Abstract:
Presented herein are biomarkers related to lung cancer. The presently identified salivary biomarkers create the basis for a lung cancer detection bioassay with sensitivity and specificity. Means and methods for evaluating the data generated using multiple biomarkers in order to validate findings and further use of the multiplexed lung cancer assay in clinical, diagnostic and therapeutic uses is also included.

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
       [0001]    The present application claims priority to provisional application U.S. Ser. No. 61/303,205, filed Feb. 10, 2010, herein incorporated by reference in its entirety. 
     
    
     STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    This invention was made with Government support under Grant No. DE016275, awarded by the National Institutes of Health. The Government has certain rights in this invention. 
     
    
     BACKGROUND 
       [0003]    According to the National Cancer Institute, lung cancer is the leading cause of cancer death (hypertext transfer protocol://www.cancer.gov/cancertopics/types/lung). In 2008, approximately 215,000 new lung cancer patients were diagnosed in the United States alone. Roughly 87% of these patients will have on-small cell lung cancer (NSCLC). 
         [0004]    Smoking, particularly of cigarettes, is by far the main contributor to lung cancer. In the U.S., it is estimated that there are 45 million current and 45 million former smokers at risk for developing lung carcinoma. Lung cancer is estimated to remain a major health problem for at least the next 50 years. 
         [0005]    Over 75% of lung cancer cases are diagnosed in late stages because there remains no practical way to screen the large numbers of people at risk. Early detection offers the promise of improved cure rates. 
         [0006]    Attempts have been made to identify and stratify individuals at high risk for developing lung cancer and early detection. Initially spectum cytology and chest X-ray were used as screening tools. Unfortunately these procedures failed to increase the number of curable cases. 
         [0007]    These procedures were supplanted by computerized tomography (CT) scans, which are more sensitive than chest X-ray. Unfortunately, broad application of CT screening of the at risk population has several drawbacks including high false positives (detection of benign lung nodules) and poor ability to identify central tumors. Because of the high false positive rate, approaching 50%, it is estimated that for every lung cancer death prevented by CT screening, two unnecessary invasive procedures will be conducted. 
         [0008]    As such, a need exists for methods useful for detecting lung cancer, and in particular biomarkers that can detect early stages of the disease and are largely non-invasive. 
       BRIEF SUMMARY OF THE INVENTION 
       [0009]    In accordance with some embodiments of the invention, a method of determining the likelihood of the presence or occurrence of lung cancer in a test subject is provided. The disclosed method includes analyzing a saliva sample from the subject with an assay that specifically detects a biomarker in the saliva sample, the biomarker selected from the group of: CCNI (Cyclin I)(SEQ ID NO: 1), EGFR(Epidermal growth factor receptor)(SEQ ID NO:2), FGF9 (Fibroblast growth factor 19)(SEQ ID NO: 3), GREB1 protein (SEQ ID NO:4), LZTS (Leucine zipper, putative suppresor I)(SEQ ID NO: 5), BRAF (v-raf murine sarcoma viral oncogene homolog B1 (SEQ ID NO: 6), FRS2 (Fibroblast growth factor receptor substrate 2)(SEQ ID NO: 7), ANXA1 (Annexin A1)(SEQ ID NO: 8), Hp2 (Haptoglobin 2)(SEQ ID NO:9), Zinc Alpha2-Glycoprotein (SEQ ID NO: 10),  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oralis  16S rRNA, and  Granulicatella adiacens  16S rRNA. The relative occurrence of the biomarkers is determined and compared to a control, thereby allowing the lung cancer status of the test subject to be determined. 
         [0010]    In some embodiments, a method of determining the likelihood of the presence or occurrence of lung cancer in a test subject is provided, the method entailing the detection of at least two biomarkers in a saliva sample. In other embodiments, a method of determining the likelihood of the presence or occurrence of lung cancer in a test subject is provided, the method entailing the detection of  Porphyromonas catoniae  16S rRNA and  Camplylobacter showae  16S rRNA. The relative occurrence of these biomarkers or these biomakers and others in these instances is determined and compared to a control, for example, thereby allowing the lung cancer status of the test subject to be determined. 
         [0011]    In some embodiments, a method of determining the likelihood of the presence or occurrence of lung cancer in a test subject is provided, the method entailing the detection of at least three biomarkers in a saliva sample. In other embodiments, a method of determining the likelihood of the presence or occurrence of lung cancer in a test subject is provided, the method entailing the detection of GREB1, CCNI and FRS. In other embodiments, a method of determining the likelihood of the presence or occurrence of lung cancer in a test subject is provided, the method entailing the detection of. The relative occurrence of these biomarkers or these biomakers and others in these instances is determined and compared to a control, for example, thereby allowing the lung cancer status of the test subject to be determined. 
         [0012]    In other embodiments, the method of determining the likelihood of the presence or occurrence of lung cancer in a test subject includes an assay in which a nucleic acid encoding at least one biomarker is detected. The nucleic acid can be detected by, for example, mass spectroscopy, polymerase chain reaction (PCR), microarray hybridization, thermal sequencing, capillary array sequencing, or solid phase sequencing. 
         [0013]    In other embodiments, the method of determining the likelihood of the presence or occurrence of lung cancer in a test subject includes an assay in which a polypeptide encoding at least one biomarker is detected. The polypeptide can be detected by, for example, enzyme-linked immunosorbent assay (ELISA), Western blot, flow cytometry, immunofluorescence, immunohistochemistry, or mass spectroscopy. 
         [0014]    In accordance with other embodiments of the invention, a method for assessing the efficacy of a therapy is disclosed. This method includes analyzing a first saliva sample from the subject with an assay that specifically detects a biomarker, the biomarker selected from the group consisting of CCNI, EGFR, FGF9, GREB1 protein, LZTS, BRAF, FRS2, ANXA1, Hp2, Zinc Alpha2-Glycoprotein,  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oxalis  16S rRNA, and  Granulicatella adiacens  16S rRNA. This first analysis provides a first expression profile. A therapy is applied to a subject. An analysis of a second saliva sample from the subject is undertaken with an assay that specifically detects at least two biomarkers selected from the group consisting of CCNI, EGFR, FGF9, GREB1 protein, LZTS, BRAF, FRS2, ANXA1, Hp2, Zinc Alpha2-Glycoprotein,  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oxalis  16S rRNA, and  Granulicatella adiacens  16S rRNA thereby providing a second expression profile. The first and second expression profiles are compared in order to assess the efficacy of a therapy. 
         [0015]    In another embodiment, a solid support is provided, wherein the solid support includes a capture binding probe selective for at least two biomarkers selected from the group of CCNI, EGFR, FGF9, GREB1 protein, LZTS, BRAF, FRS2,  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oralis  16S rRNA, and  Granulicatella adiacens  16S rRNA. In some embodiments, a first and a second solid support are provided, wherein the first solid support includes a capture binding probe selective for a biomarker selected from the group consisting of CCNI, EGFR, FGF9, GREB1 protein, LZTS, BRAF, FRS2,  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oralis  16S rRNA, and  Granulicatella adiacens  16S rRNA and wherein the second solid support includes a capture binding ligand selective for a biomarker selected from ANXA1, Hp2, Zinc Alpha2-Glycoprotein, and Calprotectin. 
         [0016]    In some embodiments, the capture binding ligand of the kit is an antibody. In another embodiment the kit provides one or more primers for the selective amplification of a biomarker, wherein the biomarker is selected from the group of: CCNI, EGFR, FGF9, GREB1 protein, LZTS, BRAF, FRS2,  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oralis  16S rRNA, and  Granulicatella adiacens  16S rRNA. In some embodiments one or more of the primers possess a detectable label. 
         [0017]    In accordance with some embodiments of the invention, a method of determining the likelihood of the presence or occurrence of lung cancer in a test subject is provided. The disclosed method includes analyzing a saliva sample from the subject with an assay that specifically detects at least seventeen biomarkers in the saliva sample. The biomarkers are selected from the group of: CCNI (Cyclin I)(SEQ ID NO: 1), EGFR(Epidermal growth factor receptor)(SEQ ID NO:2), FGF9 (Fibroblast growth factor 19)(SEQ ID NO: 3), GREB1 protein (SEQ ID NO:4), LZTS (Leucine zipper, putative suppresor I)(SEQ ID NO: 5), BRAF (v-raf murine sarcoma viral oncogene homolog B1 (SEQ ID NO: 6), FRS2 (Fibroblast growth factor receptor substrate 2)(SEQ ID NO: 7), ANXA1 (Annexin A1)(SEQ ID NO: 8), Hp2 (Haptoglobin 2)(SEQ ID NO:9), Zinc Alpha2-Glycoprotein(SEQ ID NO: 10),  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oxalis  16S rRNA, and  Granulicatella adiacens  16S rRNA. The relative occurrence of at least seventeen biomarkers is determined and compared to a control, thereby allowing the lung cancer status of the test subject to be determined. 
         [0018]    In any of the embodiments above, wherein a method for determining the likelihood of the presence or occurrence of lung cancer in a test subject, the number of biomarkers used can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more. 
         [0019]    These and other embodiments, features and potential advantages will become apparent with reference to the following description and drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  is a schematic representation of the protocol for saliva collection. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Introduction 
       [0021]    Early detection of lung cancer offers the promise of easier treatment (smaller surgeries, less radiation or chemotherapy) and improved survival. Conventional screening (CT, X-ray, sputum cytology) has a less-than desirable sensitivity and specificity. A sensitive assay to identify biomarkers using non-invasively collected specimens is therefore ideal for lung cancer detection. 
         [0022]    Biomarkers, whether produced by healthy individuals or by individuals affected by specific systemic disease, can be used as tell-tale molecules for monitoring health status, disease onset, treatment responsiveness, and outcome. 
         [0023]    Saliva is a readily accessible source of biomarkers. As a clinical diagnostic biofluid, saliva offers many advantages; sample collection is simple, non-invasive, and causes less anxiety on the part of patients. The use of saliva also offers a cost-effective approach for large-scale screens. 
         [0024]    The present invention discloses the diagnostic/prognostic significance of seventeen salivary biomarkers, including detection of the 16S rRNA indicative of seven bacteria strains. These biomarkers include CCNI (Cyclin I)(SEQ ID NO: 1), EGFR(Epidermal growth factor receptor)(SEQ ID NO:2), FGF9 (Fibroblast growth factor 19)(SEQ ID NO: 3), GREB1 protein (SEQ ID NO:4), LZTS (Leucine zipper, putative suppresor I)(SEQ ID NO: 5), BRAF (v-raf murine sarcoma viral oncogene homolog B1 (SEQ ID NO: 6), FRS2 (Fibroblast growth factor receptor substrate 2)(SEQ ID NO: 7), ANXA1 (Annexin A1)(SEQ ID NO: 8), Hp2 (Haptoglobin 2)(SEQ ID NO:9), Zinc Alpha2-Glycoprotein (SEQ ID NO:10),  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oralis  16S rRNA,  Granulicatella adiacens  16S rRNA, and combinations thereof, in lung cancer detection. Detection of these and other biomarkers in saliva are useful for diagnosis and prognosis of lung cancer. 
         [0025]    Methods for detecting salivary biomarkers (proteins and nucleic acids) include techniques such as ELISA, PCR, for example, RT-PCR or mass spectroscopy, alone or in combination with other markers. Any specific probe can be used for detection, such as an antibody, a receptor, a ligand, RT-PCR etc. Mass spectroscopy can also be used for protein detection. Thus, the present invention can be used alone or as a complement to traditional antigen analysis to enhance the diagnosis of lung and other cancers. 
       Definitions 
       [0026]    CCNI (Cyclin I)(SEQ ID NO: 1), EGFR(Epidermal growth factor receptor)(SEQ ID NO:2), FGF9 (Fibroblast growth factor 19)(SEQ ID NO: 3), GREB1 protein (SEQ ID NO:4), LZTS (Leucine zipper, putative suppresor I)(SEQ ID NO: 5), BRAF (v-raf murine sarcoma viral oncogene homolog B1 (SEQ ID NO: 6), FRS2 (Fibroblast growth factor receptor substrate 2)(SEQ ID NO: 7), ANXA1 (Annexin A1)(SEQ ID NO: 8), Hp2 (Haptoglobin 2)(SEQ ID NO:8), Zinc Alpha2-Glycoprotein (SEQ ID NO:9),  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oralis  16S rRNA,  Granulicatella adiacens  16S rRNA refer to nucleic acids, e.g., gene, DNA, pre-mRNA, mRNA, and polypeptides, polymorphic variants, alleles, mutants, and interspecies homologs that have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein. The nucleic acids and proteins of the invention include both naturally occurring or recombinant molecules. The nucleic acid or protein sequence is provided, for example, in SEQ ID NOs: 1-9. 
         [0027]      Porphyromonas catoniae, Campylobacter showae, Streptocococcus salivaris, Campylobacter rectus, Veillonella parvula, Kigella oralis, Granulicatella adiacens  refer to bacterial strains. These strains and others can be detected in a sample using any number of methods, including, for example, the detection of 16S rRNA (ribonucleic acid or deoxyribonucleic acid), the detection of chromosomal DNA, mRNA or by culturing bacteria from a sample in an appropriate growth medium under appropriate growth conditions. 
         [0028]    “Cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, kidney, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including non-Hodgkin&#39;s lymphomas (e.g., Burkitt&#39;s, Small Cell, and Large Cell lymphomas) and Hodgkin&#39;s lymphoma, leukemia, and multiple myeloma. 
         [0029]    “Therapeutic treatment” and “cancer therapies” refers to chemotherapy, hormonal therapy, radiotherapy, and immunotherapy. 
         [0030]    The terms “overexpress,” “overexpression” or “overexpressed” interchangeably refer to a protein that is transcribed or translated at a detectably greater level, usually in a cancer cell, in comparison to a normal cell. The term includes overexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g, organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a normal cell. Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e., ELISA, immunohistochemical techniques, mass spectroscopy). Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell. In certain instances, overexpression is 1-fold, 2-fold, 3-fold, 4-fold or more higher levels of transcription or translation in comparison to a normal cell. 
         [0031]    The terms “cancer-associated antigen” or “tumor-specific marker” or “tumor marker” interchangeably refers to a molecule (typically protein or nucleic acid such as RNA) that is expressed in the cell, expressed on the surface of a cancer cell or secreted by a cancer cell in comparison to a normal cell, and which is useful for the diagnosis of cancer, for providing a prognosis, and for preferential targeting of a pharmacological agent to the cancer cell. Oftentimes, a cancer-associated antigen is overexpressed in a cancer cell in comparison to a normal cell, for instance, about 1.2-fold over expression, about 2-fold overexpression, about 3-fold overexpression or more in comparison to a normal cell. Oftentimes, a cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. Oftentimes, a cancer-associated antigen will be expressed exclusively on the cell surface of a cancer cell and not synthesized or expressed on the surface of a normal cell. Exemplified cell surface tumor markers include the proteins c-erbB-2 and human epidermal growth factor receptor (HER) for breast cancer, PSMA for prostate cancer, and carbohydrate mucins in numerous cancers, including breast, ovarian and colorectal. 
         [0032]    It will be understood by the skilled artisan that markers may be used singly or in combination with other markers for any of the uses, e.g., diagnosis or prognosis of lung cancer, disclosed herein. 
         [0033]    The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site hypertext transfer protocol://www.ncbi.nlm.nih gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length. 
         [0034]    For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. 
         [0035]    A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith &amp; Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman &amp; Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson &amp; Lipman, Proc. Nat&#39;l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1987-2005, Wiley Interscience)). 
         [0036]    An example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (hypertext transfer protocol://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always &gt;0) and N (penalty score for mismatching residues; always &lt;0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &amp; Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. 
         [0037]    “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single or double-stranded form, and complements thereof. 
         [0038]    Specifically refers to ability of a ligand to bind to a target with an affinity at least 1.2, 1.5, 2, 3, 5, 8, 10, 20, 50, 100, 150, 200, 500, 1000, or more fold greater than a ligand not specific for the target. 
         [0039]    Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (for example, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. 
         [0040]    A particular nucleic acid sequence also implicitly encompasses “splice variants” and nucleic acid sequences encoding truncated forms of cancer antigens. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant or truncated form of that nucleic acid. “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. Nucleic acids can be truncated at the 5′ end or at the 3′ end. Polypeptides can be truncated at the N-terminal end or the C-terminal end. Truncated versions of nucleic acid or polypeptide sequences can be naturally occurring or recombinantly created. 
         [0041]    The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an .alpha. carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. 
         [0042]    Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. 
         [0043]    “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences. 
         [0044]    As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. 
         [0045]    The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). 
         [0046]    A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. 
         [0047]    The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. 
         [0048]    The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The T m  is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. 
         [0049]    Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al., supra. 
         [0050]    For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). 
         [0051]    “Antibody” means a protein comprising one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (κ), lambda (λ) and heavy chain genetic loci, which together compose the myriad variable region genes, and the constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε) and alpha (α), which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively. Antibody herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody or an antibody generated recombinantly for experimental, therapeutic or other purposes as further defined below. Antibody fragments include Fab, Fab′, F(ab′)2, Fv, scFv or other antigen-binding subsequences of antibodies and can include those produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. The term “antibody” refers to both monoclonal and polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing, inhibitory or stimulatory. 
       Biomarkers 
       [0052]    Biomarkers may originate from epidemiological studies, animal studies, pathophysiological considerations and end-organ experiments. Ideally, a biomarker will have a high predictive value for a meaningful outcome measure, can be or is validated in appropriately designed prospective trials, reflects therapeutic success by corresponding changes in the surrogate marker results, and should be easy to assess in clinical practice. 
         [0053]    Biomarkers can be used in conjunction with other diagnostic tools or used alone. 
         [0054]    The term “surrogate marker,” “biomolecular marker,” “biomarker” or “marker” (also sometimes referred to herein as a “target analyte,” “target species” or “target sequence”) refers to a molecule whose measurement provides information as to the state of a subject. In various exemplary embodiments, the biomarker is used to assess a pathological state. Measurements of the biomarker may be used alone or combined with other data obtained regarding a subject in order to determine the state of the subject. In one embodiment, the biomarker is “differentially present” in a sample taken from a subject of one phenotypic status (e.g., having a disease) as compared with another phenotypic status (e.g., not having the disease). In one embodiment, the biomarker is “differentially present” in a sample taken from a subject undergoing no therapy or one type of therapy as compared with another type of therapy. Alternatively, the biomarker may be “differentially present” even if there is no phenotypic difference, e.g. the biomarkers may allow the detection of asymptomatic risk. 
         [0055]    A biomarker may be over-expressed (over-abundant) or under-expressed (under abundant) relative to a control. The biomarker can be an allelic variant, truncated or mutated form of a wild-type nucelic acid or protein. The biomarker can be a splice variant. 
         [0056]    A biomarker may be determined to be “differentially present” in a variety of ways, for example, between different phenotypic statuses if the mean or median level (particularly the expression level of the associated mRNAs as described below) of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio. 
         [0057]    As described herein, a biomarker may be, for example, a small molecule, an analyte or target analyte, a nucleic acid, a protein, a metabolite or any derivative thereof or any and all combinations of these molecules, with proteins and nucleic acids finding particular use in the invention. As will be appreciated by those in the art, a large number of analytes may be detected using the present methods; basically, any biomarker for which a binding ligand, described below, may be made may be detected using the methods of the invention. 
         [0058]    In various embodiments, the biomarkers used in the panels of the invention can be detected either as proteins or as nucleic acids (e.g. mRNA or cDNA transcripts) in any combination. In various embodiments, the protein form of a biomarker is measured. As will be appreciated by those in the art, protein assays may be done using standard techniques such as ELISA assays. In various embodiments, the nucleic acid form of a biomarker (e.g., the corresponding mRNA) is measured. In various exemplary embodiments, one or more biomarkers from a particular panel are measured using a protein assay and one or more biomarkers from the same panel are measured using a nucleic acid assay. 
         [0059]    As will be appreciated by those in the art, there are a large number of possible proteinaceous target analytes and target species that may be detected using the present invention. The term “protein,” “polypeptide” or “oligopeptide” refers to at least two or more peptides or amino acids joined by one or more peptide bonds. A protein or an amino acid may be naturally or nonnaturally occurring and may be also be an analog, a derivative or a peptidomimetic structure. The term “protein” refers to wild-type sequences, variants of wild-type sequences and either of these containing analogs or derivatized amino acids. In various embodiments, variants of the sequences described herein, including proteins and nucleic acids based on e.g. splice variants, variants comprising a deletion, addition, substitution, fragments, preproprotein, processed preproprotein (e.g. without a signaling peptide), processed proprotein (e.g. resulting in an active form), nonhuman sequences and variant nonhuman sequences may be used as biomarkers. 
         [0060]    In various embodiments, the biomarker is a nucleic acid. The term “nucleic acid” or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, for example in the use of binding ligand probes, nucleic acid analogs are included that may have alternate backbones. 
         [0061]    Biomarkers can also be bacterial nucleic acids or proteins. The oral cavity is a large reservoir of bacteria, with more than 700 species or phylotypes, of which over 50% have not been cultivated. In some embodiments, assessing bacterial flora composition and dynamic changes are biomarkers for lung cancer. 
         [0062]    A recently developed 16S rRNA-based oligonucleotide microarray (Human Oral Microbe Identification Microarray) made it possible to profile the oral bacterial flora in patients with and without lung cancer. In some embodiments, the presence, absence, or level of 16S rRNA from bacteria in a sample may correlate with a disease or condition. “Bacteria” refers to small prokaryotic organisms (linear dimensions of around 1 μm) with non-compartmentalized circular DNA and ribosomes of about 70 S. “16S RNA” refers to a nucleic acid component of the 30S subunit of prokaryotic ribosomes; the gene that encodes the 16S rRNA or the 16S rRNA itself. Bacterial strains of species or phylotypes have less than about a 2% difference in 16S rRNA. Closely related species or phylotypes generally have between about a 2% and about a 4% difference in 16S rRNA, whereas a genus often has between about a 5% and about a 10% difference in 16S rRNA. 
         [0063]    To resolve the identity of bacterial populations, probes on a microarray can be designed, for example, to take advantage of conserved features of the 16S rRNA gene. For example, probes complementary to the more conserved features regions identify species in a large phylogenetic group, each group corresponding to a higher taxon (for example, domain, phylum, class, order, or family). Probes complementary to more variable regions distinguish genera and species. 
         [0064]    Biomarkers can also include micro RNAs. “MicroRNAs” (miRs) refers to a class of small naturally occurring non-coding RNAs (18-24 nucleotides) that regulate gene expression. Many microRNAs are well conserved across species and they are present in a broad range of species: plants, nematodes, fruit flies and humans. MicroRNAs have partially or perfect complementary sequence to one or more messenger RNA molecules (mRNAs) and their main function is to negatively regulate the expression of genes. In particular, microRNAs bind to the 3′ untranslated regions of mRNAs (3-UTR) thus leading to down regulation of mRNAs in a variety of ways such as mRNA cleavage, translational repression and deadenylation. 
         [0065]    A variety of experimental approaches and different techniques have been used to identify new microRNAs, as well as to study their expression pattern in the different biological processes. The cloning and identification of new microRNAs have been successfully done from size fractioned RNA samples using small RNA cloning approaches. Other approaches is as putative microRNAs homologues to microRNAs that already have been described in other species or using computational approaches alone or in combination with microarray analysis and sequence-directed cloning. 
         [0066]    One of the first techniques used for detection and profiling of microRNAs was Northern Blotting, where hybridization is done with a complementary 32P, digoxigenin-labeled oligo or modified Locked-nucleic-acid (LNA) oligonucleotides after gel separation. 
         [0067]    Other techniques that have been developed to specifically detect microRNAs are a modified invader assay (a synthetic oligonucleotide, the probe, which is in an appropriate overlap-flap structure is enzymatically cleavage by a structure-specific 5* nuclease) and in situ hybridization (using fluorescent-labeled complementary probes containing chemically modified nucleotides e.g. LNAs). Another widely used technique for detection and profiling of microRNAs is the use of oligonucleotide micro-array based detection platforms either with DNA capture probes or using modified Locked-nucleic-acid (LNA) oligonucleotides in which the ribose moiety is modified with an extra bridge that connects the 2′-0 and 4′-C atoms. 
         [0068]    In addition, quantitative real-time PCR (reverse transcriptase/polymerase chain reaction using Taqman or SYBR green technology) has been used for detection and profiling of precursor or mature microRNAs. This technique is sensitive and requires low amounts of starting material for the detection of individual mature microRNAs. Taqman microRNA arrays have been developed that provide the sensitivity of the qRT-PCR, while at the same time enables the simultaneously detection of different microRNAs in one sample. 
         [0069]    Biomarkers can also include metabolites. “Metabolite” or “small molecule” refers to organic and inorganic molecules which are present in a sample. The term does not include large macromolecules, such as large proteins (e.g., proteins with molecular weights over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weights of over 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). 
         [0070]    The metabolites of the cell are generally found free in solution. A “metabolic profile”, or “small molecule profile”, means a complete or partial inventory of small molecules within a targeted cell, tissue, organ, organism, or fraction thereof (e.g., cellular compartment). The inventory may include the quantity and/or type of small molecules present. The “small molecule profile” may be determined using a single technique or multiple different techniques. 
         [0071]    A metabolic profile can be developed by analyzing a sample using for example, techniques such as GC-MS (gas chromatography-mass spectrometry) and LC-MS (liquid chromatography-mass spectrometry). 
       Biomarker Panels 
