
Normal
75
M



0.995


B12029
Normal
71
M



0.995

## Claims

1. A method for identification of differentially methylated genomic CpG dinucleotide sequences associated with adenocarcinoma in an individual, said method comprising:
(a) obtaining a biological sample comprising genomic DNA from said individual;
(b) measuring the level of methylation in said biological sample at a CpG dinucleotide sequence in a genomic target designated as SEQ ID NO: 265 and SEQ ID NO: 293; and
(c) comparing the level of methylation at said genomic CpG dinucleotide sequences in the biological sample to a reference level of methylation of said genomic CpG dinucleotide sequences, wherein said reference level comprises the level of methylation at said genomic CpG dinucleotide sequences in a normal sample; wherein an increase in the level of methylation of said genomic CpG dinucleotide sequences in the biological sample for genomic target designated as SEQ ID NO: 265 and SEQ ID NO: 293 compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with adenocarcinoma.

2. The method of claim 1, wherein step (b) further comprises measuring the level of methylation in said biological sample at a CpG dinucleotide sequence in at least one of the genomic targets designated as SEQ ID NO: 140, 174, 305, 314, 360 or 371, wherein an increase in the level of methylation of said genomic CpG dinucleotide sequences in the biological sample for the genomic targets designated as SEQ ID NO: 140, 314 or 371, or a decrease in the level of methylation of said genomic CpG dinucleotide sequences in the biological sample for the genomic targets designated as SEQ ID NO: 174, 305 or 360, compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with adenocarcinoma.

3. The method of claim 1, wherein the level of methylation of said differentially methylated genomic CpG dinucleotide sequences is used to diagnose adenocarcinoma in the individual.

4. A method for identification of differentially methylated genomic CpG dinucleotide sequences associated with squamous cell carcinoma in an individual, said method comprising:
(a) obtaining a biological sample comprising genomic DNA from said individual;
(b) measuring the level of methylation in said biological sample at a CpG dinucleotide sequence in a genomic target designated as SEQ ID NO: 265 and SEQ ID NO. 315; and
(c) comparing the level of methylation at said genomic CpG dinucleotide sequences in the biological sample to a reference level of methylation of said genomic CpG dinucleotide sequences, wherein said reference level comprises the level of methylation at said genomic CpG dinucleotide sequences in a normal sample; wherein an increase in the level of methylation of said genomic CpG dinucleotide sequences in the biological sample for genomic target designated as SEQ ID NO: 265 and SEQ ID NO. 315 compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with squamous cell carcinoma.

5. The method of claim 4, wherein step (b) further comprises measuring the level of methylation in said biological sample at a CpG dinucleotide sequence in at least one of the genomic targets designated as SEQ ID NO: 140, 174, 305, 314, 360 or 371, wherein an increase in the level of methylation of said genomic CpG dinucleotide sequences for the genomic targets designated as SEQ ID NO: 140, 314 or 371, or a decrease in the level of methylation of said genomic CpG dinucleotide sequences for the genomic targets designated as SEQ ID NO: 174, 305 or 360, compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with squamous cell carcinoma.

6. The method of claim 4, wherein the level of methylation of said differentially methylated genomic CpG dinucleotide sequences is used to diagnose squamous cell carcinoma in the individual.

7. A method for identification of differentially methylated genomic CpG dinucleotide sequences associated with adenocarcinoma and squamous cell carcinoma in an individual, said method comprising:
(a) obtaining a biological sample comprising genomic DNA from said individual;
(b) measuring the level of methylation in said biological sample at a CpG dinucleotide sequence in a genomic target designated as SEQ ID NO: 265 and at least one of SEQ ID NO: 140, 174, 305, 314, 360 or 371; and
(c) comparing the level of methylation at said genomic CpG dinucleotide sequences in the biological sample to a reference level of methylation of said genomic CpG dinucleotide sequences, wherein said reference level comprises the level of methylation at said genomic CpG dinucleotide sequences in a normal sample; wherein an increase in the level of methylation of said genomic CpG dinucleotide sequences in the biological sample for the genomic targets designated as SEQ ID NO: 140, 265, 314 or 371, or a decrease in the level of methylation of said genomic CpG dinucleotide sequences in the biological sample for the genomic targets designated as SEQ ID NO: 174, 305 or 360, compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with adenocarcinoma and squamous cell carcinoma.

8. The method of claim 7, wherein step (b) further comprises measuring the level of methylation in said biological sample at a CpG dinucleotide sequence in at least two of the genomic target designated as SEQ ID NO: 140, 174, 305, 314, 360 or 371.

9. The method of claim 7, wherein the level of methylation of said differentially methylated genomic CpG dinucleotide sequences is used to diagnose adenocarcinoma or squamous cell carcinoma in the individual.