       [0072]    Any combination of the biomarkers described herein is used to assemble a biomarker panel, which is detected or measured as described herein. As is generally understood in the art, a combination may refer to an entire set or any subset or subcombination thereof. The term “biomarker panel,” “biomarker profile,” or “biomarker fingerprint” refers to a set of biomarkers. As used herein, these terms can also refer to any form of the biomarker that is measured. Thus, if CCNI is part of a biomarker panel, then either CCNI mRNA, for example, or protein, for example, could be considered to be part of the panel. While individual biomarkers are useful as diagnostics, combination of biomarkers can sometimes provide greater value in determining a particular status than single biomarkers alone. Specifically, the detection of a plurality of biomarkers in a sample can increase the sensitivity and/or specificity of the test. Thus, in various embodiments, a biomarker panel may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more types of biomarkers. In various exemplary embodiments, the biomarker panel consists of a minimum number of biomarkers to generate a maximum amount of information. Thus, in various embodiments, the biomarker panel consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more types of biomarkers. Where a biomarker panel “consists of a set of biomarkers, no biomarkers other than those of the set are present. In exemplary embodiments, the biomarker panel consists of 1 biomarkers disclosed herein. In various embodiments, the biomarker panel consists of 2 biomarkers disclosed herein. In various embodiments, the biomarker panel consists of 3 biomarkers disclosed herein. In various embodiments, the biomarker panel consists of 4 or more biomarkers disclosed herein. 
         [0073]    In various exemplary embodiments, the biomarker panel comprises or consists of a biomarker selected from the group of CCNI (Cyclin I)(SEQ ID NO: 1), EGFR(Epidermal growth factor receptor)(SEQ ID NO:2), FGF9 (Fibroblast growth factor 19)(SEQ ID NO: 3), GREB1 protein (SEQ ID NO:4), LZTS (Leucine zipper, putative suppresor I)(SEQ ID NO: 5), BRAF (v-raf murine sarcoma viral oncogene homolog B1 (SEQ ID NO: 6), FRS2 (Fibroblast growth factor receptor substrate 2)(SEQ ID NO: 7), ANXA1 (Annexin A1)(SEQ ID NO: 8), Hp2 (Haptoglobin 2)(SEQ ID NO:8), Chain B, Crystal Structure of the Complex formed between MHc-Like Zinc Alpha2-Glycoprotein and Pro1,  Porphyromonas catoniae  16S rRNA,  Campylobacter showae  16S rRNA,  Streptocococcus salivaris  16S rRNA,  Campylobacter rectus  16S rRNA,  Veillonella parvula  16S rRNA,  Kigella oralis  16S rRNA. 
         [0074]    A biomarker can also be a clinical parameter. The term “clinical parameter” refers to all non-sample or non-analyte biomarkers of subject health status or other characteristics, such as, without limitation, age, ethnicity, gender, family history, height, and weight. 
         [0075]    The biomarkers of the invention show a statistically significant difference in lung cancer diagnsosis. In various embodiments, diagnostic tests that use these biomarkers alone or in combination show a sensitivity and specificity of at least about 85%, at least about 90%, at least about 95%, at least about 98% and about 100%. 
       Measurement and Detection of Biomarkers 
       [0076]    Biomarkers generally can be measured and detected through a variety of assays, methods and detection systems known to one of skill in the art. The term “measuring,” “detecting,” or “taking a measurement” refers to a quantitative or qualitative determination of a property of an entity, for example, quantifying the amount or concentration of a molecule or the activity level of a molecule. The term “concentration” or “level” can refer to an absolute or relative quantity. Measuring a molecule may also include determining the absence or presence of the molecule. Various methods include but are not limited to refractive index spectroscopy (RI), ultra-violet spectroscopy (UV), fluorescence analysis, electrochemical analysis, radiochemical analysis, near-infrared spectroscopy (near-IR), infrared (IR) spectroscopy, nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, pyrolysis mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas chromatography, liquid chromatography, gas chromatography combined with mass spectrometry, liquid chromatography combined with mass spectrometry, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) combined with mass spectrometry, ion spray spectroscopy combined with mass spectrometry, capillary electrophoresis, colorimetry and surface plasmon resonance (such as according to systems provided by Biacore Life Sciences). See also PCT Publications WO/2004/056456 and WO/2004/088309. In this regard, biomarkers can be measured using the above-mentioned detection methods, or other methods known to the skilled artisan. Other biomarkers can be similarly detected using reagents that are specifically designed or tailored to detect them. 
         [0077]    Different types of biomarkers and their measurements can be combined in the compositions and methods of the present invention. In various embodiments, the protein form of the biomarkers is measured. In various embodiments, the nucleic acid form of the biomarkers is measured. In exemplary embodiments, the nucleic acid form is mRNA. In various embodiments, measurements of protein biomarkers are used in conjunction with measurements of nucleic acid biomarkers. 
         [0078]    Methods for detecting mRNA, such as RT-PCR, real time PCR, branch DNA, NASBA and others, are well known in the art. Using sequence information provided by the database entries for the biomarker sequences, expression of the biomarker sequences can be detected (if present) and measured using techniques well known to one of ordinary skill in the art. For example, sequences in sequence database entries or sequences disclosed herein can be used to construct probes for detecting biomarker RNA sequences in, e.g., Northern blot hybridization analyses or methods which specifically, and, preferably, quantitatively amplify specific nucleic acid sequences. As another example, the sequences can be used to construct primers for specifically amplifying the biomarker sequences in, e.g., amplification-based detection methods such as reverse-transcription based polymerase chain reaction (RT-PCR). When alterations in gene expression are associated with gene amplification, deletion, polymorphisms and mutations, sequence comparisons in test and reference populations can be made by comparing relative amounts of the examined DNA sequences in the test and reference cell populations. In addition to Northern blot and RT-PCR, RNA can also be measured using, for example, other target amplification methods (e.g., TMA, SDA, NASBA), signal amplification methods (e.g., bDNA), nuclease protection assays, in situ hybridization and the like. 
         [0079]    In one embodiment in the present invention are biochip assays. By “biochip” or “chip” herein is meant a composition generally comprising a solid support or substrate to which a capture binding ligand (also called an adsorbent, affinity reagent or binding ligand, or when nucleic acid is measured, a capture probe) is attached and can bind either proteins, nucleic acids or both. Generally, where a biochip is used for measurements of protein and nucleic acid biomarkers, the protein biomarkers are measured on a chip separate from that used to measure the nucleic acid biomarkers. For nonlimiting examples of additional platforms and methods useful for measuring nucleic acids, see Publications US/2006/0275782, US/2005/0064469 and DE10201463. In various embodiments, biomarkers are measured on the same platform, such as on one chip. In various embodiments, biomarkers are measured using different platforms and/or different experimental runs. 
         [0080]    By “binding ligand,” “capture binding ligand,” “capture binding species,” “capture probe” or grammatical equivalents herein is meant a compound that is used to detect the presence of or to quantify, relatively or absolutely, a target analyte, target species or target sequence (all used interchangeably) and that will bind to the target analyte, target species or target sequence. Generally, the capture binding ligand or capture probe allows the attachment of a target species or target sequence to a solid support for the purposes of detection as further described herein. Attachment of the target species to the capture binding ligand may be direct or indirect. In exemplary embodiments, the target species is a biomarker. As will be appreciated by those in the art, the composition of the binding ligand will depend on the composition of the biomarker. Binding ligands for a wide variety of biomarkers are known or can be readily found using known techniques. For example, when the biomarker is a protein, the binding ligands include proteins (particularly including antibodies or fragments thereof (F ab s, etc.) as discussed further below) or small molecules. The binding ligand may also have cross-reactivity with proteins of other species. Antigen-antibody pairs, receptor-ligands, and carbohydrates and their binding partners are also suitable analyte-binding ligand pairs. In various embodiments, the binding ligand may be nucleic acid. Nucleic acid binding ligands find particular use when proteins are the targets; alternatively, as is generally described in U.S. Pat. Nos. 5,270,163; 5,475,096; 5,567,588; 5,595,877; 5,637,459; 5,683,867; 5,705,337 and related patents, hereby incorporated by reference, nucleic acid “aptamers” can be developed for binding to virtually any biomarker. Nucleic acid binding ligands also find particular use when nucleic acids are binding targets. There is a wide body of literature relating to the development of binding partners based on combinatorial chemistry methods. In these embodiments, when the binding ligand is a nucleic acid, preferred compositions and techniques are outlined in PCT Publication WO/1998/020162, hereby incorporated by reference. 
         [0081]    In various exemplary embodiments, the capture binding ligand is an antibody. These embodiments are particularly useful for the detection of the protein form of a biomarker. 
         [0082]    Detecting or measuring the level (e.g. the transcription level) of a biomarker involves binding of the biomarker to a capture binding ligand, generally referred to herein as a “capture probe” when the mRNA of the biomarker is to be detected on a solid support. In that sense, the biomarker is a target sequence. The term “target sequence” or “target nucleic acid” or grammatical equivalents herein means a nucleic acid sequence that may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others. As is outlined herein, the target sequence may be a target sequence from a sample, or a secondary target such as a product of an amplification reaction such as PCR etc. In some embodiments, measuring a nucleic acid can thus refer to measuring the complement of the nucleic acid. It may be any length, with the understanding that longer sequences are more specific. 
         [0083]    The target sequence may also comprise different target domains; for example, a first target domain of the sample target sequence may hybridize to a first capture probe, a second target domain may hybridize to a label probe (e.g. a “sandwich assay” format), etc. The target domains may be adjacent or separated as indicated. Unless specified, the terms “first” and “second” are not meant to confer an orientation of the sequences with respect to the 5′-3′ orientation of the target sequence. For example, assuming a 5′-3′ orientation of the target sequence, the first target domain may be located either 5′ to the second domain, or 3′ to the second domain. 
         [0084]    When nucleic acids are used as the target analyte, the assays of the invention can take on a number of embodiments. In one embodiment, the assays are done in solution format, using any number of solution based formats. In one embodiment, end-point or real time PCR formats are used, as are well known in the art. These assays can be done either as a panel, in individual tubes or wells, or as multiplex assays, using sets of primers and different labels within a single tube or well. In addition to PCR-based solution formats, other formats can be utilized, including, but not limited to for example ligation based assays utilizing FRET dye pairs. In this embodiment, only upon ligation of two (or more) probes hybridized to the target sequence is a signal generated. 
         [0085]    In many embodiments, the assays are done on a solid support, utilizing a capture probe associated with the surface. As discussed herein, the capture probes (or capture binding ligands, as they are sometimes referred to) can be covalently attached to the surface, for example using capture probes terminally modified with functional groups, for example amino groups, that are attached to modified surfaces such as silanized glass. Alternatively, non-covalent attachment, such as electrostatic, hydrophobic/hydrophilic adhesion can be utilized. As is appreciated by those in the art and discussed herein, a large number of attachments are possible on a wide variety of surfaces. 
         [0086]    In this embodiment, the assays can take on a number of formats. In one embodiment, the target sequence comprises a detectable label, as described herein. In this embodiment, the label is generally added to the target sequence during amplification of the target in one of two ways: either labeled primers are utilized during the amplification step or labeled dNTPs are used, both of which are well known in the art. The label can either be a primary or secondary label as discussed herein. For example, in one embodiment, the label on the primer and/or a dNTP is a primary label such as a fluorophore. Alternatively, the label may be a secondary label such as biotin or an enzyme; for example, in one embodiment, the primers or dNTPs are labeled with biotin, and then a streptavidin/label complex is added. In one embodiment, the streptavidin/label complex contains a label such as a fluorophore. In an alternative embodiment, the streptavidin/label complex comprises an enzymatic label. For example, the complex can comprise horseradish peroxidase, and upon addition of TMB, the action of the horseradish peroxidase causes the TMB to precipitate, causing an optically detectable event. This has a particular benefit in that the optics for detection does not require the use of a fluorimeter. 
         [0087]    In alternate embodiments, the solid phase assay relies on the use of a labeled soluble capture ligand, sometimes referred to as a “label probe” or “signaling probe” when the target analyte is a nucleic acid. In this format, the assay is a “sandwich” type assay, where the capture probe binds to a first domain of the target sequence and the label probe binds to a second domain. In this embodiment, the label probe can also be either a primary (e.g. a fluorophore) or a secondary (biotin or enzyme) label. In one embodiment, the label probe comprises biotin, and a streptavidin/enzyme complex is used, as discussed herein. As above, for example, the complex can comprise horseradish peroxidase, and upon addition of TMB, the action of the horseradish peroxidase causes the TMB to precipitate, causing an optically detectable event. 
         [0088]    Detection of a target species in some embodiments requires a “label” or “detectable marker” (as described below) that can be incorporated in a variety of ways. Thus, in various embodiments, the composition comprises a “label” or a “detectable marker.” In one embodiment, the target species (or target analyte or target sequence) is labeled; binding of the target species thus provides the label at the surface of the solid support. 
         [0089]    In embodiments finding particular use herein, a sandwich format is utilized, in which target species are unlabeled. In these embodiments, a “capture” or “anchor” binding ligand is attached to the detection surface as described herein, and a soluble binding ligand (frequently referred to herein as a “signaling probe,” “label probe” or “soluble capture ligand”) binds independently to the target species and either directly or indirectly comprises at least one label or detectable marker. 
         [0090]    By “label” or “labeled” herein is meant that a compound has at least one molecule, element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into four classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal; c) colored or luminescent dyes; and d) enzymes; although labels include particles such as magnetic particles as well. The dyes may be chromophores or phosphors but are preferably fluorescent dyes, which due to their strong signals provide a good signal-to-noise ratio for decoding. Suitable dyes for use in the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue, Texas Red, Alexa dyes and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference. Additional labels include nanocrystals or Q-dots as described in U.S. Pat. No. 6,544,732 incorporated by reference. 
         [0091]    In various embodiments, a secondary detectable label is used. A secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, can act on an additional product to generate a primary label (e.g. enzymes), or may allow the separation of the compound comprising the secondary label from unlabeled materials, etc. Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, enzymes such as horseradish peroxidase, alkaline phosphatases, lucifierases, etc. Secondary labels can also include additional labels. 
         [0092]    In various embodiments, the secondary label is a binding partner pair. For example, the label may be a hapten or antigen, which will bind its binding partner. For example, suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides)) and antibodies (including fragments thereof (F ab s, etc.)); proteins and small molecules, including biotin/streptavidin; enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners. Nucleic acid-nucleic acid binding proteins pairs are also useful. In general, the smaller of the pair is attached to the NTP for incorporation into the primer. Preferred binding partner pairs include, but are not limited to, biotin (or imino-biotin) and streptavidin, digeoxinin and Abs, and Prolinx™ reagents. 
         [0093]    In the sandwich formats of the invention, an enzyme serves as the secondary label, bound to the soluble capture ligand. Of particular use in some embodiments is the use of horseradish peroxidase, which when combined with 3,3′,5,5′-tetramethylbenzidine (TMB) forms a colored precipitate which is then detected. In some cases, the soluble capture ligand comprises biotin, which is then bound to a enzyme-streptavidin complex and forms a colored precipitate with the addition of TMB. 
         [0094]    In various embodiments, the label or detectable marker is a conjugated enzyme (for example, horseradish peroxidase). In various embodiments, the system relies on detecting the precipitation of a reaction product or on a change in, for example, electronic properties for detection. In various embodiments, none of the compounds comprises a label. 
         [0095]    As used herein, the term “fluorescent signal generating moiety” or “fluorophore” refers to a molecule or part of a molecule that absorbs energy at one wavelength and re-emits energy at another wavelength. Fluorescent properties that can be measured include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer, and the like. 
         [0096]    Signals from single molecules can be generated and detected by a number of detection systems, including, but not limited to, scanning electron microscopy, near field scanning optical microscopy (NSOM), total internal reflection fluorescence microscopy (TIRFM), and the like. Abundant guidance is found in the literature for applying such techniques for analyzing and detecting nanoscale structures on surfaces, as evidenced by the following references that are incorporated by reference: Reimer et al, editors,  Scanning Electron Microscopy: Physics of Image Formation and Microanalysis,  2nd Edition (Springer, 1998); Nie et al,  Anal. Chem.,  78: 1528-1534 (2006); Hecht et al,  Journal Chemical Physics,  112: 7761-7774 (2000); Zhu et al, editors,  Near - Field Optics: Principles and Applications  (World Scientific Publishing, Singapore, 1999); Drmanac, PCT Publication WO/2004/076683; Lehr et al,  Anal. Chem.,  75: 2414-2420 (2003); Neuschafer et al,  Biosensors  &amp;  Bioelectronics,  18: 489-497 (2003); Neuschafer et al, U.S. Pat. No. 6,289,144; and the like. 
         [0097]    Thus, a detection system for fluorophores includes any device that can be used to measure fluorescent properties as discussed above. In various embodiments, the detection system comprises an excitation source, a fluorophore, a wavelength filter to isolate emission photons from excitation photons and a detector that registers emission photons and produces a recordable output, in some embodiments as an electrical signal or a photographic image. Examples of detection devices include without limitation spectrofluorometers and microplate readers, fluorescence microscopes, fluorescence scanners (including e.g. microarray readers) and flow cytometers. 
         [0098]    In various exemplary embodiments, the binding of the biomarker to the binding ligand is specific or selective, and the binding ligand is part of a binding pair. By “specifically bind” or “selectively bind” or “selective for” a biomarker herein is meant that the ligand binds the biomarker with specificity sufficient to differentiate between the biomarker and other components or contaminants of the test sample. 
         [0099]    The term “solid support” or “substrate” refers to any material that can be modified to contain discrete individual sites appropriate for the attachment or association of a capture binding ligand. Suitable substrates include metal surfaces such as gold, electrodes, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes, Teflon, derivatives thereof, etc.), polysaccharides, nylon or nitrocellulose, resins, mica, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, fiberglass, ceramics, GETEK (a blend of polypropylene oxide and fiberglass) and a variety of other polymers. Of particular use in the present invention are the ClonDiag materials described below. 
         [0100]    Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which comprises a capture binding ligand. An “array location,” “addressable location,” “pad” or “site” herein means a location on the substrate that comprises a covalently attached capture binding ligand. An “array” herein means a plurality of capture binding ligands in a regular, ordered format, such as a matrix. The size of the array will depend on the composition and end use of the array. Arrays containing from about two or more different capture binding ligands to many thousands can be made. Generally, the array will comprise 3, 4, 5, 6, 7 or more types of capture binding ligands depending on the end use of the array. In the present invention, the array can include controls, replicates of the markers and the like. Exemplary ranges are from about 3 to about 50. In some embodiments, the compositions of the invention may not be in array format; that is, for some embodiments, compositions comprising a single capture ligand may be made as well. In addition, in some arrays, multiple substrates may be used, either of different or identical compositions. Thus for example, large arrays may comprise a plurality of smaller substrates. 
         [0101]    Accordingly, in one aspect, the invention provides a composition comprising a solid support comprising a capture binding ligand for each biomarker of a biomarker panel. In various embodiments, the capture ligand is a nucleic acid. In various embodiments, the capture binding ligand is an antibody. In various embodiments, the composition further comprises a soluble binding ligand for each biomarker of a biomarker panel. 
         [0102]    A number of different biochip array platforms as known in the art may be used. For example, the compositions and methods of the present invention can be implemented with array platforms such as GeneChip® (Affymetrix), CodeLink™ Bioarray (Amersham), Expression Array System (Applied Biosystems), SurePrint microarrays (Agilent), Sentrix® LD BeadChip or Sentrix® Array Matrix (Illumina) and Verigene (Nanosphere). 
         [0103]    In various exemplary embodiments, detection and measurement of biomarkers utilizes colorimetric methods and systems in order to provide an indication of binding of a target analyte or target species. In colorimetric methods, the presence of a bound target species such as a biomarker will result in a change in the absorbance or transmission of light by a sample or substrate at one or more wavelengths. Detection of the absorbance or transmission of light at such wavelengths thus provides an indication of the presence of the target species. 
         [0104]    A detection system for colorimetric methods includes any device that can be used to measure colorimetric properties as discussed above. Generally, the device is a spectrophotometer, a colorimeter or any device that measures absorbance or transmission of light at one or more wavelengths. In various embodiments, the detection system comprises a light source; a wavelength filter or monochromator; a sample container such as a cuvette or a reaction vial; a detector, such as a photoresistor, that registers transmitted light; and a display or imaging element. 
         [0105]    In various exemplary embodiments, a ClonDiag chip platform is used for the colorimetric detection of biomarkers. In various embodiments, a ClonDiag ArrayTube (AT) is used. One unique feature of the ArrayTube is the combination of a micro probe array (the biochip) and micro reaction vial. In various embodiments, where a target sequence is a nucleic acid, detection of the target sequence is done by amplifying and biotinylating the target sequence contained in a sample and optionally digesting the amplification products. The amplification product is then allowed to hybridize with probes contained on the ClonDiag chip. A solution of a streptavidin-enzyme conjugate, such as Poly horseradish peroxidase (HRP) conjugate solution, is contacted with the ClonDiag chip. After washing, a dye solution such as o-dianisidine substrate solution is contacted with the chip. Oxidation of the dye results in precipitation that can be detected colorimetrically. Further description of the ClonDiag platform is found in Monecke S, Slickers P, Hotzel H et al.,  Clin Microbiol Infect  2006, 12: 718-728; Monecke S, Berger-Bashi B, Coombs C et al.,  Clin Microbiol Infect  2007, 13: 236-249; Monecke S, Leube I and Ehricht R,  Genome Lett  2003, 2: 106-118; Monecke S and Ehricht R,  Clin Microbiol Infect  2005, 11: 825-833; German Patent DE 10201463; US Publication US/2005/0064469 and ClonDiag,  ArrayTube  ( AT )  Experiment Guideline for DNA - Based Applications,  version 1.2, 2007, all incorporated by reference in their entirety. One of skill in the art will appreciate that numerous other dyes that react with a peroxidase can be utilized to produce a colorimetric change, such as 3,3′,5,5′-tetramethylbenzidine (TMB). For information on specific assay protocols, see www.clondiag.com/technologies/publications.php. 
         [0106]    In various embodiments, where a target species is a protein, the ArrayTube biochip comprises capture binding ligands such as antibodies. A sample is contacted with the biochip, and any target species present in the sample is allowed to bind to the capture binding ligand antibodies. A soluble capture binding ligand or a detection compound such as a horseradish peroxidase conjugated antibody is allowed to bind to the target species. A dye, such as TMB, is then added and allowed to react with the horseradish peroxidase, causing precipitation and a color change that is detected by a suitable detection device. Further description of protein detection using ArrayTube is found in, for example, Huelseweh B, Ehricht R and Marschall H-J,  Proteomics,  2006, 6, 2972-2981; and ClonDiag,  ArrayTube  ( AT )  Experiment Guideline for Protein - Based Applications,  version 1.2, 2007, all incorporated by reference in their entirety. 
         [0107]    Transmission detection and analysis is performed with a ClonDiag AT reader instrument. Suitable reader instruments and detection devices include the ArrayTube Workstation ATS and the ATR 03. 
         [0108]    In addition to ArrayTube, the ClonDiag ArrayStrip (AS) can be used. The ArrayStrip provides a 96-well format for high volume testing. Each ArrayStrip consists of a standard 8-well strip with a microarray integrated into the bottom of each well. Up to 12 ArrayStrips can be inserted into one microplate frame enabling the parallel multiparameter testing of up to 96 samples. The ArrayStrip can be processed using the ArrayStrip Processor ASP, which performs all liquid handling, incubation, and detection steps required in array based analysis. In various embodiments, where a protein is detected, a method of using the ArrayStrip to detect the protein comprises conditioning the AS array with buffer or blocking solution; loading of up to 96 sample solutions in the AS wells to allow for binding of the protein; 3× washing; conjugating with a secondary antibody linked to HRP; 3× washing; precipitation staining with TMB; and AS array imaging and optional data storage. 
         [0109]    Those skilled in the art will be familiar with numerous additional immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein. See generally E. Maggio,  Enzyme - Immunoassay,  (CRC Press, Inc., Boca Raton, Fla., 1980); see also U.S. Pat. Nos. 4,727,022; 4,659,678; 4,376,110; 4,275,149; 4,233,402; and 4,230,767. 
         [0110]    In general, immunoassays carried out in accordance with the present invention may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves the specific antibody (e.g., anti-biomarker protein antibody), a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof can be carried out in a homogeneous solution. Immunochemical labels which may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, or coenzymes. 
         [0111]    In a heterogeneous assay approach, the reagents are usually the sample, the antibody, and means for producing a detectable signal. Samples as described above may be used. The antibody can be immobilized on a support, such as a bead (such as protein A and protein G agarose beads), plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the sample. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, or enzyme labels. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays include immunoblotting, immunofluorescence methods, immunoprecipitation, chemiluminescence methods, electrochemiluminescence (ECL) or enzyme-linked immunoassays. 
         [0112]    Antibodies can be conjugated to a solid support suitable for a diagnostic assay (e.g., beads such as protein A or protein G agarose, microspheres, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as passive binding. Antibodies as described herein may likewise be conjugated to detectable labels or groups such as radiolabels (e.g.,  35 S,  125 I,  131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein, Alexa, green fluorescent protein, rhodamine) in accordance with known techniques. 
         [0113]    Using any of the methods and compositions described herein, a sample can be assayed to determine levels of a biomarker panel. Thus, in one aspect, the invention provides a method of assaying a sample from a patient to determine concentrations of a biomarker panel in the sample. In some embodiments, the method comprises contacting the sample with a composition comprising a solid support comprising a capture binding ligand or capture probe for each biomarker of a biomarker panel. 
         [0114]    The invention further provides kits for use in determining lung health or lung cancer status for a number of medical (including diagnostic and therapeutic), industrial, forensic and research applications. Kits may comprise a carrier, such as a box, carton, tube or the like, having in close confinement therein one or more containers, such as vials, tubes, ampoules, bottles, pouches, envelopes and the like. In various embodiments, the kits comprise one or more components selected from one or more media or media ingredients and reagents for the measurement of the various biomarkers and biomarker panels disclosed herein. For example, kits of the invention may also comprise, in the same or different containers, one or more DNA polymerases, one or more primers, one or more suitable buffers, one or more nucleotides (such as deoxynucleoside triphosphates (dNTPs) and preferably fluorescently labeled dNTPs) and labeling components. The one or more components may be contained within the same container, or may be in separate containers to be admixed prior to use. The kits of the present invention may also comprise one or more instructions or protocols for carrying out the methods of the present invention. The kits may also comprise a computer or a component of a computer, such as a computer-readable storage medium or device. Examples of storage media include, without limitation, optical disks such as CD, DVD and Blu-ray Discs (BD); magneto-optical disks; magnetic media such as magnetic tape and internal hard disks and removable disks; semi-conductor memory devices such as EPROM, EEPROM and flash memory; and RAM. The computer-readable storage medium may comprise software encoding references to the various therapies and treatment regimens disclosed herein. The software may be interpreted by a computer to provide the practitioner with treatments according to various measured concentrations of biomarkers as provided herein. In various embodiments, the kit comprises a biomarker assay involving a lateral-flow-based point-of-care rapid test with detection of risk thresholds, or a biochip with quantitative assays for the constituent biomarkers. 
       Methods of Diagnosing and Treating 
       [0115]    The compositions and methods of the present invention can be used in the prognosis, diagnosis and treatment of disease in a subject. The invention provides compositions and methods for laboratory and point-of-care tests for measuring biomarkers in a sample from a subject. The invention can be generally applied for a number of different diseases. In exemplary embodiments, the disease is lung cancer. 
         [0116]    The biomarkers and biomarker panels disclosed herein can be used in methods to diagnose, identify or screen subjects that have, do not have or are at risk for having disease; to monitor subjects that are undergoing therapies for disease; to determine or suggest a new therapy or a change in therapy; to differentially diagnose disease states associated with the disease from other diseases or within sub-classifications of disease; to evaluate the severity or changes in severity of disease in a patient; to stage a subject with the disease and to select or modify therapies or interventions for use in treating subjects with the disease. In an exemplary embodiment, the methods of the present invention are used to identify and/or diagnose subjects who are asymptomatic or presymptomatic for a disease. In this context, “asymptomatic” or “presymptomatic” means not exhibiting the traditional symptoms or enough abnormality for disease. 
         [0117]    In various embodiments, a method of determining a prognosis of a disease in a subject, diagnosing a disease in a subject, or treating a disease in a subject comprises taking a measurement of a biomarker panel in a sample from the subject. In various exemplary embodiments, the biomarker panel consists of two or more of CCNI (Cyclin I)(SEQ ID NO: 1), EGFR(Epidermal growth factor receptor)(SEQ ID NO:2), FGF9 (Fibroblast growth factor 19)(SEQ ID NO: 3), GREB1 protein (SEQ ID NO:4), LZTS (Leucine zipper, putative suppresor I)(SEQ ID NO: 5), BRAF (v-raf murine sarcoma viral oncogene homolog B1 (SEQ ID NO: 6), FRS2 (Fibroblast growth factor receptor substrate 2)(SEQ ID NO: 7), ANXA1 (Annexin A1)(SEQ ID NO: 8), Hp2 (Haptoglobin 2)(SEQ ID NO:8), Chain B, Crystal Structure of the Complex formed between MHc-Like Zinc Alpha2-Glycoprotein and Pro1,  Porphyromonas catoniae, Campylobacter showae, Streptocococcus salivaris, Campylobacter rectus, Veillonella parvula, Kigella oralis.    
         [0118]    The term “disease status” includes any distinguishable manifestation of the disease, including non-disease. For example, disease status includes, without limitation, the presence or absence of disease, the risk of developing disease, the stage of the disease, the progression of disease (e.g., progress of disease or remission of disease over time), the severity of disease and the effectiveness or response to treatment of disease. 
         [0119]    A “subject” in the context of the present invention is an animal, preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In various exemplary embodiments, a subject is human and may be referred to as a patient. Mammals other than humans can be advantageously used as subjects that represent animal models of a disease or for veterinarian applications. A subject can be one who has been previously diagnosed or identified as having a disease, and optionally has already undergone, or is undergoing, a therapeutic intervention for a disease. Alternatively, a subject can also be one who has not been previously diagnosed as having a disease. For example, a subject can be one who exhibits one or more risk factors for a disease, or one who does not exhibit a disease risk factor, or one who is asymptomatic for a disease. A subject can also be one who is suffering from or at risk of developing a disease. In certain embodiments, the subject can be already undergoing therapy or can be a candidate for therapy. 
         [0120]    As will be appreciated by those in the art, the biomarkers may be measured in using several techniques designed to achieve more predictable subject and analytical variability. 
         [0121]    The term “sample” refers to a specimen or culture obtained from a subject and includes fluids, gases and solids including for example tissue. In various exemplary embodiments, the sample comprises saliva. As will be appreciated by those in the art, virtually any experimental manipulation or sample preparation steps may have been done on the sample. For example, wash steps and/or fragmentation may be applied to a sample. In various embodiments, a biomarker panel is measured directly in a subject without the need to obtain a separate sample from the patient. 
         [0122]    In one aspect, the invention provides a method of diagnosing a subject for a disease comprising taking a measurement of a biomarker panel; and correlating the measurement with the disease. The term “correlating” generally refers to determining a relationship between one type of data with another or with a state. In various embodiments, correlating the measurement with disease comprises comparing the measurement with a reference biomarker profile or some other reference value. In various embodiments, correlating the measurement with disease comprises determining whether the subject is currently in a state of disease. 
         [0123]    The quantity or activity measurements of a biomarker panel can be compared to a reference value. Differences in the measurements of biomarkers in the subject sample compared to the reference value are then identified. In exemplary embodiments, the reference value is given by a risk category as described further below. 
         [0124]    In various embodiments, the reference value is a baseline value. A baseline value is a composite sample of an effective amount of biomarkers from one or more subjects who do not have a disease, who are asymptomatic for a disease or who have a certain level of a disease. A baseline value can also comprise the amounts of biomarkers in a sample derived from a subject who has shown an improvement in risk factors of a disease as a result of treatments or therapies. In these embodiments, to make comparisons to the subject-derived sample, the amounts of biomarkers are similarly calculated. A reference value can also comprise the amounts of biomarkers derived from subjects who have a disease confirmed by an invasive or non-invasive technique, or are at high risk for developing a disease. Optionally, subjects identified as having a disease, or being at increased risk of developing a disease are chosen to receive a therapeutic regimen to slow the progression of a disease, or decrease or prevent the risk of developing a disease. A disease is considered to be progressive (or, alternatively, the treatment does not prevent progression) if the amount of biomarker changes over time relative to the reference value, whereas a disease is not progressive if the amount of biomarkers remains constant over time (relative to the reference population, or “constant” as used herein). The term “constant” as used in the context of the present invention is construed to include changes over time with respect to the reference value. 
         [0125]    The biomarkers of the present invention can be used to generate a “reference biomarker profile” of those subjects who do not have a disease according to a certain threshold, are not at risk of having a disease or would not be expected to develop a disease. The biomarkers disclosed herein can also be used to generate a “subject biomarker profile” taken from subjects who have a disease or are at risk for having a disease. The subject biomarker profiles can be compared to a reference biomarker profile to diagnose or identify subjects at risk for developing a disease, to monitor the progression of disease, as well as the rate of progression of disease, and to monitor the effectiveness of disease treatment modalities. The reference and subject biomarker profiles of the present invention can be contained in a machine-readable medium, such as but not limited to, analog tapes like those readable by a VCR; optical media such as CD-ROM, DVD-ROM and the like; and solid state memory, among others. 
         [0126]    Measurements of the biomarker panels of the invention can lead a practitioner to affect a therapy with respect to a subject. Thus, the invention provides methods of treating a disease in a subject comprising taking a measurement of a biomarker panel in a sample from the subject, and affecting a therapy with respect to the subject. The terms “therapy” and “treatment” may be used interchangeably. In certain embodiments, the therapy can be selected from, without limitation, initiating therapy, continuing therapy, modifying therapy or ending therapy. A therapy also includes any prophylactic measures that may be taken to prevent disease. 
         [0127]    In certain embodiments, treatment comprises administering a disease-modulating drug to a subject. The drug can be a therapeutic or prophylactic used in subjects diagnosed or identified with a disease or at risk of having the disease. In certain embodiments, modifying therapy refers to altering the duration, frequency or intensity of therapy, for example, altering dosage levels. 
         [0128]    In various embodiments, effecting a therapy comprises causing a subject to or communicating to a subject the need to make a change in lifestyle, for example, increasing exercise, changing diet, reducing or eliminating smoking and so on. The therapy can also include surgery, for example, mastectomy. 
         [0129]    Measurement of biomarker levels allow for the course of treatment of a disease to be monitored. The effectiveness of a treatment regimen for a disease can be monitored by detecting one or more biomarkers in an effective amount from samples obtained from a subject over time and comparing the amount of biomarkers detected. For example, a first sample can be obtained prior to the subject receiving treatment and one or more subsequent samples are taken after or during treatment of the subject. Changes in biomarker levels across the samples may provide an indication as to the effectiveness of the therapy. 
         [0130]    To identify therapeutics or drugs that are appropriate for a specific subject, a test sample from the subject can also be exposed to a therapeutic agent or a drug, and the level of one or more biomarkers can be determined. Biomarker levels can be compared to a sample derived from the subject before and after treatment or exposure to a therapeutic agent or a drug, or can be compared to samples derived from one or more subjects who have shown improvements relative to a disease as a result of such treatment or exposure. Thus, in one aspect, the invention provides a method of assessing the efficacy of a therapy with respect to a subject comprising taking a first measurement of a biomarker panel in a first sample from the subject; effecting the therapy with respect to the subject; taking a second measurement of the biomarker panel in a second sample from the subject and comparing the first and second measurements to assess the efficacy of the therapy. 
         [0131]    Additionally, therapeutic or prophylactic agents suitable for administration to a particular subject can be identified by detecting a biomarker (which may be two or more) in an effective amount from a sample obtained from a subject and exposing the subject-derived sample to a test compound that determines the amount of the biomarker(s) in the subject-derived sample. Accordingly, treatments or therapeutic regimens for use in subjects having a disease or subjects at risk for developing a disease can be selected based on the amounts of biomarkers in samples obtained from the subjects and compared to a reference value. Two or more treatments or therapeutic regimens can be evaluated in parallel to determine which treatment or therapeutic regimen would be the most efficacious for use in a subject to delay onset, or slow progression of a disease. In various embodiments, a recommendation is made on whether to initiate or continue treatment of a disease. 
       Drug Treatments 
       [0132]    In various exemplary embodiments, effecting a therapy comprises administering a disease-modulating drug to the subject. The subject may be treated with one or more disease-modulating drugs until altered levels of the measured biomarkers return to a baseline value measured in a population not suffering from the disease, experiencing a less severe stage or form of a disease or showing improvements in disease biomarkers as a result of treatment with a disease-modulating drug. Additionally, improvements related to a changed level of a biomarker or clinical parameter may be the result of treatment with a disease-modulating drug. 
         [0133]    A number of compounds such as a disease-modulating drug may be used to treat a subject and to monitor progress using the methods of the invention. In certain embodiments, the disease-modulating drug comprises 
         [0134]    The beneficial effects of these and other drugs can be visualized by assessment of clinical and laboratory biomarkers. 
         [0135]    Any drug or combination of drugs disclosed herein may be administered to a subject to treat a disease. The drugs herein can be formulated in any number of ways, often according to various known formulations in the art or as disclosed or referenced herein. 
         [0136]    In various embodiments, any drug or combination of drugs disclosed herein is not administered to a subject to treat a disease. In these embodiments, the practitioner may refrain from administering the drug or combination of drugs, may recommend that the subject not be administered the drug or combination of drugs or may prevent the subject from being administered the drug or combination of drugs. 
         [0137]    In various embodiments, one or more additional drugs may be optionally administered in addition to those that are recommended or have been administered. An additional drug will typically not be any drug that is not recommended or that should be avoided. In exemplary embodiments, one or more additional drugs comprise one or more glucose lowering drugs. 
       Decision Matrices 
       [0138]    The therapy chosen by a practitioner can depend on the concentrations of biomarkers determined in a sample. In various exemplary embodiments, the therapy depends on which category from a range of categories particular to each biomarker the measured concentration of each biomarker falls in. In various exemplary embodiments, the therapy depends on the combination of risk levels for different symptoms or diseases that are indicated by a biomarker panel. 
         [0139]    With respect to concentration measurements of a biomarker, the term “category” refers to a subset of a partition of the possible concentrations that a biomarker may have. Each category may be associated with a label or classification chosen by the practitioner. The labels may be refer to, for example, the risk level of an individual for having or being subject to a disease state. The categories and labels may be derived from the current literature or according to the findings of the practitioner. 
         [0140]    Each biomarker of a biomarker panel can thus be associated with a discrete set of categories, for example, risk categories. Combining one category from each biomarker forms a “decision point.” In various exemplary embodiments, the complete set of decision points comprises all possible n-tuples of categories, wherein n is the number of biomarkers in the biomarker panel. This complete set will have m 1 ×m 2 × . . . m n  possible decision points, wherein m i  is the number of categories for biomarker i. 
         [0141]    Every decision point can be associated with a condition or a disease state, which is not necessarily unique. That is, one or more decision points can be associated with the same disease state. The association of every possible decision point with a condition or disease state can be referred to as a “disease classification matrix” or a “disease classification tree.” Thus, by correlating a measurement of a biomarker panel with a decision point, the practitioner can classify the condition or disease state of a patient. 
         [0142]    Every decision point can also be associated with a particular therapy, which is not necessarily unique. That is, one or more decision points can be associated with the same therapy. The association of every possible decision point with one or more therapies can be referred to as a “therapy decision matrix” or “therapy decision tree.” 
         [0143]    Each decision point can be associated with more than one type of information. For example, both disease state and therapy can be indicated by a decision point. 
         [0144]    The articles “a,” “an” and “the” as used herein do not exclude a plural number of the referent, unless context clearly dictates otherwise. The conjunction “or” is not mutually exclusive, unless context clearly dictates otherwise. The term “include” is used to refer to non-limiting examples. 
       EXAMPLES 
       [0145]    The following examples are offered to illustrate, but not to limit the invention. 
       Example 1 
     Salivary Transciptomic Profiling and Analysis 
     Saliva Collection 
       [0146]    Unstimulated whole saliva samples were collected with previously established protocols. Subjects were asked to refrain from eating, drinking, smoking, or oral hygiene procedures for at least 30 minutes before the collection. Lipstick was wiped off, and the subject rinsed her mouth once with plain water. Typically, patients donated approximately 5-10 ml of saliva. Samples were then centrifuged at 2,600 g for 15 minutes at 4° C. The supernatant was then stored at −80° C. until use. Of note, protease inhibitors cocktail, containing 1 μl aprotinin, 10 μl PMSF (phenylmethanesulfonyl fluoride) and 3 μl sodium orthovanadate (all from Sigma, St. Louis, Mo.) were added to each 1 ml saliva sample. 
         [0000]    mRNA Isolation and Analysis 
         [0147]    RNA was isolated from 330 μl of saliva supernatant using MagMax™ Viral RNA Isolation Kit (Ambion, Austin, Tex.). This process was automated using KingFisher® mL technology (Thermo Fisher Scientific, Waltham, Mass.), followed by TURBO™ DNase treatment (Ambion, Austin, Tex.) to remove contaminating DNA. 90 μl of extracted RNA (out of 100 μl) was concentrated to 11 μl and was linearly amplified using the RiboAmp® RNA Amplification kit (Molecular Devices, Sunnyvale, Calif.). After purification, cDNA was transcribed and biotinylated using GeneChip® Expression 3′-Amplification Reagents for in vitro transcription labeling (Affymetrix, Santa Clara, Calif.). Approximately 20 μg of labeled RNA were subsequently submitted for GeneChip® analysis using an Affymetrix Human Genome U133 Plus 2.0 Array. Chip hybridization and scanning were performed. 
       Gene Array Statistical Analysis 
       [0148]    The CEL files from all databases were imported into the statistical R 2.7.0 (hypertext transfer protocol://www.r-project.org) using Bioconductor 2.2 (hypertext transfer protocol://www.bioconductor.org. The Probe Logarithmic Intensity Error Estimation (PLIER) expression measures were computed after background correction and quantile normalization for each microarray dataset. Probeset-level quantile normalization was performed across all samples to make the effect sizes similar among all datasets. Finally, for every probeset, two-sample t-test was applied to identify differential expression. After obtaining the estimates and the p-values of each probset, p-values of each probeset was corrected for false discovery rate (FDR). Genes were selected at the FDR level of 0.05, and with cancer effect size &gt;2 fold change between cancer and normal samples. 
       Screening of Biomarker Candidates 
       [0149]    The biomarker candidates generated by microarray profiling were subjected to further screening by real-time quantitative RT-PCR (qPCR) on the same set of samples used for the microarray analysis. To accomplish this, total RNA was reverse-transcribed using reverse transcriptase and gene-specific primers using the following thermal cycling conditions: 1 min at 60° C., 30 min at 50° C., 2 min at 95° C., followed by 15 cycles of 15 s at 95° C., 30 s at 50° C., 10 s at 72° C. These steps were followed with a final extension of 5 min. at 72° C. and then cooling to 4° C. The preamplified product was cleaned using ExoSAP-IT (USB Corporation) and diluted 1/40 in water. 2 μl of the cDNA was used for qPCR. 
         [0150]    qPCR was carried out in a 96-well plate in a reation volume of 10 μl using power SYBR®-Green Master Mix (Applied Biosystems, Foster City, Calif.) for 15 min at 95° C. for initial denaturing, followed by 40 cycles of 95° C. for 30 s and 60° C. for 30 s in the ABI 7500HT Fast Real Time PCR system (Applied Biosystems, Foster City, Calif.). All qPCRS were performed in duplicate for all candidate mRNA. The specificity of the PCR was confirmed according to the melting curve of each gene, and the average threshold cycle (Ct) was examined. 
         [0151]    Amplicon lengths were around 100-130 by for the outer primer pairs used in preamplification and 60-80 by for the inner primer pairs used in qPCR. RT-qPCR primers were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, Calif.). All primers were synthesized by Sigma-Genosys (Woodlands, Tex.), and the amplicons were intron spanning whenever possible. 
         [0152]    Raw data were normalized by subtracting GAPDH Ct values from the biomarker Ct values to generate ΔCt. The Mann-Whitney rank sum test was used for between-group biomarker comparisons. 
         [0153]    The data analysis for qPCR was performed using the 2 −Ct  method, where GAPDH is used as the reference gene. The qPCR based gene expression values between two groups were compared using the non-parametric Wilcoxon test. To normalize for RNA input, qPCR was also performed for GAPDH. Raw data were normalized by subtracting GAPDH Ct values from the marker Ct values to provide ΔCt and then analyze with the stats, utilities packages from R 2.7.0 (world wide web.r-project.org) and the ROC package from Bioconductor 2.2 (world wide web.bioconductor.org). Statistical comparisons were made with the use of the Mann-Whitney U test with consideration of two different distributions for control and pancreatic cancer groups. Biomarkers that differentiated between groups of subjects (P value &lt;0.05) were identified and compared by Area Under Curve (AUC) value. The AUC is based on constructing a receiver operating characteristic (ROC) curve which plots the sensitivity versus one minus the specificity. The AUC value is computed by numerical integration of the ROC curve. The range of this value can be 0.5 to 1.0. A value of 0.5 indicates that the biomarker is no better that a coin toss, while 1.0 indicates the relatively best diagnostic accuracy. 
         [0154]    Seven mRNA biomarkers were identified as differentially expressed between lung cancer subjects and control subjects. The logistic regression model, with the combination of three mRNA biomarkers (GREB1, CCNI, and FRS2) differentiated lung cancer patients from control subjects, yielding a ROC-plot AUC value of approximately 0.91 with approximately 91% sensitivity and approximately 88% specificity. 
       Example 2 
     Salivary Proteomic Profiling and Analysis 
     Protein Isolation and Analysis 
       [0155]    Saliva from 10 healthy control subjects and 10 lung cancer subjects were collected and centrifuged at 2600 g at 4° C. for 15 minutes. Saliva supernatant from the 10 health control subjects and 10 lung cancer subjects were pooled to form a control sample and a cancer sample for proteomic profiling. 250 μg of proteins in the pooled saliva samples were precipitated by methanol and then resuspended in 2-D cell lysis buffer (30 mM Tris-HCl, pH 8.8, containing 7M urea, 2M thiourea and 4% CHAPS detergent). The total proteins of each pooled sample, lung cancer and control, were labeled with the cyanine dyes Cy2 and Cy5 respectively. The two labeled sample sets were then combined and subjected to two-dimensional difference gel electrophoresis. After loading the labeled samples, isoelectric focusing (IEF) (pH3-10) was run following the protocol provided by Amersham BioSciences. The IPG strips were rinses in the SDS-gel running buffer before transferring to 13.5% SDS-gels. The SDS-gels were run at 15° C. until the dye front ran out of the gels. Gel images were scanned immediately following the SDS-PAGE using Typhoon TRIO™ (Amersham BioSciences). The fold change of the protein expression levels was obtained from in-gel DeCyder™ analysis. 
         [0156]    Spots with fold changes larger than 1.5 on the gel were cut and then were washed multiple times to remove staining dye and other chemicals. Gel spots were dried to absorb maximum volume of digestion buffer. Dried 2D gel spots were rehydrated in digestion buffer containing sequencing grade modified trypsin (Promega, USA). Proteins were digested in-gel at 37° C. overnight. Digested peptides were extracted from the gel with TFA extraction buffer and with shaking. The digested tryptic peptides were desalted using C-18 Zip-tips (Millipore). The desalted peptides were mixed with CHCA matrix (α-cyano-4-hydroxycinnamic acid) and spotted into wells of a MALDI plate for MALDI-TOF MS (ABI4800) identification. Protein identification was based on peptide fingerprint mass mapping (using MS spectra) and peptide fragmentation mapping (using MS/MS spectra). Combined MS and MS/MS spectra were submitted for database search using GPS Explorer software equipped with the MASCOT search engine to identify proteins from primary sequence databases. 
       Screening of Biomarker Candidates 
       [0157]    Four proteins (Annexin A1, Haptoglobin HP2, Interleukin 1 receptor antagonsit, Zinc Alpha2-glycoprotein). Calprotectin was verified by commercial ELISA kit. The distribution of Haptoglobin HP2, Zinc Alpha2-glycoprotein and Calprotectin in the cancer group and control shows significant difference with a p value of 0.00903, 0.05, and 0.048, respectively. 
         [0158]    The four proteins identified in the 2-D gel analysis (above) were subjected to Western blot analysis on the original sample set. Reduced protein samples (15 μg total protein per lane) were loaded onto a 10% bis-Tris gel and run at 150V in MES SDS running buffer for one hour. Pre-stained protein standard (Invitrogen) was used to track protein migration. The proteins were transferred to nitrocellulose membrane by using iBlot® (Invitrogen). The membrane was then washed in wash buffer containing 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% (v/v) Tween®-20 (Sigma-Aldrich) before blocking for one hour in wash buffer containing 5% non-fat dry milk. After further washes in wash buffer, the membrane was incubated with primary antibody (mouse anti-Annexin A1 (Abcam), mouse anti-haptoglobin (Lifespan Bioscience), mouse anti-ZAG (Santa Cruz Biotech), mouse anti-actin (Sigma-Aldrich), all according to manufacturers instructions in blocking buffer at room temperature for 2 h. The membrane was then washed before applying the secondary antibody (anti-mouse IgG peroxidase-linked species specific whole antibody from sheep, GE Healthcare) according to manufacturer&#39;s instructions for one hour at room temperature. Finally, the membrane was washed and visualized using ECL Plus™ detection kit (GE Healthcare). The signal intensity of the bands was measured using Image J software (NIH, Bethesda, Md., USA). The intensity of a band representing the protein of interest was divided by the intensity of it corresponding β-actin expression on the same blot for normalization. 
         [0159]    The ELISA for calprotectin was done according to manufacturers instructions (Cell Sciences). All saliva samples were diluted 100 fold in sample diluent. All samples were loaded in duplicate. 
       Example 3 
     Salivary Microbial Biomarkers 
       [0160]    Saliva from 10 healthy control subjects and 10 lung cancer subjects were collected and centrifuged at 2600 g at 4° C. for 15 minutes. The supernatant was decanted and the pellet was used for DNA isolation. 
         [0161]    DNA was extracted from the pellet using UltraClean® Microbial DNA Isolation Kit (MO Bio Laboratories) following the manufacturer&#39;s instructions. Isolated DNA was then submitted to the Forsyth Institute (Cambridge, Mass., USA) for microbe analysis using their Human Oral Microbe Identification Microarray service. Based on the microarray profiles from control and lung cancer subjects, eleven microbes were identified as being differentially present among the two subject groups. 
         [0162]    Quantities of bacterial strains in the original DNA samples were determined using real-time PCR. Specific primers were designed for all species and real-time PCR was performed to quantify bacterial quantity between lung cancer samples and control samples. Seven bacteria were identified by real-time PCR as differentially present among the sample groups. Two bacteria,  Porphyromonas catoniae  and  Campylobacter showae,  were identified as being more abundant in lung cancer subject relative to controls. 
       Example 4 
     Screening Method 
       [0163]    A patient undergoing routine dental care is screened during the visit. For example, a 62 year old female patient, and former smoker, prior to oral exam is asked to provide a saliva sample. A saliva sample is collected and analyzed either at the point of care or is submitted for analysis by a reference laboratory. The saliva sample is tested for the biomarkers of the instant invention and optionally other biomarkers. Results from the analysis are provided to the dental professional and the patient is informed as to whether she has lung cancer. 
         [0000]    
       
         
               
               
             
           
               
                   
                 SEQ ID NO: 1 
               
               
                   
                 Cyclin I (NM_001237.3) 
               
               
                   
                 ccatttcaat agtcgcggga tacttgaact gcaagaacag 
               
               
                   
                   
               
               
                   
                 ccgccgctcc ggcgggctgc tcgctgcatc tctgggcgtc 
               
               
                   
                   
               
               
                   
                 tttggctcgc cacgctgggc agtgcctgcc tgcgcctttc 
               
               
                   
                   
               
               
                   
                 gcaacctcct cggccctgcg tggtctcgag ctgggtgagc 
               
               
                   
                   
               
               
                   
                 gagcgggcgg gctggtaggc tggcctgggc tgcgaccggc 
               
               
                   
                   
               
               
                   
                 ggctacgact attctttggc cgggtcggtg cgagtggtcg 
               
               
                   
                   
               
               
                   
                 gctgggcaga gtgcacgctg cttggcgccg caggctgatc 
               
               
                   
                   
               
               
                   
                 ccgccgtcca ctcccgggag cagtgatgtt gggcaactct 
               
               
                   
                   
               
               
                   
                 gcgccggggc ctgcgacccg cgaggcgggc tcggcgctgc 
               
               
                   
                   
               
               
                   
                 tagcattgca gcagacggcg ctccaagagg accaggagaa 
               
               
                   
                   
               
               
                   
                 tatcaacccg gaaaaggcag cgcccgtcca acaaccgcgg 
               
               
                   
                   
               
               
                   
                 acccgggccg cgctggcggt actgaagtcc gggaacccgc 
               
               
                   
                   
               
               
                   
                 ggggtctagc gcagcagcag aggccgaaga cgagacgggt 
               
               
                   
                   
               
               
                   
                 tgcacccctt aaggatcttc ctgtaaatga tgagcatgtc 
               
               
                   
                   
               
               
                   
                 accgttcctc cttggaaagc aaacagtaaa cagcctgcgt 
               
               
                   
                   
               
               
                   
                 tcaccattca tgtggatgaa gcagaaaaag aagctcagaa 
               
               
                   
                   
               
               
                   
                 gaagccagct gaatctcaaa aaatagagcg tgaagatgcc 
               
               
                   
                   
               
               
                   
                 ctggctttta attcagccat tagtttacct ggacccagaa 
               
               
                   
                   
               
               
                   
                 aaccattggt ccctcttgat tatccaatgg atggtagttt 
               
               
                   
                   
               
               
                   
                 tgagtcacca catactatgg acatgtcaat tgtattagaa 
               
               
                   
                   
               
               
                   
                 gatgaaaagc cagtgagtgt taatgaagta ccagactacc 
               
               
                   
                   
               
               
                   
                 atgaggatat tcacacatac cttagggaaa tggaggttaa 
               
               
                   
                   
               
               
                   
                 atgtaaacct aaagtgggtt acatgaagaa acagccagac 
               
               
                   
                   
               
               
                   
                 atcactaaca gtatgagagc tatcctcgtg gactggttag 
               
               
                   
                   
               
               
                   
                 ttgaagtagg agaagaatat aaactacaga atgagaccct 
               
               
                   
                   
               
               
                   
                 gcatttggct gtgaactaca ttgataggtt cctgtcttcc 
               
               
                   
                   
               
               
                   
                 atgtcagtgc tgagaggaaa acttcagctt gtgggcactg 
               
               
                   
                   
               
               
                   
                 ctgctatgct gttagcctca aagtttgaag aaatataccc 
               
               
                   
                   
               
               
                   
                 cccagaagta gcagagtttg tgtacattac agatgatacc 
               
               
                   
                   
               
               
                   
                 tacaccaaga aacaagttct gagaatggag catctagttt 
               
               
                   
                   
               
               
                   
                 tgaaagtcct tacttttgac ttagctgctc caacagtaaa 
               
               
                   
                   
               
               
                   
                 tcagtttctt acccaatact ttctgcatca gcagcctgca 
               
               
                   
                   
               
               
                   
                 aactgcaaag ttgaaagttt agcaatgttt ttgggagaat 
               
               
                   
                   
               
               
                   
                 taagtttgat agatgctgac ccatacctca agtatttgcc 
               
               
                   
                   
               
               
                   
                 atcagttatt gctggagctg cctttcattt agcactctac 
               
               
                   
                   
               
               
                   
                 acagtcacgg gacaaagctg gcctgaatca ttaatacgaa 
               
               
                   
                   
               
               
                   
                 agactggata taccctggaa agtcttaagc cttgtctcat 
               
               
                   
                   
               
               
                   
                 ggaccttcac cagacctacc tcaaagcacc acagcatgca 
               
               
                   
                   
               
               
                   
                 caacagtcaa taagagaaaa gtacaaaaat tcaaagtatc 
               
               
                   
                   
               
               
                   
                 atggtgtttc tctcctcaac ccaccagaga cactaaatct 
               
               
                   
                   
               
               
                   
                 gtaacaatga aagactgcct ttgttttcta agatgtaaat 
               
               
                   
                   
               
               
                   
                 cactcaaagt atatggtgta cagtttttaa cttaggtttt 
               
               
                   
                   
               
               
                   
                 aattttacaa tcatttctga atacagaagt tgtggccaag 
               
               
                   
                   
               
               
                   
                 tacaaattat ggtatctatt actttttaaa tggttttaat 
               
               
                   
                   
               
               
                   
                 ttgtatatct tttgtatatg tatctgtctt agatatttgg 
               
               
                   
                   
               
               
                   
                 ctaattttaa gtggttttgt taaagtatta atgatgccag 
               
               
                   
                   
               
               
                   
                 ctgtcaggat aataaattga tttggaaaac tttgcaagtc 
               
               
                   
                   
               
               
                   
                 aaatttaact tcttcaggat tttgcttagt aaagaagttt 
               
               
                   
                   
               
               
                   
                 acttggttta ctatataatg ggaagtgaaa agccttcctc 
               
               
                   
                   
               
               
                   
                 taaaattaaa gtaggtttag gaaaacagac cctcaaattc 
               
               
                   
                   
               
               
                   
                 tgacattcat tttcctaagc aactggatca atttgctgac 
               
               
                   
                   
               
               
                   
                 ttgggcataa tctaatctaa gcatatctga atacagtatt 
               
               
                   
                   
               
               
                   
                 cagagataga tacagtagag attccccaga ctttttcgct 
               
               
                   
                   
               
               
                   
                 ctttgtaaaa cctgtttgtt taggttttgc gaggtaaact 
               
               
                   
                   
               
               
                   
                 caacagaggt tgggagtgga agagggtggg aagcttatat 
               
               
                   
                   
               
               
                   
                 gcaaattaac agacgagaaa tgctccagaa ggtttattat 
               
               
                   
                   
               
               
                   
                 tttaaagcac attaaaaaca aaaaactatt tttaaaatcc 
               
               
                   
                   
               
               
                   
                 tgctagattt tataatggat ttgtgaataa aaaataccca 
               
               
                   
                   
               
               
                   
                 gggttctcag aatggaataa atatcccttt taatagttat 
               
               
                   
                   
               
               
                   
                 atatacagat atacaactgt tagctttaat tggcagctct 
               
               
                   
                   
               
               
                   
                 cttctttttt cttcttttca ctggcttttt acttggtgct 
               
               
                   
                   
               
               
                   
                 ttttcttgtt ttgcactggt ggtctgtgtt ctgtgaataa 
               
               
                   
                   
               
               
                   
                 agcaaagtaa gaatttacta agagtatgtt aagttttgga 
               
               
                   
                   
               
               
                   
                 ttattgaaat aagaggcatt tcttagtttt ccagtaggat 
               
               
                   
                   
               
               
                   
                 ctaaaatgtg tcagctatga gtaagactgg catccaagaa 
               
               
                   
                   
               
               
                   
                 gtttatatta tagatttagg tcctaatttt tataaatcac 
               
               
                   
                   
               
               
                   
                 aaggtaaaaa aatcacagaa cagatggatc tctaatgaaa 
               
               
                   
                   
               
               
                   
                 aagggatgtc tttttgttta tagtcatgtg gcaagatgag 
               
               
                   
                   
               
               
                   
                 agtaaaacca gagagcaaac ctctataagt gttgagtata 
               
               
                   
                   
               
               
                   
                 tgtatacatt tgaaataaac cagaaatttg ttaccttaaa 
               
               
                   
                   
               
               
                   
                 aaaaaaaaaa a 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 2 
               
               
                   
                 EGFR(NM_005228.3) 
               
               
                   
                 ccccggcgca gcgcgggcgc agcagcctcc gccccccgca 
               
               
                   
                   
               
               
                   
                 cggtgtgagc gcccgacgcg gccgaggcgg ccggagtccc 
               
               
                   
                   
               
               
                   
                 gagctagccc cggcggccgc cgccgcccag accggacgac 
               
               
                   
                   
               
               
                   
                 aggccacctc gtcggcgtcc gcccgagtcc ccgcctcgcc 
               
               
                   
                   
               
               
                   
                 gccaacgcca caaccaccgc gcacggcccc ctgactccgt 
               
               
                   
                   
               
               
                   
                 ccagtattga tcgggagagc cggagcgagc tcttcgggga 
               
               
                   
                   
               
               
                   
                 gcagcgatgc gaccctccgg gacggccggg gcagcgctcc 
               
               
                   
                   
               
               
                   
                 tggcgctgct ggctgcgctc tgcccggcga gtcgggctct 
               
               
                   
                   
               
               
                   
                 ggaggaaaag aaagtttgcc aaggcacgag taacaagctc 
               
               
                   
                   
               
               
                   
                 acgcagttgg gcacttttga agatcatttt ctcagcctcc 
               
               
                   
                   
               
               
                   
                 agaggatgtt caataactgt gaggtggtcc ttgggaattt 
               
               
                   
                   
               
               
                   
                 ggaaattacc tatgtgcaga ggaattatga tctttccttc 
               
               
                   
                   
               
               
                   
                 ttaaagacca tccaggaggt ggctggttat gtcctcattg 
               
               
                   
                   
               
               
                   
                 ccctcaacac agtggagcga attcctttgg aaaacctgca 
               
               
                   
                   
               
               
                   
                 gatcatcaga ggaaatatgt actacgaaaa ttcctatgcc 
               
               
                   
                   
               
               
                   
                 ttagcagtct tatctaacta tgatgcaaat aaaaccggac 
               
               
                   
                   
               
               
                   
                 tgaaggagct gcccatgaga aatttacagg aaatcctgca 
               
               
                   
                   
               
               
                   
                 tggcgccgtg cggttcagca acaaccctgc cctgtgcaac 
               
               
                   
                   
               
               
                   
                 gtggagagca tccagtggcg ggacatagtc agcagtgact 
               
               
                   
                   
               
               
                   
                 ttctcagcaa catgtcgatg gacttccaga accacctggg 
               
               
                   
                   
               
               
                   
                 cagctgccaa aagtgtgatc caagctgtcc caatgggagc 
               
               
                   
                   
               
               
                   
                 tgctggggtg caggagagga gaactgccag aaactgacca 
               
               
                   
                   
               
               
                   
                 aaatcatctg tgcccagcag tgctccgggc gctgccgtgg 
               
               
                   
                   
               
               
                   
                 caagtccccc agtgactgct gccacaacca gtgtgctgca 
               
               
                   
                   
               
               
                   
                 ggctgcacag gcccccggga gagcgactgc ctggtctgcc 
               
               
                   
                   
               
               
                   
                 gcaaattccg agacgaagcc acgtgcaagg acacctgccc 
               
               
                   
                   
               
               
                   
                 cccactcatg ctctacaacc ccaccacgta ccagatggat 
               
               
                   
                   
               
               
                   
                 gtgaaccccg agggcaaata cagctttggt gccacctgcg 
               
               
                   
                   
               
               
                   
                 tgaagaagtg tccccgtaat tatgtggtga cagatcacgg 
               
               
                   
                   
               
               
                   
                 ctcgtgcgtc cgagcctgtg gggccgacag ctatgagatg 
               
               
                   
                   
               
               
                   
                 gaggaagacg gcgtccgcaa gtgtaagaag tgcgaagggc 
               
               
                   
                   
               
               
                   
                 cttgccgcaa agtgtgtaac ggaataggta ttggtgaatt 
               
               
                   
                   
               
               
                   
                 taaagactca ctctccataa atgctacgaa tattaaacac 
               
               
                   
                   
               
               
                   
                 ttcaaaaact gcacctccat cagtggcgat ctccacatcc 
               
               
                   
                   
               
               
                   
                 tgccggtggc atttaggggt gactccttca cacatactcc 
               
               
                   
                   
               
               
                   
                 tcctctggat ccacaggaac tggatattct gaaaaccgta 
               
               
                   
                   
               
               
                   
                 aaggaaatca cagggttttt gctgattcag gcttggcctg 
               
               
                   
                   
               
               
                   
                 aaaacaggac ggacctccat gcctttgaga acctagaaat 
               
               
                   
                   
               
               
                   
                 catacgcggc aggaccaagc aacatggtca gttttctctt 
               
               
                   
                   
               
               
                   
                 gcagtcgtca gcctgaacat aacatccttg ggattacgct 
               
               
                   
                   
               
               
                   
                 ccctcaagga gataagtgat ggagatgtga taatttcagg 
               
               
                   
                   
               
               
                   
                 aaacaaaaat ttgtgctatg caaatacaat aaactggaaa 
               
               
                   
                   
               
               
                   
                 aaactgtttg ggacctccgg tcagaaaacc aaaattataa 
               
               
                   
                   
               
               
                   
                 gcaacagagg tgaaaacagc tgcaaggcca caggccaggt 
               
               
                   
                   
               
               
                   
                 ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg 
               
               
                   
                   
               
               
                   
                 gagcccaggg actgcgtctc ttgccggaat gtcagccgag 
               
               
                   
                   
               
               
                   
                 gcagggaatg cgtggacaag tgcaaccttc tggagggtga 
               
               
                   
                   
               
               
                   
                 gccaagggag tttgtggaga actctgagtg catacagtgc 
               
               
                   
                   
               
               
                   
                 cacccagagt gcctgcctca ggccatgaac atcacctgca 
               
               
                   
                   
               
               
                   
                 caggacgggg accagacaac tgtatccagt gtgcccacta 
               
               
                   
                   
               
               
                   
                 cattgacggc ccccactgcg tcaagacctg cccggcagga 
               
               
                   
                   
               
               
                   
                 gtcatgggag aaaacaacac cctggtctgg aagtacgcag 
               
               
                   
                   
               
               
                   
                 acgccggcca tgtgtgccac ctgtgccatc caaactgcac 
               
               
                   
                   
               
               
                   
                 ctacggatgc actgggccag gtcttgaagg ctgtccaacg 
               
               
                   
                   
               
               
                   
                 aatgggccta agatcccgtc catcgccact gggatggtgg 
               
               
                   
                   
               
               
                   
                 gggccctcct cttgctgctg gtggtggccc tggggatcgg 
               
               
                   
                   
               
               
                   
                 cctcttcatg cgaaggcgcc acatcgttcg gaagcgcacg 
               
               
                   
                   
               
               
                   
                 ctgcggaggc tgctgcagga gagggagctt gtggagcctc 
               
               
                   
                   
               
               
                   
                 ttacacccag tggagaagct cccaaccaag ctctcttgag 
               
               
                   
                   
               
               
                   
                 gatcttgaag gaaactgaat tcaaaaagat caaagtgctg 
               
               
                   
                   
               
               
                   
                 ggctccggtg cgttcggcac ggtgtataag ggactctgga 
               
               
                   
                   
               
               
                   
                 tcccagaagg tgagaaagtt aaaattcccg tcgctatcaa 
               
               
                   
                   
               
               
                   
                 ggaattaaga gaagcaacat ctccgaaagc caacaaggaa 
               
               
                   
                   
               
               
                   
                 atcctcgatg aagcctacgt gatggccagc gtggacaacc 
               
               
                   
                   
               
               
                   
                 cccacgtgtg ccgcctgctg ggcatctgcc tcacctccac 
               
               
                   
                   
               
               
                   
                 cgtgcagctc atcacgcagc tcatgccctt cggctgcctc 
               
               
                   
                   
               
               
                   
                 ctggactatg tccgggaaca caaagacaat attggctccc 
               
               
                   
                   
               
               
                   
                 agtacctgct caactggtgt gtgcagatcg caaagggcat 
               
               
                   
                   
               
               
                   
                 gaactacttg gaggaccgtc gcttggtgca ccgcgacctg 
               
               
                   
                   
               
               
                   
                 gcagccagga acgtactggt gaaaacaccg cagcatgtca 
               
               
                   
                   
               
               
                   
                 agatcacaga ttttgggctg gccaaactgc tgggtgcgga 
               
               
                   
                   
               
               
                   
                 agagaaagaa taccatgcag aaggaggcaa agtgcctatc 
               
               
                   
                   
               
               
                   
                 aagtggatgg cattggaatc aattttacac agaatctata 
               
               
                   
                   
               
               
                   
                 cccaccagag tgatgtctgg agctacgggg tgaccgtttg 
               
               
                   
                   
               
               
                   
                 ggagttgatg acctttggat ccaagccata tgacggaatc 
               
               
                   
                   
               
               
                   
                 cctgccagcg agatctcctc catcctggag aaaggagaac 
               
               
                   
                   
               
               
                   
                 gcctccctca gccacccata tgtaccatcg atgtctacat 
               
               
                   
                   
               
               
                   
                 gatcatggtc aagtgctgga tgatagacgc agatagtcgc 
               
               
                   
                   
               
               
                   
                 ccaaagttcc gtgagttgat catcgaattc tccaaaatgg 
               
               
                   
                   
               
               
                   
                 cccgagaccc ccagcgctac cttgtcattc agggggatga 
               
               
                   
                   
               
               
                   
                 aagaatgcat ttgccaagtc ctacagactc caacttctac 
               
               
                   
                   
               
               
                   
                 cgtgccctga tggatgaaga agacatggac gacgtggtgg 
               
               
                   
                   
               
               
                   
                 atgccgacga gtacctcatc ccacagcagg gcttcttcag 
               
               
                   
                   
               
               
                   
                 cagcccctcc acgtcacgga ctcccctcct gagctctctg 
               
               
                   
                   
               
               
                   
                 agtgcaacca gcaacaattc caccgtggct tgcattgata 
               
               
                   
                   
               
               
                   
                 gaaatgggct gcaaagctgt cccatcaagg aagacagctt 
               
               
                   
                   
               
               
                   
                 cttgcagcga tacagctcag accccacagg cgccttgact 
               
               
                   
                   
               
               
                   
                 gaggacagca tagacgacac cttcctccca gtgcctgaat 
               
               
                   
                   
               
               
                   
                 acataaacca gtccgttccc aaaaggcccg ctggctctgt 
               
               
                   
                   
               
               
                   
                 gcagaatcct gtctatcaca atcagcctct gaaccccgcg 
               
               
                   
                   
               
               
                   
                 cccagcagag acccacacta ccaggacccc cacagcactg 
               
               
                   
                   
               
               
                   
                 cagtgggcaa ccccgagtat ctcaacactg tccagcccac 
               
               
                   
                   
               
               
                   
                 ctgtgtcaac agcacattcg acagccctgc ccactgggcc 
               
               
                   
                   
               
               
                   
                 cagaaaggca gccaccaaat tagcctggac aaccctgact 
               
               
                   
                   
               
               
                   
                 accagcagga cttctttccc aaggaagcca agccaaatgg 
               
               
                   
                   
               
               
                   
                 catctttaag ggctccacag ctgaaaatgc agaataccta 
               
               
                   
                   
               
               
                   
                 agggtcgcgc cacaaagcag tgaatttatt ggagcatgac 
               
               
                   
                   
               
               
                   
                 cacggaggat agtatgagcc ctaaaaatcc agactctttc 
               
               
                   
                   
               
               
                   
                 gatacccagg accaagccac agcaggtcct ccatcccaac 
               
               
                   
                   
               
               
                   
                 agccatgccc gcattagctc ttagacccac agactggttt 
               
               
                   
                   
               
               
                   
                 tgcaacgttt acaccgactagccaggaagt acttccacct 
               
               
                   
                   
               
               
                   
                 cgggcacatt ttgggaagtt gcattccttt 
               
               
                   
                   
               
               
                   
                 gtcttcaaactgtgaagcat ttacagaaac gcatccagca 
               
               
                   
                   
               
               
                   
                 agaatattgt ccctttgagc agaaatttat ctttcaaaga 
               
               
                   
                   
               
               
                   
                 ggtatatttg aaaaaaaaaa aaagtatatg tgaggatttt 
               
               
                   
                   
               
               
                   
                 tattgattgg ggatcttgga gtttttcatt gtcgctattg 
               
               
                   
                   
               
               
                   
                 atttttactt caatgggctc ttccaacaag gaagaagctt 
               
               
                   
                   
               
               
                   
                 gctggtagca cttgctaccc tgagttcatc caggcccaac 
               
               
                   
                   
               
               
                   
                 tgtgagcaag gagcacaagc cacaagtctt ccagaggatg 
               
               
                   
                   
               
               
                   
                 cttgattcca gtggttctgc ttcaaggctt ccactgcaaa 
               
               
                   
                   
               
               
                   
                 acactaaaga tccaagaagg ccttcatggc cccagcaggc 
               
               
                   
                   
               
               
                   
                 cggatcggta ctgtatcaag tcatggcagg tacagtagga 
               
               
                   
                   
               
               
                   
                 taagccactc tgtcccttcc tgggcaaaga agaaacggag 
               
               
                   
                   
               
               
                   
                 gggatggaat tcttccttag acttactttt gtaaaaatgt 
               
               
                   
                   
               
               
                   
                 ccccacggta cttactcccc actgatggac cagtggtttc 
               
               
                   
                   
               
               
                   
                 cagtcatgag cgttagactg acttgtttgt cttccattcc 
               
               
                   
                   
               
               
                   
                 attgttttga aactcagtat gctgcccctg tcttgctgtc 
               
               
                   
                   
               
               
                   
                 atgaaatcag caagagagga tgacacatca aataataact 
               
               
                   
                   
               
               
                   
                 cggattccag cccacattgg attcatcagc atttggacca 
               
               
                   
                   
               
               
                   
                 atagcccaca gctgagaatg tggaatacct aaggatagca 
               
               
                   
                   
               
               
                   
                 ccgcttttgt tctcgcaaaa acgtatctcc taatttgagg 
               
               
                   
                   
               
               
                   
                 ctcagatgaa atgcatcagg tcctttgggg catagatcag 
               
               
                   
                   
               
               
                   
                 aagactacaa aaatgaagct gctctgaaat ctcctttagc 
               
               
                   
                   
               
               
                   
                 catcacccca accccccaaa attagtttgt gttacttatg 
               
               
                   
                   
               
               
                   
                 gaagatagtt ttctcctttt acttcacttc aaaagctttt 
               
               
                   
                   
               
               
                   
                 tactcaaaga gtatatgttc cctccaggtc agctgccccc 
               
               
                   
                   
               
               
                   
                 aaaccccctc cttacgcttt gtcacacaaa aagtgtctct 
               
               
                   
                   
               
               
                   
                 gccttgagtc atctattcaa gcacttacag ctctggccac 
               
               
                   
                   
               
               
                   
                 aacagggcat tttacaggtg cgaatgacag tagcattatg 
               
               
                   
                   
               
               
                   
                 agtagtgtgg aattcaggta gtaaatatga aactagggtt 
               
               
                   
                   
               
               
                   
                 tgaaattgat aatgctttca caacatttgc agatgtttta 
               
               
                   
                   
               
               
                   
                 gaaggaaaaa agttccttcc taaaataatt tctctacaat 
               
               
                   
                   
               
               
                   
                 tggaagattg gaagattcag ctagttagga gcccaccttt 
               
               
                   
                   
               
               
                   
                 tttcctaatc tgtgtgtgcc ctgtaacctg actggttaac 
               
               
                   
                   
               
               
                   
                 agcagtcctt tgtaaacagt gttttaaact ctcctagtca 
               
               
                   
                   
               
               
                   
                 atatccaccc catccaattt atcaaggaag aaatggttca 
               
               
                   
                   
               
               
                   
                 gaaaatattt tcagcctaca gttatgttca gtcacacaca 
               
               
                   
                   
               
               
                   
                 catacaaaat gttccttttg cttttaaagt aatttttgac 
               
               
                   
                   
               
               
                   
                 tcccagatca gtcagagccc ctacagcatt gttaagaaag 
               
               
                   
                   
               
               
                   
                 tatttgattt ttgtctcaat gaaaataaaa ctatattcat 
               
               
                   
                   
               
               
                   
                 ttccactcta aaaaaaaaaa aaaaaa 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 3 
               
               
                   
                 FGF19 (NM_002010.2) 
               
               
                   
                 actctgcgcg ccggcggggg ctgcgcagga ggagcgctcc 
               
               
                   
                   
               
               
                   
                 gcccggctac aacgctccgc gagccggcgc ggcaacacct 
               
               
                   
                   
               
               
                   
                 gttcgcggca gcctgggcgg cacgcgagct cccggacgcg 
               
               
                   
                   
               
               
                   
                 gctctcctcg ctcgccgctc gccacccgtt ctaagccaat 
               
               
                   
                   
               
               
                   
                 ggacatctgc cgagcctctg gagaatcctg gatactagct 
               
               
                   
                   
               
               
                   
                 ttggacgcct aaagtttctt cttctttttg ttttattatt 
               
               
                   
                   
               
               
                   
                 attatcattt tttggagggg ggaccgggag gggagatttg 
               
               
                   
                   
               
               
                   
                 tcgccgccac caacgtgaga tttttttttc cccttgaagg 
               
               
                   
                   
               
               
                   
                 attcatgctg atgtctgcag agtcggttag agagtaaaaa 
               
               
                   
                   
               
               
                   
                 cagcgcatgc cttcctggag tcaggatccg taaattctga 
               
               
                   
                   
               
               
                   
                 cgtagcccgt gcatcttaaa aatccctata ataacgccta 
               
               
                   
                   
               
               
                   
                 ggcatttaag ttgctatggt cattctgatc tcaaaccaaa 
               
               
                   
                   
               
               
                   
                 tggagaaact acggattttt tttccttatt acggtcggat 
               
               
                   
                   
               
               
                   
                 gggatgaaga ccttcctgcc tgctaagagc tggggatcta 
               
               
                   
                   
               
               
                   
                 tctatagaga tacatagata tgtttatcaa tatgtcagtg 
               
               
                   
                   
               
               
                   
                 tgtgagtata aagtggtggt ttcttagact atcagtggtt 
               
               
                   
                   
               
               
                   
                 tgaccttgaa cctgtgccag tgaaacagca gattactttt 
               
               
                   
                   
               
               
                   
                 atttatgcat ttaatggatt gaagaaaaga accttttttt 
               
               
                   
                   
               
               
                   
                 tctctctctc tctgcaactg cagtaaggga ggggagttgg 
               
               
                   
                   
               
               
                   
                 atatacctcg cctaatatct cctgggttga caccatcatt 
               
               
                   
                   
               
               
                   
                 attgtttatt cttgtgctcc aaaagccgag tcctctgatg 
               
               
                   
                   
               
               
                   
                 gctcccttag gtgaagttgg gaactatttc ggtgtgcagg 
               
               
                   
                   
               
               
                   
                 atgcggtacc gtttgggaat gtgcccgtgt tgccggtgga 
               
               
                   
                   
               
               
                   
                 cagcccggtt ttgttaagtg accacctggg tcagtccgaa 
               
               
                   
                   
               
               
                   
                 gcaggggggc tccccagggg acccgcagtc acggacttgg 
               
               
                   
                   
               
               
                   
                 atcatttaaa ggggattctc aggcggaggc agctatactg 
               
               
                   
                   
               
               
                   
                 caggactgga tttcacttag aaatcttccc caatggtact 
               
               
                   
                   
               
               
                   
                 atccagggaa ccaggaaaga ccacagccga tttggcattc 
               
               
                   
                   
               
               
                   
                 tggaatttat cagtatagca gtgggcctgg tcagcattcg 
               
               
                   
                   
               
               
                   
                 aggcgtggac agtggactct acctcgggat gaatgagaag 
               
               
                   
                   
               
               
                   
                 ggggagctgt atggatcaga aaaactaacc caagagtgtg 
               
               
                   
                   
               
               
                   
                 tattcagaga acagttcgaa gaaaactggt ataatacgta 
               
               
                   
                   
               
               
                   
                 ctcatcaaac ctatataagc acgtggacac tggaaggcga 
               
               
                   
                   
               
               
                   
                 tactatgttg cattaaataa agatgggacc ccgagagaag 
               
               
                   
                   
               
               
                   
                 ggactaggac taaacggcac cagaaattca cacatttttt 
               
               
                   
                   
               
               
                   
                 acctagacca gtggaccccg acaaagtacc tgaactgtat 
               
               
                   
                   
               
               
                   
                 aaggatattc taagccaaag ttgacaaaga cagtttcttc 
               
               
                   
                   
               
               
                   
                 acttgagccc ttaaaaaagt aaccactata aaggtttcac 
               
               
                   
                   
               
               
                   
                 gcggtgggtt cttattgatt cgctgtgtca tcacatcagc 
               
               
                   
                   
               
               
                   
                 tccactgttg ccaaactttg tcgcatgcat aatgtatgat 
               
               
                   
                   
               
               
                   
                 ggaggcttgg atgggaatat gctgattttg ttctgcactt 
               
               
                   
                   
               
               
                   
                 aaaggcttct cctcctggag ggctgcctag ggccacttgc 
               
               
                   
                   
               
               
                   
                 ttgatttatc atgagagaag aggagagaga gagagactga 
               
               
                   
                   
               
               
                   
                 gcgctaggag tgtgtgtatg tgtgtgtgtg tgtgtgtgtg 
               
               
                   
                   
               
               
                   
                 tgtgtgtgta tgtgtgtagc gggagatgtg ggcggagcga 
               
               
                   
                   
               
               
                   
                 gagcaaaagg actgcggcct gatgcatgct ggaaaaagac 
               
               
                   
                   
               
               
                   
                 acgcttttca tttctgatca gttgtacttc atcctatatc 
               
               
                   
                   
               
               
                   
                 agcacagctg ccatacttcg acttatcagg attctggctg 
               
               
                   
                   
               
               
                   
                 gtggcctgcg cgagggtgca gtcttactta aaagactttc 
               
               
                   
                   
               
               
                   
                 agttaattct cactggtatc atcgcagtga acttaaagca 
               
               
                   
                   
               
               
                   
                 aagacctctt agtaaaaaat aaaaaaaaat aaaaaataaa 
               
               
                   
                   
               
               
                   
                 aataaaaaaa gttaaattta tttatagaaa ttccaaaggc 
               
               
                   
                   
               
               
                   
                 aacattttat ttattttata tatttattta ttatatagag 
               
               
                   
                   
               
               
                   
                 tttattttta atgaaacatg tacaggccag ataggcattt 
               
               
                   
                   
               
               
                   
                 tggaagcttt aggctctgta agcattaaat ggcaaagtcc 
               
               
                   
                   
               
               
                   
                 gctatgaacc tgtggtaaat tcatgcaagt agatataatg 
               
               
                   
                   
               
               
                   
                 gtgcatggat ataagaaatt ctaatgaccc taatgtacta 
               
               
                   
                   
               
               
                   
                 aaggcgacaa tctcttttgt gcccatatta ttgtaaactt 
               
               
                   
                   
               
               
                   
                 atgcacatcg ctcatgacac tgagtattca ctcttcagac 
               
               
                   
                   
               
               
                   
                 tgcttgtttc atagcttatc ccagaggatt aaagataaac 
               
               
                   
                   
               
               
                   
                 tgggtctcaa actttgattc tgtgtctgca atatttcctc 
               
               
                   
                   
               
               
                   
                 tctcataagt gactccacta ttgtaacttc atggttggaa 
               
               
                   
                   
               
               
                   
                 aatatgaggg ttgatatatg tcttacttgt ttaaatctgt 
               
               
                   
                   
               
               
                   
                 cgcagaatat accaaagcta aataataact atgctttcat 
               
               
                   
                   
               
               
                   
                 tttagccgat ctccagaatg acagtattaa catcaaacat 
               
               
                   
                   
               
               
                   
                 tgtattgatt tagaattctc aaaaaaggaa aaaaaagtac 
               
               
                   
                   
               
               
                   
                 atagcacaga ctattttttt taaagacgta agaatcagat 
               
               
                   
                   
               
               
                   
                 taacaggatc atacttgtaa actttttttg gttcacttgg 
               
               
                   
                   
               
               
                   
                 ctatcaaata tgaaattata gaagtatcat aggggtcatt 
               
               
                   
                   
               
               
                   
                 gtaacatctt ttagagaaaa tggctatcag tgtgaactgt 
               
               
                   
                   
               
               
                   
                 cataattacg tggtaatag acccttagta aaacttgcaa 
               
               
                   
                   
               
               
                   
                 aatgaaacta ataaatcgtt atcaataatg acaatgaggg 
               
               
                   
                   
               
               
                   
                 ggaaagtatt atacttgttg actgtgtttt gttttttaaa 
               
               
                   
                   
               
               
                   
                 atggtctcca caagcgctca atttttttag aggggatatt 
               
               
                   
                   
               
               
                   
                 actatataga atatctttta caaggctttt ataacatttt 
               
               
                   
                   
               
               
                   
                 atgctgaaaa gcataagaat acgtatttct ttagtagcaa 
               
               
                   
                   
               
               
                   
                 taattttgga acttgccctt gggcaagcga gactatttct 
               
               
                   
                   
               
               
                   
                 tactatatac taaggagaaa agagccaaat tcttaaagca 
               
               
                   
                   
               
               
                   
                 atatttaaga aaaaaggaat ttataacaaa ttctcatcta 
               
               
                   
                   
               
               
                   
                 catatgacac tttctagcca gttgtgttga gaagtgcaaa 
               
               
                   
                   
               
               
                   
                 gtgacggttt aaacatgtgt tgggatttat tgaactaatt 
               
               
                   
                   
               
               
                   
                 ttaaaattta ctattcaaac tttattttgc tctgatgcac 
               
               
                   
                   
               
               
                   
                 attctctatg aaaaataaaa gtgtgtcact ggtgagtgac 
               
               
                   
                   
               
               
                   
                 agctgttatg agctagaagc gcatgactta ttgtgacgat 
               
               
                   
                   
               
               
                   
                 gtcttgcctt tctgtggtcc aagttggagt acatggcaat 
               
               
                   
                   
               
               
                   
                 gccctcctgc tgatgtgcat taaggaaaat ctaagtctaa 
               
               
                   
                   
               
               
                   
                 tatttggaat taagatatat tttaggggga ggggacagaa 
               
               
                   
                   
               
               
                   
                 gcaatgtaaa atagttgatt tatgataaag ctcagaatgt 
               
               
                   
                   
               
               
                   
                 cctcttcatt tattttcttg ttttattttc ctttctaaac 
               
               
                   
                   
               
               
                   
                 agaaactgca tttaattcca aaaagtagta ttcttattta 
               
               
                   
                   
               
               
                   
                 ttatttaacc ctttgctgct gctaaaatgt gcacatattc 
               
               
                   
                   
               
               
                   
                 aggctttagt ttttccaaaa ggcatttttt ttttggctga 
               
               
                   
                   
               
               
                   
                 aaaatattaa acatttgacc acagggaaga atcaagtttc 
               
               
                   
                   
               
               
                   
                 taggatgtca taggtatact atgtagcact gaaaaaattg 
               
               
                   
                   
               
               
                   
                 attttaggtg acagccaaaa gtagtcttaa agtagcatga 
               
               
                   
                   
               
               
                   
                 gaccttagat aatcgaccta aaagaaagaa aattgtgaaa 
               
               
                   
                   
               
               
                   
                 aagacaaaaa tcttcatgca ttcctataaa acgctacttt 
               
               
                   
                   
               
               
                   
                 aaggtctact tttggagtta attttgtttg gtactttttt 
               
               
                   
                   
               
               
                   
                 tttttttaag acgagcaaat tgttatatgc ttttggcaat 
               
               
                   
                   
               
               
                   
                 tgatacaata aactgtaatg gtctgtaaat aaataaatat 
               
               
                   
                   
               
               
                   
                 tgactcatgc gatttatgta aatagtggaa ctgggagagt 
               
               
                   
                   
               
               
                   
                 ggatggctca gggtttcggt gtgggcattg tctcttgggc 
               
               
                   
                   
               
               
                   
                 agtagagtga gtcatcccca gctcatgggt ttgcatccag 
               
               
                   
                   
               
               
                   
                 ttcttgtctt aagagaccca aagcccagtg aatggcagcc 
               
               
                   
                   
               
               
                   
                 ctgagccact gtggaatggg ggttctggtt tcacaaacag 
               
               
                   
                   
               
               
                   
                 atgcttagat agccaaacca ctgtcttgtt ggtgccaaca 
               
               
                   
                   
               
               
                   
                 cttgcactgt ggtcaaagac ttaccgagca tgggctgaac 
               
               
                   
                   
               
               
                   
                 aaccttccca tctgtcatgt gaatgtcccc aagcagtggt 
               
               
                   
                   
               
               
                   
                 gaaggacatg ctaggtcagt gttggggaac ctgccctgcc 
               
               
                   
                   
               
               
                   
                 aggtcctgtt ttgtagataa acaaatggct gccttctggt 
               
               
                   
                   
               
               
                   
                 gtttttattc tatttcatct cattaacact acaaccttgt 
               
               
                   
                   
               
               
                   
                 gttatttact tgataatctg taattgtatg taaatacata 
               
               
                   
                   
               
               
                   
                 caggattatg taatttgtgt aaatacataa ttacagagtt 
               
               
                   
                   
               
               
                   
                 ttgaaaactg aaaaaaaaaa aaaaa 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 4 
               
               
                   
                 GREBI (NM_033090.2) 
               
               
                   
                 gaaagaatca aagcgcacat cttgtacttt gatgcccata 
               
               
                   
                   
               
               
                   
                 ggaagggctc tcctctggcc cctctggctt tgtttggagc 
               
               
                   
                   
               
               
                   
                 agaaaacaac aaactgcagc tgaggacagc caccctttct 
               
               
                   
                   
               
               
                   
                 tcgtctctgc tgagcgaagg ctacacggcc cttcctcctt 
               
               
                   
                   
               
               
                   
                 gcagctgttt caccttctac cttgcgtgga gccaggcttt 
               
               
                   
                   
               
               
                   
                 tgcaccgaat ctgagatgcc attttaaaca gaagactcca 
               
               
                   
                   
               
               
                   
                 tcctcttgaa gatgggaaat tcttacgctg gacagctgaa 
               
               
                   
                   
               
               
                   
                 gacgacacgc tttgaagagg tcttgcacaa ttccatcgag 
               
               
                   
                   
               
               
                   
                 gcatccctgc ggtccaacaa cctggtgccc aggcccatct 
               
               
                   
                   
               
               
                   
                 tttcccagct gtacctggaa gctgagcagc agcttgccgc 
               
               
                   
                   
               
               
                   
                 tctagaaggt ggtagccgag tggacaatga ggaagaggaa 
               
               
                   
                   
               
               
                   
                 gaagagggag aaggagggct ggaaacaaat ggccccccaa 
               
               
                   
                   
               
               
                   
                 accctttcca gctgcaccct ctgcctgaag gatgctgtac 
               
               
                   
                   
               
               
                   
                 cacagacggg ttttgccagg ccgggaagga cctgcgcctt 
               
               
                   
                   
               
               
                   
                 gtctccattt ccaacgagcc catggatgtc cctgcgggct 
               
               
                   
                   
               
               
                   
                 ttctcctcgt gggggtcaag tcccccagcc tgccggacca 
               
               
                   
                   
               
               
                   
                 tctcctggtg tgcgccgttg acaagaggtt cttgccagat 
               
               
                   
                   
               
               
                   
                 gacaatggcc acaatgctct tcttggtttc tctgggaatt 
               
               
                   
                   
               
               
                   
                 gtgttggctg tggaaagaaa ggcttctgtt acttcacgga 
               
               
                   
                   
               
               
                   
                 attctccaat catataaatc tgaaactgac cactcaaccc 
               
               
                   
                   
               
               
                   
                 aagaagcaga aacacttgaa gtattacctg gtccgtaatg 
               
               
                   
                   
               
               
                   
                 cacaagggac tctaaccaaa ggacctttaa tctgttggaa  
               
               
                   
                   
               
               
                   
                 aggctcagag tttagaagcc ggcagatccc cgccagtact 
               
               
                   
                   
               
               
                   
                 tgttccagtt ccctcttccc agccctggag agcacggct 
               
               
                   
                   
               
               
                   
                 ccttccccag cgagcccgtt cctgggacga accccagcat 
               
               
                   
                   
               
               
                   
                 cctgatggga gctcagcagg caggaccagc ttctgatcac 
               
               
                   
                   
               
               
                   
                 ccctcactaa acgcagcaat gggtccggct gttttcaacg 
               
               
                   
                   
               
               
                   
                 gcaaagattc cccgaagtgc caacaactgg caaagaataa 
               
               
                   
                   
               
               
                   
                 cctgttggcc ctgccgcgac catcggcttt aggtatcttg 
               
               
                   
                   
               
               
                   
                 tcaaactccg ggccccccaa aaaacgccac aaagggtggt 
               
               
                   
                   
               
               
                   
                 ctccagaatc tccatcagct ccagatggtg gctgccccca 
               
               
                   
                   
               
               
                   
                 aggtggtggg aacagagcta agtatgagag cgcaggcatg 
               
               
                   
                   
               
               
                   
                 tcctgcgtgc cgcaggttgg cttggtggga ccagcttcag 
               
               
                   
                   
               
               
                   
                 tcacctttcc agtggtggcc tctggagaac cagtgtctgt 
               
               
                   
                   
               
               
                   
                 tcctgacaac ttgctgaaaa tatgcaaggc caagccagtg 
               
               
                   
                   
               
               
                   
                 atatttaaag gccatgggaa cttcccttac ctctgtggga 
               
               
                   
                   
               
               
                   
                 acctgaatga cgtcgtggtc agccccctct tgtacacgtg 
               
               
                   
                   
               
               
                   
                 ctaccagaat tcccagtctg tctcacgggc atacgagcag 
               
               
                   
                   
               
               
                   
                 tacggcgcct ctgccatcca gcccatctcc gaggagatgc 
               
               
                   
                   
               
               
                   
                 agctcctgct taccgtctac tacctggtcc agctggcaca 
               
               
                   
                   
               
               
                   
                 tcaaatacga aatccggacg tataaaccag acaattcata 
               
               
                   
                   
               
               
                   
                 aaagagaaaa taccagaagg aaacaaatat tgaaaagtat 
               
               
                   
                   
               
               
                   
                 tcagtctcac tcatcatcaa agacatgcaa atcacaaatc 
               
               
                   
                   
               
               
                   
                 aagtcatctt tttctgaatt aataacccaa atgatggtac 
               
               
                   
                   
               
               
                   
                 ccagtggtgc ttaggtgcgg taaaaccagc gcttgtccga 
               
               
                   
                   
               
               
                   
                 tgcaccgttc gcgtggtaaa ctgacgcatt caggctcttg 
               
               
                   
                   
               
               
                   
                 ggaagcaatc tgatgatatg tagtagctgg ttctgtaaat 
               
               
                   
                   
               
               
                   
                 ccacctttga gaatttaggc gaaggaagta acgttctata 
               
               
                   
                   
               
               
                   
                 tgtcaagatg tgcattgcag agggatttat aaaggtgaat 
               
               
                   
                   
               
               
                   
                 atttggaaga aataagagaa tccacaatag agaccttgct 
               
               
                   
                   
               
               
                   
                 aagtaaacgg tggtgctcac gtatgatggg acattatgga 
               
               
                   
                   
               
               
                   
                 cacactaaca gcactcttta tgttggctga aaatggcact 
               
               
                   
                   
               
               
                   
                 gaaactgatg atagggtcat ggttaaggaa agaatgcaag 
               
               
                   
                   
               
               
                   
                 acccaaagtt tatactgaca atcattgcag ctatttgtaa 
               
               
                   
                   
               
               
                   
                 ggacagtttt aatactaatt ccagcaatac atgtttttat 
               
               
                   
                   
               
               
                   
                 tccctgtcct ggagtaggag aaagcctata ttcccaggct 
               
               
                   
                   
               
               
                   
                 gaatgttcta cacatttacc actgtatatg cacataggga 
               
               
                   
                   
               
               
                   
                 cagtgtaacc tgtctatacc accgtagttc cagtcctaac 
               
               
                   
                   
               
               
                   
                 tttctgaatt ctgtttaaag accttct 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 5 
               
               
                   
                 LZTS1 (NM_021020.2) 
               
               
                   
                 tgagggcttt gctatgacct cagtcccctc acggagccac 
               
               
                   
                   
               
               
                   
                 gactgcccct tgctgccaca gcctttccaa gaccctgccc 
               
               
                   
                   
               
               
                   
                 ggccctgccc catcctcagc cccgagtcac catgggcagc 
               
               
                   
                   
               
               
                   
                 gtcagtagcc tcatctccgg ccacagcttc cacagcaagc 
               
               
                   
                   
               
               
                   
                 actgccgggc ttcgcagtac aagctgcgca agtcctccca 
               
               
                   
                   
               
               
                   
                 cctcaagaag ctcaaccggt attccgacgg gctgctgagg 
               
               
                   
                   
               
               
                   
                 tttggcttct cccaggactc cggtcacggc aagtccagct 
               
               
                   
                   
               
               
                   
                 ccaaaatggg caagagcgaa gacttcttct acatcaaggt 
               
               
                   
                   
               
               
                   
                 cagccagaaa gcccggggct cccatcaccc agattacacg 
               
               
                   
                   
               
               
                   
                 gcactgtcca gcggggattt agggggccag gctggggtgg 
               
               
                   
                   
               
               
                   
                 actttgaccc gtccacaccc cccaagctca tgcccttctc 
               
               
                   
                   
               
               
                   
                 caatcagcta gaaatgggct ccgagaaggg tgcagtgagg 
               
               
                   
                   
               
               
                   
                 cccacagcct tcaagcctgt gctgccacgg tcaggagcca 
               
               
                   
                   
               
               
                   
                 tcctgcactc ctccccggag agtgccagcc accagctgca 
               
               
                   
                   
               
               
                   
                 ccccgcccct ccagacaagc ccaaggagca ggagctgaag 
               
               
                   
                   
               
               
                   
                 cctggcctgt gctctggggc gctgtcagac tccggccgga 
               
               
                   
                   
               
               
                   
                 actccatgtc cagcctgccc acacacagca ccagcagcag 
               
               
                   
                   
               
               
                   
                 ctaccagctg gacccgctgg tcacacccgt gggacccaca 
               
               
                   
                   
               
               
                   
                 agccgttttg ggggctccgc ccacaacatc acccagggca 
               
               
                   
                   
               
               
                   
                 tcgtcctcca ggacagcaac atgatgagcc tgaaggctct 
               
               
                   
                   
               
               
                   
                 gtccttctcc gacggaggta gcaagctggg ccactcgaac 
               
               
                   
                   
               
               
                   
                 aaggcagaca agggcccctc gtgtgtccgc tcccccatct 
               
               
                   
                   
               
               
                   
                 ccacggacga gtgcagcatc caggagctgg agcagaagct 
               
               
                   
                   
               
               
                   
                 gttggagagg gagggcgccc tccagaagct gcagcgcagc 
               
               
                   
                   
               
               
                   
                 tttgaggaga aggagcttgc ctccagcctg gcctacgagg 
               
               
                   
                   
               
               
                   
                 agcggccgcg gcgctgcagg gacgagctgg agggcccgga 
               
               
                   
                   
               
               
                   
                 gcccaaaggc ggcaacaagc tcaagcaggc ctcgcagaag 
               
               
                   
                   
               
               
                   
                 agccagcgcg cgcagcaggt cctgcacctg caggtactgc 
               
               
                   
                   
               
               
                   
                 agcttcagca ggagaagcgg cagctccggc aggagctcga 
               
               
                   
                   
               
               
                   
                 gagcctcatg aaggagcagg acctgctgga gaccaagctc 
               
               
                   
                   
               
               
                   
                 aggtcctacg agagggagaa gaccagcttc ggccccgcgc 
               
               
                   
                   
               
               
                   
                 tggaggagac ccagtgggag gtgtgccaga agtcaggcga 
               
               
                   
                   
               
               
                   
                 gatctccctc ctgaagcagc agctgaagga gtcccagacg 
               
               
                   
                   
               
               
                   
                 gaggtgaacg ccaaggctag cgagatcctg ggtctcaagg 
               
               
                   
                   
               
               
                   
                 cacagctgaa ggacacgcgg ggcaagctgg agggcctgga 
               
               
                   
                   
               
               
                   
                 gctgaggacc caggacctgg agggcgccct gcgcaccaag 
               
               
                   
                   
               
               
                   
                 ggcctggagc tggaggtctg tgagaatgag ctgcagcgca 
               
               
                   
                   
               
               
                   
                 agaagaacga ggcggagctg ctgcgggaga aggtgaacct 
               
               
                   
                   
               
               
                   
                 gctggagcag gagctgcagg agctgcgggc ccaggccgcc 
               
               
                   
                   
               
               
                   
                 ctggcccgcg acatggggcc gcccaccttc cccgaggacg 
               
               
                   
                   
               
               
                   
                 tccctgccct gcagcggga ctggagcggc tgcgggccga 
               
               
                   
                   
               
               
                   
                 gctgcgggag gagcggcaag gccatgacca gatgtcctcg 
               
               
                   
                   
               
               
                   
                 ggcttccagc atgagcggct cgtgtggaag gaggagaagg 
               
               
                   
                   
               
               
                   
                 agaaggtgat tcagtaccag aaacagctgc agcagagcta 
               
               
                   
                   
               
               
                   
                 cgtggccatg taccagcgga accagcgcct ggagaaggcc 
               
               
                   
                   
               
               
                   
                 ctgcagcagc tggcacgtgg ggacagcgcc ggggagccct 
               
               
                   
                   
               
               
                   
                 tggaggttga cctggaaggg gctgacatcc cctacgagga 
               
               
                   
                   
               
               
                   
                 catcatagcc actgagatct gaggggctgc ctgggaaggc 
               
               
                   
                   
               
               
                   
                 gagtctgggg acctggcact gggaggcagg gctctcccgt 
               
               
                   
                   
               
               
                   
                 gcatcccccc tgctcagcaa ttcagacccc tttgagagac 
               
               
                   
                   
               
               
                   
                 gccactccct gggacacaga cccaggaccc ccgaggggag 
               
               
                   
                   
               
               
                   
                 ggcaggatgg cctttccttc cctctctgat gtcccagtgc 
               
               
                   
                   
               
               
                   
                 tcaccagccc tgcagcccac cagacgtcag gccctgactc 
               
               
                   
                   
               
               
                   
                 ctctggcttt cccaggagat gggtccaggg gtctgtctgc 
               
               
                   
                   
               
               
                   
                 tttggttaag ggctccctaa actttggcct ttgttcgaaa 
               
               
                   
                   
               
               
                   
                 tagatatcct ctccccctcc tccagggaag gtggccacag 
               
               
                   
                   
               
               
                   
                 caagtacagc ggctcccctc tgcttctcat cccaacctct 
               
               
                   
                   
               
               
                   
                 ttttcctcct ggacacattg gaatgccttg gaaatagaaa 
               
               
                   
                   
               
               
                   
                 gaagccatat atgaccagaa gccttggaac cagccccatc 
               
               
                   
                   
               
               
                   
                 agaacctgag ctattttcct ctggccgcag aggtgtagg 
               
               
                   
                   
               
               
                   
                 gtggaatgag ccgcggggaa gctggctttg aaacctcagg 
               
               
                   
                   
               
               
                   
                 gctgtcccag ccccggcaag ccacaggaag gaggggagag 
               
               
                   
                   
               
               
                   
                 acaggcagcc cagcagtgtg gagaccctgc cacagccaga 
               
               
                   
                   
               
               
                   
                 ggagggcaga gggagaatcc aagggttgag agccagtggc 
               
               
                   
                   
               
               
                   
                 gggtgatggc cagcccctgg ggcccagccc ctgtttactg 
               
               
                   
                   
               
               
                   
                 gttcttgcaa atgggagctg agcagcctct ggacagccag 
               
               
                   
                   
               
               
                   
                 tgacctttga cctcggtgac cactcttctt taagccatag 
               
               
                   
                   
               
               
                   
                 accctgaggc cctgggctgg gtgctgggaa gggagggttg 
               
               
                   
                   
               
               
                   
                 aaaccaccgt gaaccagagg gtgtggcttt ccaggcaccc 
               
               
                   
                   
               
               
                   
                 tcagggagcc tccccatctg tccagctggg gccagaggct 
               
               
                   
                   
               
               
                   
                 gggagtccct acctgcttca cgttggccgg cggctactct 
               
               
                   
                   
               
               
                   
                 ggaatgtttt tccctcccca gaatcaagct tttgcttgat 
               
               
                   
                   
               
               
                   
                 ccagaagagc ccatatcact aagatggcat atatgtgatc 
               
               
                   
                   
               
               
                   
                 tgggcatttt cctcctctgc ctacagccag gtttagcggc 
               
               
                   
                   
               
               
                   
                 aaacctttcc cccttagcac cttcagggct gagttctggg 
               
               
                   
                   
               
               
                   
                 tttctagagg tcaggacggc tcctcagagc gccaggaagc 
               
               
                   
                   
               
               
                   
                 cagagcccca agcaggacga aaaagaggca tacacacagc 
               
               
                   
                   
               
               
                   
                 agtgtgaata gcctggccac cagccatcct ccctccacct 
               
               
                   
                   
               
               
                   
                 caagaccccc atttgtccca gactaaagga tccagagagc 
               
               
                   
                   
               
               
                   
                 agctcccttt ctcaggagct tgggcagtgc cccagggagt 
               
               
                   
                   
               
               
                   
                 ccagggtttc tctgcagatg tgcggagcgg gaggcggtgg 
               
               
                   
                   
               
               
                   
                 tagagagaga taaaaggtgg agtttctctg ttgtttggtt 
               
               
                   
                   
               
               
                   
                 cagggatttt atttttaatt ttatgagaca gggtcttgct 
               
               
                   
                   
               
               
                   
                 ctgtccccca ggctggagtg cagtggcatg atcatagctc 
               
               
                   
                   
               
               
                   
                 actgcagcct catactcctg ggctcaagca atcctcctgc 
               
               
                   
                   
               
               
                   
                 ctcagccttc caactagctg ggactacagg tgcgcgccac 
               
               
                   
                   
               
               
                   
                 cgtgcctggc taacttttca ttttttttgt agggacgggg 
               
               
                   
                   
               
               
                   
                 tctcgttttg ttgccaaagc tggtctcaaa cttgtggcct 
               
               
                   
                   
               
               
                   
                 caagcaatcc acctgccttg gcctcccaaa gtgctgagat 
               
               
                   
                   
               
               
                   
                 tgcagatgtg agccaccgtg cctggccaga tttttctttt 
               
               
                   
                   
               
               
                   
                 attcttcttt ctttttcttt tttgctttct tgtcttttca 
               
               
                   
                   
               
               
                   
                 gaagcaagcc agacctagca ggctgttcca tgttctattt 
               
               
                   
                   
               
               
                   
                 ttgactgtag ccacagctgc tgttctcagg acagcatccc 
               
               
                   
                   
               
               
                   
                 ttcccacatg cctgcgcctg ctgcctgctg agatgaggag 
               
               
                   
                   
               
               
                   
                 gggagcgtct gggaacttgc gagtccaagg ccagtcccca 
               
               
                   
                   
               
               
                   
                 tttctgcctc gctcaccgct ggcccttaga gaccccgagg 
               
               
                   
                   
               
               
                   
                 taggggtggg gagatgcttc tctccttgcc ccccgccctc 
               
               
                   
                   
               
               
                   
                 atgggtccta gcccttccct gagtgcgggc tgaggccaga 
               
               
                   
                   
               
               
                   
                 gtcacctttt ctgtggctgg ctctaccttc ctgtccctga 
               
               
                   
                   
               
               
                   
                 ggttaaacgg tgcccatcct gccatcctca aacgacagag 
               
               
                   
                   
               
               
                   
                 gagcttttct ggaatttcaa accattgctc ttagtcccaa 
               
               
                   
                   
               
               
                   
                 gctaggctta aacctggaat ctacaagcca aaagtccctc 
               
               
                   
                   
               
               
                   
                 cctgcctgag ggcagtaccc tccattgggc acagtccaga 
               
               
                   
                   
               
               
                   
                 cccaagtcaa agatgcccca ttccttgcgc ctcagccctc 
               
               
                   
                   
               
               
                   
                 agttccttca tttccaccag gccgtgcctt gtttgagttt 
               
               
                   
                   
               
               
                   
                 ttcctcccag tgagactgcc ccacggagac agaggaaagg 
               
               
                   
                   
               
               
                   
                 gctggctccc cctccccagg ctggagaccc ccccccaact 
               
               
                   
                   
               
               
                   
                 ccaggaaaga gcagtcagag tccagtgctc tgcctcagac 
               
               
                   
                   
               
               
                   
                 gttgcctgag aagaagtggc tgccacaccc aggggaaggc 
               
               
                   
                   
               
               
                   
                 cctgaggcgg aggctgtgct ccgccatggt gtcccggtac 
               
               
                   
                   
               
               
                   
                 cttccataca cagaggagtg cagccttctc catatctcc 
               
               
                   
                   
               
               
                   
                 tggccctgtc ccaggccggc ccagatgtgt cccccccagg 
               
               
                   
                   
               
               
                   
                 ccttgtccta cgtccaaggt ggcagatgtc ttccctgggc 
               
               
                   
                   
               
               
                   
                 tgccaccagc ccccgcccca gagtggccca ccgtggcact 
               
               
                   
                   
               
               
                   
                 agaatgcaag tatcctgcga ccttgcaacc tcaccttcct 
               
               
                   
                   
               
               
                   
                 gtgggtgttc tttcctgccc tgtccaaaag cgccctcact 
               
               
                   
                   
               
               
                   
                 attcttggac catgccagat tctgcctctc tggaaagagg 
               
               
                   
                   
               
               
                   
                 ctctggacag cagaagcctc caagcacaga gcctggcccc 
               
               
                   
                   
               
               
                   
                 aggccccaga cagggtgggc ttcctgccct tccctctggg 
               
               
                   
                   
               
               
                   
                 cacgcctgct ggccgaccca ctgacccact cggatggacc 
               
               
                   
                   
               
               
                   
                 aacctgctct gtccccaaag gacgcctgca ggagagagca 
               
               
                   
                   
               
               
                   
                 gcactccgca tcacctcacc aaggatcgga ctctgcccct 
               
               
                   
                   
               
               
                   
                 ggacctggga acgactggac tgtcacgggg ttccctccta 
               
               
                   
                   
               
               
                   
                 gctctcccag tgaactcctg ccaggcacac acagccccta 
               
               
                   
                   
               
               
                   
                 tagcactgag ctcacatggg actgggatat gggggcatct 
               
               
                   
                   
               
               
                   
                 cttccccaga gaggcactca gtgagcctcc tgtgcctggc 
               
               
                   
                   
               
               
                   
                 cccagcctgg gccatctctt aggtgagaca gttgcccgaa 
               
               
                   
                   
               
               
                   
                 actaagccag gcctggctgg aggagcagca gcttggggag 
               
               
                   
                   
               
               
                   
                 agggatttcc ctgcagacct caagccatca tgcggtgggt 
               
               
                   
                   
               
               
                   
                 gctgccatga cagaggctgc acccctgggc cagcggggct 
               
               
                   
                   
               
               
                   
                 gctcacccac ctcttgtgca aggtggcctt tgtgctgcgc 
               
               
                   
                   
               
               
                   
                 ctgcaggcag agctggagcc cccagcagag gcaggctggg 
               
               
                   
                   
               
               
                   
                 acggaccagc atctggaaga tgtacatagt tatttttctc 
               
               
                   
                   
               
               
                   
                 tttgtggttt cttgtttggt ttggtttgct tttgacagct 
               
               
                   
                   
               
               
                   
                 tcattttatt tttgacgtca ctttttggcc atgtaaacta 
               
               
                   
                   
               
               
                   
                 tttgtggcaa ttttatgttt ttatttatga ataaagaatg 
               
               
                   
                   
               
               
                   
                 ccatttctca cgccctcta 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 6 
               
               
                   
                 BRAF (NM_004333.4) 
               
               
                   
                 cgcctccctt ccccctcccc gcccgacagc ggccgctcgg 
               
               
                   
                   
               
               
                   
                 gccccggctc tcggttataa gatggcggcg ctgagcggtg 
               
               
                   
                   
               
               
                   
                 gcggtggtgg cggcgcggag ccgggccagg ctctgttcaa 
               
               
                   
                   
               
               
                   
                 cggggacatg gagcccgagg ccggcgccgg cgccggcgcc 
               
               
                   
                   
               
               
                   
                 gcggcctctt cggctgcgga ccctgccatt ccggaggagg 
               
               
                   
                   
               
               
                   
                 tgtggaatat caaacaaatg attaagttga cacaggaaca 
               
               
                   
                   
               
               
                   
                 tatagaggcc ctattggaca aatttggtgg ggagcataat 
               
               
                   
                   
               
               
                   
                 ccaccatcaa tatatctgga ggcctatgaa gaatacacca 
               
               
                   
                   
               
               
                   
                 gcaagctaga tgcactccaa caaagagaac aacagttatt 
               
               
                   
                   
               
               
                   
                 ggaatctctg gggaacggaa ctgatttttc tgtttctagc 
               
               
                   
                   
               
               
                   
                 tctgcatcaa tggataccgt tacatcttct tcctatcta 
               
               
                   
                   
               
               
                   
                 gcctttcagt gctaccttca tctctttcag tttttcaaaa 
               
               
                   
                   
               
               
                   
                 tcccacagat gtggcacgga gcaaccccaa gtcaccacaa 
               
               
                   
                   
               
               
                   
                 aaacctatcg ttagagtctt cctgcccaac aaacagagga 
               
               
                   
                   
               
               
                   
                 cagtggtacc tgcaaggtgt ggagttacag tccgagacag 
               
               
                   
                   
               
               
                   
                 tctaaagaaa gcactgatga tgagaggtct aatcccagag 
               
               
                   
                   
               
               
                   
                 tgctgtgctg tttacagaat tcaggatgga gagaagaaac 
               
               
                   
                   
               
               
                   
                 caattggttg ggacactgat atttcctggc ttactggaga 
               
               
                   
                   
               
               
                   
                 agaattgcat gtggaagtgt tggagaatgt tccacttaca 
               
               
                   
                   
               
               
                   
                 acacacaact ttgtacgaaa aacgtttttc accttagcat 
               
               
                   
                   
               
               
                   
                 tttgtgactt ttgtcgaaag ctgcttttcc agggtttccg 
               
               
                   
                   
               
               
                   
                 ctgtcaaaca tgtggttata aatttcacca gcgttgtagt 
               
               
                   
                   
               
               
                   
                 acagaagttc cactgatgtg tgttaattat gaccaacttg 
               
               
                   
                   
               
               
                   
                 atttgctgtt tgtctccaag ttctttgaac accacccaa 
               
               
                   
                   
               
               
                   
                 accacaggaa gaggcgtcct tagcagagac tgccctaaca 
               
               
                   
                   
               
               
                   
                 tctggatcat ccccttccgc acccgcctcg gactctattg 
               
               
                   
                   
               
               
                   
                 ggccccaaat tctcaccagt ccgtctcctt caaaatccat 
               
               
                   
                   
               
               
                   
                 tccaattcca cagcccttcc gaccagcaga tgaagatcat 
               
               
                   
                   
               
               
                   
                 cgaaatcaat ttgggcaacg agaccgatcc tcatcagctc 
               
               
                   
                   
               
               
                   
                 ccaatgtgca tataaacaca atagaacctg tcaatattga 
               
               
                   
                   
               
               
                   
                 tgacttgatt agagaccaag gatttcgtgg tgatggagga 
               
               
                   
                   
               
               
                   
                 tcaaccacag gtttgtctgc taccccccct gcctcattac 
               
               
                   
                   
               
               
                   
                 ctggctcact aactaacgtg aaagccttac agaaatctcc 
               
               
                   
                   
               
               
                   
                 aggacctcag cgagaaagga agtcatcttc atcctcagaa 
               
               
                   
                   
               
               
                   
                 gacaggaatc gaatgaaaac acttggtaga cgggactcga 
               
               
                   
                   
               
               
                   
                 gtgatgattg ggagattcct gatgggcaga ttacagtgg 
               
               
                   
                   
               
               
                   
                 acaaagaatt ggatctggat catttggaac agtctacaag 
               
               
                   
                   
               
               
                   
                 ggaaagtggc atggtgatgt ggcagtgaaa atgttgaatg 
               
               
                   
                   
               
               
                   
                 tgacagcacc tacacctcag cagttacaag ccttcaaaaa 
               
               
                   
                   
               
               
                   
                 tgaagtagga gtactcagga aaacacgaca tgtgaatatc 
               
               
                   
                   
               
               
                   
                 ctactcttca tgggctattc cacaaagcca caactggcta 
               
               
                   
                   
               
               
                   
                 ttgttaccca gtggtgtgag ggctccagct tgtatcacca 
               
               
                   
                   
               
               
                   
                 tctccatatc attgagacca aatttgagat gatcaaactt 
               
               
                   
                   
               
               
                   
                 atagatattg cacgacagac tgcacagggc atggattact 
               
               
                   
                   
               
               
                   
                 tacacgccaa gtcaatcatc cacagagacc tcaagagtaa 
               
               
                   
                   
               
               
                   
                 taatatattt cttcatgaag acctcacagt aaaaataggt 
               
               
                   
                   
               
               
                   
                 gattttggtc tagctacagt gaaatctcga tggagtgggt 
               
               
                   
                   
               
               
                   
                 cccatcagtt tgaacagttg tctggatcca ttttgtggat 
               
               
                   
                   
               
               
                   
                 ggcaccagaa gtcatcagaa tgcaagataa aaatccatac 
               
               
                   
                   
               
               
                   
                 agctttcagt cagatgtata tgcatttgga attgttctgt 
               
               
                   
                   
               
               
                   
                 atgaattgat gactggacag ttaccttatt caaacatcaa 
               
               
                   
                   
               
               
                   
                 caacagggac cagataattt ttatggtggg acgaggatac 
               
               
                   
                   
               
               
                   
                 ctgtctccag atctcagtaa ggtacggagt aactgtccaa 
               
               
                   
                   
               
               
                   
                 aagccatgaa gagattaatg gcagagtgcc tcaaaaagaa 
               
               
                   
                   
               
               
                   
                 aagagatgag agaccactct ttccccaaat tctcgcctct 
               
               
                   
                   
               
               
                   
                 attgagctgc tggcccgctc attgccaaaa attcaccgca 
               
               
                   
                   
               
               
                   
                 gtgcatcaga accctccttg aatcgggctg gtttccaaac 
               
               
                   
                   
               
               
                   
                 agaggatttt agtctatatg cttgtgcttc tccaaaaaca 
               
               
                   
                   
               
               
                   
                 cccatccagg cagggggata tggtgcgttt cctgtccact 
               
               
                   
                   
               
               
                   
                 gaaacaaatg agtgagagag ttcaggagag tagcaacaaa 
               
               
                   
                   
               
               
                   
                 aggaaaataa atgaacatat gtttgcttat atgttaaatt 
               
               
                   
                   
               
               
                   
                 gaataaaata ctctcttttt ttttaaggtg aaccaaagaa 
               
               
                   
                   
               
               
                   
                 cacttgtgtg gttaaagact agatataatt tttccccaaa 
               
               
                   
                   
               
               
                   
                 ctaaaattta tacttaacat tggattttta acatccaagg 
               
               
                   
                   
               
               
                   
                 gttaaaatac atagacattg ctaaaaattg gcagagcctc 
               
               
                   
                   
               
               
                   
                 ttctagaggc tttactttct gttccgggtt tgtatcattc 
               
               
                   
                   
               
               
                   
                 acttggttat tttaagtagt aaacttcagt ttctcatgca 
               
               
                   
                   
               
               
                   
                 acttttgttg ccagctatca catgtccact agggactcca 
               
               
                   
                   
               
               
                   
                 gaagaagacc ctacctatgc ctgtgtttgc aggtgagaag 
               
               
                   
                   
               
               
                   
                 ttggcagtcg gttagcctgg gttagataag gcaaactgaa 
               
               
                   
                   
               
               
                   
                 cagatctaat ttaggaagtc agtagaattt aataattcta 
               
               
                   
                   
               
               
                   
                 ttattattct taataatttt tctataacta tttcttttta 
               
               
                   
                   
               
               
                   
                 taacaatttg gaaaatgtgg atgtctttta tttccttgaa 
               
               
                   
                   
               
               
                   
                 gcaataaact aagtttcttt ttataaaaa 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 7 
               
               
                   
                 FRS2 (NM_006654.3) 
               
               
                   
                 aaaacccttc cctcccccgc tcccccggaa gtgcttttcc 
               
               
                   
                   
               
               
                   
                 aagattcggg ccggagagag gccttgtagg cacagcggct 
               
               
                   
                   
               
               
                   
                 gagactcgat ctgctccaag taggggctcc agcgcgggtc 
               
               
                   
                   
               
               
                   
                 ggagtctggg ggttcgcgcc cgccgacccg cgccctgctc 
               
               
                   
                   
               
               
                   
                 cctctcagca cctgggcgga cgaaatgacc attaagaagt 
               
               
                   
                   
               
               
                   
                 agatgcccag atgcaaaagt gatgaaacag tccatttgtc 
               
               
                   
                   
               
               
                   
                 ataaagtaag atgcagctgt ggcatgtcaa ccagcttgga 
               
               
                   
                   
               
               
                   
                 acaaaattgt atctgttttt ctcagaagag aattccacaa 
               
               
                   
                   
               
               
                   
                 ggagattttc ttctttctac catcatcaag atcaagcagg 
               
               
                   
                   
               
               
                   
                 caagtttact tgctgtcatc ttctgcaagg ttaaatcagc 
               
               
                   
                   
               
               
                   
                 aaacaaagaa aacatggtat tttgaaatat gattaaactc 
               
               
                   
                   
               
               
                   
                 ctgatgctgc agcagaggct aagaatatta atggccagat 
               
               
                   
                   
               
               
                   
                 ctagtgcaca catggtcttc tgaagaagcc atgggtagct 
               
               
                   
                   
               
               
                   
                 gttgtagctg tccagataaa gacactgtcc cagataacca 
               
               
                   
                   
               
               
                   
                 tcggaacaag tttaaggtca ttaatgtgga tgatgatggg 
               
               
                   
                   
               
               
                   
                 aatgagttag gttctggcat aatggaactt acagacacag 
               
               
                   
                   
               
               
                   
                 aactgatttt atacacccgc aaacgtgact cagtaaaatg 
               
               
                   
                   
               
               
                   
                 gcactacctc tgcctgcgac gctatggcta tgactcgaat 
               
               
                   
                   
               
               
                   
                 ctcttttctt ttgaaagtgg tcgaaggtgt caaactggac 
               
               
                   
                   
               
               
                   
                 aaggaatctt tgcctttaag tgtgcccgtg cagaagaatt 
               
               
                   
                   
               
               
                   
                 atttaacatg ttgcaagaga ttatgcaaaa taatagtata 
               
               
                   
                   
               
               
                   
                 aatgtggtgg aagagccagt tgtagaaaga aataatcatc 
               
               
                   
                   
               
               
                   
                 agacagaatt ggaagtccct agaacacctc gaacacctac 
               
               
                   
                   
               
               
                   
                 aactccagga tttgctgctc agaacttacc taatggatat 
               
               
                   
                   
               
               
                   
                 ccccgatatc cctcatttgg agatgcttca tcccatccgt 
               
               
                   
                   
               
               
                   
                 caagcagaca tccttctgtg ggaagtgctc gcctgccttc 
               
               
                   
                   
               
               
                   
                 agtaggggaa gaatctacac atcctttgct tgtggctgag 
               
               
                   
                   
               
               
                   
                 gaacaagtac atacctatgt caacactaca ggtgtgcaag 
               
               
                   
                   
               
               
                   
                 aagagcggaa aaaccgcaca agtgtgcatg ttccattgga 
               
               
                   
                   
               
               
                   
                 ggcgagggtt tctaacgctg aaagcagcac accaaaagaa 
               
               
                   
                   
               
               
                   
                 gaaccaagta gtattgagga cagggatcct cagattcttc 
               
               
                   
                   
               
               
                   
                 ttgaacctga aggagtcaaa tttgttttag ggccaacccc 
               
               
                   
                   
               
               
                   
                 tgttcaaaag cagttaatgg aaaaagagaa actggagcaa 
               
               
                   
                   
               
               
                   
                 cttggaagag atcaagttag tggaagtgga gcaaataaca 
               
               
                   
                   
               
               
                   
                 cagaatggga cactggctat gacagtgatg aacgaagaga 
               
               
                   
                   
               
               
                   
                 tgcaccctct gttaacaaac tggtgtatga aaatataaat 
               
               
                   
                   
               
               
                   
                 gggctatcta tccctagtgc ctcaggggtc aggagaggtc 
               
               
                   
                   
               
               
                   
                 gtctgacatc caccagtacc tcagataccc agaatatcaa 
               
               
                   
                   
               
               
                   
                 caactcagct cagagaagaa ctgcattatt aaactatgaa 
               
               
                   
                   
               
               
                   
                 aatctaccat ctttgcctcc tgtttgggaa gcccgcaagc 
               
               
                   
                   
               
               
                   
                 taagtaggga tgaagatgac aatttaggac caaagacccc 
               
               
                   
                   
               
               
                   
                 atctctaaat ggctaccata ataatctaga tccaatgcat 
               
               
                   
                   
               
               
                   
                 aactatgtaa atacagagaa tgtaacagtg ccagcaagtg 
               
               
                   
                   
               
               
                   
                 ctcacaaaat agaatattca aggcgtcggg actgtacacc 
               
               
                   
                   
               
               
                   
                 aacagtcttt aactttgata tcagacgccc aagtttagaa 
               
               
                   
                   
               
               
                   
                 cacaggcagc ttaattacat acaggttgac ttggaaggtg 
               
               
                   
                   
               
               
                   
                 gcagtgactc tgacaaccct cagactccaa aaacgcctac 
               
               
                   
                   
               
               
                   
                 aactcccctt ccacaaaccc ctaccaggcg cacagagctg 
               
               
                   
                   
               
               
                   
                 tatgccgtga tagacatcga gagaactgct gctatgtcaa 
               
               
                   
                   
               
               
                   
                 atttgcagaa agcactgcca cgagatgatg gtacatctag 
               
               
                   
                   
               
               
                   
                 gaaaactaga cacaatagta ctgatctgcc catgtgagcc 
               
               
                   
                   
               
               
                   
                 tggaaagcat tgtgttgttt gcacctttgt gaagttttta 
               
               
                   
                   
               
               
                   
                 aaaatgaaga tgcaagtgct tcattttcat ttctaaacac 
               
               
                   
                   
               
               
                   
                 taactccttt tatagactga taaaattttt ttctgaatat 
               
               
                   
                   
               
               
                   
                 ttcatgtgca tctttaacta aagggaatta atgtagagca 
               
               
                   
                   
               
               
                   
                 ggtactcctt aaagaacact aatttcatta tatactactc 
               
               
                   
                   
               
               
                   
                 gttgtacagc agcattcccg ttttcacagt gcctatttaa 
               
               
                   
                   
               
               
                   
                 aatgagagtt gaagtaaatg acatgctggt tgatttttat 
               
               
                   
                   
               
               
                   
                 caatattctg gacttaacgc atacctttca tgtctaagtc 
               
               
                   
                   
               
               
                   
                 atggttggct tttaaaactt tttataaagc ctcttgacaa 
               
               
                   
                   
               
               
                   
                 tgtacattgc taacaggtaa ctataggctt tgaaagtaat 
               
               
                   
                   
               
               
                   
                 gctcgtagat tcagtgttca cagtatgtgg cctccagcat 
               
               
                   
                   
               
               
                   
                 gtaacatgag gaatccttta tttcattaat taatggcttt 
               
               
                   
                   
               
               
                   
                 ttgacttgag ccaaaacata tgtaaaggaa acagaagtac 
               
               
                   
                   
               
               
                   
                 cgcacctcct cttacaccag tcagctcctt tgccttcagt 
               
               
                   
                   
               
               
                   
                 gttactagaa agcggcctgt gtccatgagt gtgctttgct 
               
               
                   
                   
               
               
                   
                 gttggtgcac tgaaaggcag gaaggagaca agattttcta 
               
               
                   
                   
               
               
                   
                 tttactcatc tcatgatgtc atttgaaggg catgtccaga 
               
               
                   
                   
               
               
                   
                 tatcttaaaa ttataatagg ctcaagaatc agtctcaggt 
               
               
                   
                   
               
               
                   
                 cactttaccc aaaaacattt gaaaatctga accacaatct 
               
               
                   
                   
               
               
                   
                 cctgaaagtt tttctcctat agattgttga caacacattg 
               
               
                   
                   
               
               
                   
                 ttttctggag gcatttgtgc cattaggttt ccatttatct 
               
               
                   
                   
               
               
                   
                 tcagtttttt tctttggtgt ttgggatgtc ttattttgtt 
               
               
                   
                   
               
               
                   
                 gccttatgtc cttttcaatttaaaatgttt gagtttgtat 
               
               
                   
                   
               
               
                   
                 atagttttga aattggatta tgtgttcatt gttgtttagt 
               
               
                   
                   
               
               
                   
                 ttgcattttt gtcaaattat ggttttgaag gttcatttgg 
               
               
                   
                   
               
               
                   
                 aacttactgt tagtctgtaa cagggttgcc cttgtccagt 
               
               
                   
                   
               
               
                   
                 atttatttat aagctgttta cttttcaagt tgataaaaac 
               
               
                   
                   
               
               
                   
                 attctccaat tctaaatttg cttgtgtcca taggtgatct 
               
               
                   
                   
               
               
                   
                 ctttagcaaa ctgagaaaaa aaggaagcta cttttaacat 
               
               
                   
                   
               
               
                   
                 gcaaagttcc ctcaaggtgt accgtgttgt ctctgtgggc 
               
               
                   
                   
               
               
                   
                 actcttcccc agcactttag cagtaattcc cccagctaca 
               
               
                   
                   
               
               
                   
                 cgctgcagtt gtactctgcc cactctagtg ttcctcagct 
               
               
                   
                   
               
               
                   
                 ctgctgtcct tttacttgta gctggatctt tgattatcct 
               
               
                   
                   
               
               
                   
                 tcgatttcca tgaaatatta atattgttgc cagcatagca 
               
               
                   
                   
               
               
                   
                 ggtacagtgg aagtcttgt gcagtgagat tgtatcataa 
               
               
                   
                   
               
               
                   
                 tttaggattt aaaatgaatt aaagtttata 
               
               
                   
                   
               
               
                   
                 taaactgaagagtctccata tgtcaaactc ttggaaaatc 
               
               
                   
                   
               
               
                   
                 aaagatgttc caatttccta aacactagag aatacgagag 
               
               
                   
                   
               
               
                   
                 aaggtagagt ggaaaaggtt aggtaacctt gcaaaatatt 
               
               
                   
                   
               
               
                   
                 ttactatttt ctctaaatat gaggaagttt gagattatga 
               
               
                   
                   
               
               
                   
                 tctggatcta ccagatataa ctaaggttaa tttagcatga 
               
               
                   
                   
               
               
                   
                 aaaagtttta gtcatattgg catccaacct attcagtaac 
               
               
                   
                   
               
               
                   
                 cgaatcatag gacaatgatg gattaggaga acaatagagt 
               
               
                   
                   
               
               
                   
                 gggatcatta taaagaaaat aaattattaaaggtgtcttt 
               
               
                   
                   
               
               
                   
                 atcgttttag tgccattttt agtgtcttta ctataaatca 
               
               
                   
                   
               
               
                   
                 atatcagtgtattttatcat tctatgtgca tagcagaatt 
               
               
                   
                   
               
               
                   
                 ttcttttctc ccttttgttc ccctgtgaac ttggtgctta 
               
               
                   
                   
               
               
                   
                 ttaaagtgct cactgttctc ttaaaagaga gcagtggtat 
               
               
                   
                   
               
               
                   
                 aggtgtgcag tttccatgat gcaggttcca tttttaatat 
               
               
                   
                   
               
               
                   
                 attgttccac ttatcctttc ttctgagtaa attgctaatt 
               
               
                   
                   
               
               
                   
                 gtgccaaatt tatgtaatag tttttgtaat gtggaataag 
               
               
                   
                   
               
               
                   
                 aattatgatg gaaccattgc acattttttt ctgaaacagc 
               
               
                   
                   
               
               
                   
                 cagtcaaggc agaacattaa tctccaaatg caagggctga 
               
               
                   
                   
               
               
                   
                 tctatttatt cattttggag gttgggtact ttattctttc 
               
               
                   
                   
               
               
                   
                 tttccgtcat ccttttcatt gtttcccccg gattctaatt 
               
               
                   
                   
               
               
                   
                 agtttttatt ttttttagat aactccaata taatcattac 
               
               
                   
                   
               
               
                   
                 agtttatgct ttaaatacta tgtgctttaa aaaggaaaat 
               
               
                   
                   
               
               
                   
                 gggaccaatt tgtctgctaa gaatttgatt ttaggtacta 
               
               
                   
                   
               
               
                   
                 taagagtatt aggaaaatat atacaactgg tgttaatttc 
               
               
                   
                   
               
               
                   
                 tagatatttt ctagaaatca cttgtgttcc tatttaataa 
               
               
                   
                   
               
               
                   
                 aaggtaattt agaatactac ttgtcctttg cagtagttta 
               
               
                   
                   
               
               
                   
                 gtaatgggca ttaagctgtg tcctcgaagg atgtacctat 
               
               
                   
                   
               
               
                   
                 tactaggtgc attttagaat gaaatattga tattttatta 
               
               
                   
                   
               
               
                   
                 gcatataatt gtggccatat atctcagatt ttctgaggca 
               
               
                   
                   
               
               
                   
                 gatctaattt tagataattc tgttggtaga ccatgtgatc 
               
               
                   
                   
               
               
                   
                 cttctttttg gttttggaaa tataatcatt gttaatgttt 
               
               
                   
                   
               
               
                   
                 tccctccaaa tagaatactg ttttatccat acaaatcata 
               
               
                   
                   
               
               
                   
                 acagcatcta tcccatgcta gggttggaaa ctgatattgg 
               
               
                   
                   
               
               
                   
                 tattacttgt gttttttctt agtgtgtttt atttcccagt 
               
               
                   
                   
               
               
                   
                 ttcatcttct tctaaaaatg aaaatatggt gccttccctc 
               
               
                   
                   
               
               
                   
                 cctccaggaa gactggcaaa tatttccttt tatttactgc 
               
               
                   
                   
               
               
                   
                 tgctgtggag tgatgagata tgcactttac tctttaagat 
               
               
                   
                   
               
               
                   
                 tcagcaaaaa gcttttcact tctcagtata tccagaatac 
               
               
                   
                   
               
               
                   
                 atcatatctg ggacttagga aaatttgcca agcaatcttt 
               
               
                   
                   
               
               
                   
                 gtttttatag atactaatgt tgaccctctc cagcgttcaa 
               
               
                   
                   
               
               
                   
                 tgttataaat agaacaagtc aagctagtgt ttatctcctc 
               
               
                   
                   
               
               
                   
                 cccctcccca aaactgtggc acagcatat aaaatgtacc 
               
               
                   
                   
               
               
                   
                 tcaataatgt tctattaaaa atgggacagg ggccttatgt 
               
               
                   
                   
               
               
                   
                 tttcataatt tcccaacaat gtgccgccat atttttgcct 
               
               
                   
                   
               
               
                   
                 caaggtaaag gttttaacag atgaaaaagt acttcccaat 
               
               
                   
                   
               
               
                   
                 tcccccgtgc tattcctaac ctataatgcc caaatgtttt 
               
               
                   
                   
               
               
                   
                 gtgcaatgtg tagtgtgtgt gtataaatac atatattctt 
               
               
                   
                   
               
               
                   
                 gaaatagaca taccatcaga gacatcattc acaagtaact 
               
               
                   
                   
               
               
                   
                 gatgtattgg catctcattc atatttctga tgtgtgaggt 
               
               
                   
                   
               
               
                   
                 atatggtact aattaccttt tccttgatgt ttgccaaatt 
               
               
                   
                   
               
               
                   
                 tgaataaagg cattggtacg aaattacaga atgtaaagaa 
               
               
                   
                   
               
               
                   
                 aatgtttttg gcttgaaaaa ttaacatatt ttatgacgta 
               
               
                   
                   
               
               
                   
                 ccacagtata ctctgcccaa accagcaccc tatctatctt 
               
               
                   
                   
               
               
                   
                 tcctgttctt tacatccctg ttccccatcc ctacttcctc 
               
               
                   
                   
               
               
                   
                 atttttggta taacacagtt cttttgtagc atcattataa 
               
               
                   
                   
               
               
                   
                 ttgcagttct atggcaattg gacagttata gcatggaaac 
               
               
                   
                   
               
               
                   
                 agactggtat aagtagtaca gtagtcacca gtgtgccaca 
               
               
                   
                   
               
               
                   
                 tttgcattag taatgcaaaa tatacatttt ataaaggaca 
               
               
                   
                   
               
               
                   
                 aactttgtgt tatgttttat tttcattaca ttgtataata 
               
               
                   
                   
               
               
                   
                 ttgtaagact attgtatgtc ctaatttgca ttataaatgt 
               
               
                   
                   
               
               
                   
                 ttttttccta cgtaaaggca taaatatagc aactttgtat 
               
               
                   
                   
               
               
                   
                 aaaggtagct tattagattt ttaatttttt cttttataaa 
               
               
                   
                   
               
               
                   
                 aaattgtcca acagtgggac taccattgcc aaattgtata 
               
               
                   
                   
               
               
                   
                 tgaaatatga attttacccc catggttaat ttcttttata 
               
               
                   
                   
               
               
                   
                 aacattccat atttctctaa taaaaagaca taagtgatac 
               
               
                   
                   
               
               
                   
                 tgtactatgc atacattgta tcttaatgct gtttcagatc 
               
               
                   
                   
               
               
                   
                 agcattttaa attttggttt gcatttttaa tattggcaaa 
               
               
                   
                   
               
               
                   
                 acgtaaccac tgttaattaa aataaaacct tgttgtatat 
               
               
                   
                   
               
               
                   
                 gtaacaacat aattttccct ctatcccttc ccaccctttg 
               
               
                   
                   
               
               
                   
                 ttctctattt ctccctatca gtgccaactt catacatttt 
               
               
                   
                   
               
               
                   
                 gtagcatggc aataaaatat aacttttaca ctgaggccga 
               
               
                   
                   
               
               
                   
                 gtgtggcttt ttggaggaag tggggatggg acgattgccc 
               
               
                   
                   
               
               
                   
                 tctagttgtc ctttgcatat gactgttttt tgccatataa 
               
               
                   
                   
               
               
                   
                 gccatgtcat caggcatgaa aagttttctc atatatgatg 
               
               
                   
                   
               
               
                   
                 taaacttgct tttaaggaca agtgtgaatg tgctttttaa 
               
               
                   
                   
               
               
                   
                 gcttaatttt tgtcatgaca actaattttt tttatctttg 
               
               
                   
                   
               
               
                   
                 gagaagtcag agttctttac aatcaaacgt ttattaactg 
               
               
                   
                   
               
               
                   
                 gagtacttag aataagctag taattgaatt tagttcaagg 
               
               
                   
                   
               
               
                   
                 gctaagcaac acatttttaa atccttattt attgtagagt 
               
               
                   
                   
               
               
                   
                 attagtatac tgtcctacaa attatgtaaa atatggttta 
               
               
                   
                   
               
               
                   
                 atattagatg actttggatt ttgcaatgcc ttactgttgt 
               
               
                   
                   
               
               
                   
                 cattctagca taaatatcca taatgaggta ctcaagttga 
               
               
                   
                   
               
               
                   
                 tactggaagctgagctgatc atacactgac ctgaagcatt 
               
               
                   
                   
               
               
                   
                 catgaaaagc tgctttattg aataaagtct gattggagtt 
               
               
                   
                   
               
               
                   
                 cttttcatgc tcactttccc cttattgctg aaagtagatt 
               
               
                   
                   
               
               
                   
                 gcaataaaaccccaataaaa cgtttggtcg gataaaaaaa 
               
               
                   
                   
               
               
                   
                 aaaaaaaaaa aa 
               
             
          
         
       
     
         [0164]    SEQ ID NO: 8 ANXA1 (NP — 000691.1) mamvseflkq awfieneeqe yvqtvksskg gpgsayspyp tfnpssdvaa lhkaimvkgvdeatiidilt krnnaqrqqi kaaylqetgk pldetlkkal tghleevvla llktpaqfda delraamkgl gtdedtliei lasrtnkeir dinrvyreel krdlakdits dtsgdfrnal lslakgdrse dfgvnedlad sdaralyeag errkgtdvnv fntilttrsy pqlrrvfqky tkyskhdmnk vldlelkgdi ekcltaivkc atskpaffae klhqamkgvg trhkalirim vsrseidmnd ikafyqkmyg islcqailde tkgdyekilv alcggn 
         [0165]    SEQ ID NO: 9 Hp2 (NP — 001119574.1) msalgavial llwgqlfavd sgndvtdiad dgcpkppeia hgyvehsvry qcknyyklrt egdgvytlnn ekqwinkavg dklpeceavc gkpknpanpv qrilgghlda kgsfpwqakm vshhnittga tlineqwllt taknlflnhs enatakdiap tltlyvgkkq lveiekvvlh pnysqvdigl iklkqkvsvn ervmpiclps kdyaevgrvg yvsgwgrnan fkftdhlkyv mlpvadqdqc irhyegstvp ekktpkspvg vqpilnehtf cagmskyqed tcygdagsaf avhdleedtw yatgilsfdk scavaeygvy vkvtsiqdwv qktiaen 
         [0166]    SEQ ID NO: 10 Zinc alpha2-glycoprotein (Chain B, Crystal structure of the complex formed between Mhc-Like Zinc Alpha2-glycoprotein and Prol) qdntrkiiik nfdipksvrp ndevtavlav qtelkecmvv ktylissipl qgafnykyt cicddnpktf ywdfytnrtv qiaavvdvir elgicpddaa vipiknnrfy tieilkve 
         [0167]    SEQ ID NO: 11 calprotectin mltelekaln siidvyhkys likgnfhavy rddlkkllet ecpqyirkkg advwfkeldi ntdgavnfqe flilvikmgv aahkkshees like 
         [0168]    All references, publications, patent applications, issued patents, accession records and databases cited herein, including in any appendices, are incorporated by reference in their entirety for all purposes.