CROSS-RELATED REFERENCES

[0001] This application claims priority to United States provisional patent application no. 61/637, 152 entitled "Biomarkers for Hepatocellular Carcinoma," which was filed on July 24, 2012. The contents of the provisional application are incorporated herein by reference in its entirety as if set forth verbatim.

TECHNICAL FIELD

[0002] The present invention relates generally to the discovery of autoantibodies to Wolf-Hirschhorn syndrome candidate 2 (WHSC2) and (inosine 5 ' -monophosphate) dehydrogenase 2 (IMPDH2) as biomarkers of hepatocellular carcinoma (HCC). More specifically, the present invention relates to the detection and quantitation of WSHC2 and IMPDH2 specific autoantibodies for diagnosing, monitoring or evaluating the status of HCC in a subject.

BACKGROUND OF THE INVENTION

[0003] Hepatocellular carcinoma (HCC) is the most common type of liver cancer, being responsible for almost 700,00 deaths in 2008 (Ferlay, J. et al., (2010), GLOBOCAN 2008 v l .2, Cancer Incidence and Mortality Worldwide: I ARC CancerBase No. 10, Lyon, France: International Agency for Research on Cancer, [http://globocan.iarc.fr]; see also, Parkin, et al., (1999) C A. Cancer J. Clin.. 49:33-64, and Okuda, et al., (1993), Neoplasms of the Liver in Diseases of the Liver, 7th edition 1236). In the United States, the 5-year overall survival (1992-1996) rate is 5% (El-Serag et al, Hepatol ogy 33:62-65 (2001 )). The development of HCC is generally believed to be caused by chronic liver injury, such as from viral infection, e.g., from hepatitis B or C. alcoholic liver damage and aflatoxin B exposure.

[0004] HCC is usually not discovered in subjects until the disease is in its advanced stages, resulting in ineffective treatment and poor prognosis. Historically, only 10-20% of primary HCCs are found to be resectable at the time of diagnosis, where the majority of unresectable HCC subjects die within one year (Fong, et al., (2001 ), Cancer of the Liver and Biliary Tree in Cancer: Principles & Practice of Oncology, 1 162-1 199). Thus, the development of HCC screening tests has been the focus of a significant amount of research in efforts to improve the detection of HCC in early stages.

[0005] Biomarkers are measurable biological and physiological parameters that can serve as indices for health-related assessments. Protein biomarkers are particularly powerful because they are amenable to simple blood or bodily fluid tests and, once successfully developed, can benefit the cancer subjects as simple clinical tools. In terms of identifying protein biomarkers for cancer detection, either a body fluid or tissue biopsy approach can be utilized to conduct diagnostic screening for cancers such as HCC.

[0006] The primary biomarker that has been widely used for the screening and diagnosis of HCC is alpha-fetoprotein (AFP). As such, current HCC screening tests measure the levels of AFP in the blood serum, where elevated serum levels of AFP may indicate HCC. However, a major disadvantage of using screening tests that measure AFP levels in subjects stems from the fact that detecting elevated serum levels of AFP has poor specificity for HCC. since elevated AFP levels are also detected in a wide variety of non-hepatic malignancies and benign conditions, including acute and chronic hepatitis (Mclntire KR et al., (1975), Cancer Res. 35, 991 -996; Liaw, Y. P., (1986), Liver 6, 133-137). Further, AFP screening tests can miss up to half of HCC cases due to its poor sensitivity and specificity, in particular because 30-50% of HCC cases do not present with elevated AFP levels (Johnson, P. J., (2001 ), Clin. Liver Dis. 5, 145-149).

[0007] In recent years, other potential biomarkers for HCC have been investigated but nothing superior to AFP has been found (Seow et al., (2001), Proteomics, 1 : 1249-1263). In view of unreliable non-invasive techniques for screening for HCC. alternative methods such as biopsies, ultrasonography and computed tomography have also been employed, but these methods also suffer from issues with low sensitivity or specificity and thus are less than desirable. For example, only 25-28% of HCC nodules smaller than 2 cm can be detected by ultrasonography and CT scan during arterial portography.

[0008] Accordingly, there is a need for the identi ication of a biomarker or biomarkers that can be used to diagnose, monitor and/or assess the status in a subject for HCC that exhibits good sensitivity and/or specificity. The present invention fulfills this need by providing a method of detecting autoantibodies to WSHC2 and IMPDH2 to assist diagnosing, monitoring and/or evaluating the status of a subject having or suspected of having HCC.

SUMMARY OF THE INVENTION

[0009] The embodiments disclosed below satisfy this need. The following simplified summary is prov ided in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview, and is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0010] The present invention provides a method for detecting WSHC2 and IMPDH2 specific autoantibodies. In one embodiment, the present invention relates to a method for detecting Wolf-Hirschhorn syndrome candidate 2 (WHSC2) autoantibodies in a subject comprising the steps of: (a) preparing a WHSC2 protein; (b) reacting the WHSC2 protein with a sample from the subject; and (c) detecting the WHSC2 autoantibodies in the sample.

[001 1] In another embodiment, the present invention relates to a method for detecting Wolf-Hirschhorn syndrome candidate 2 (WHSC2) autoantibodies in a subject comprising the steps of: (a) preparing a WHSC2 protein; (b) preparing antibodies to the WHSC2 protein; (c) reacting the antibodies to the WHSC2 protein with a sample from the subject; and (d) detecting the presence of WHSC2 autoantibodies in the subject sample.

[0012] In yet another embodiment, the present invention relates to a method for detecting (inosine 5 ' -monophosphate) dehydrogenase 2 (IMPDH2) autoantibodies in a subject comprising the steps of: (a) preparing a 1 PDH2 protein; (b) reacting the IMPDH2 protein with a sample from the subject; and (c) detecting the IMPDH2 autoantibodies in the sample.

[0013] In another embodiment, the present invention relates to a method for detecting (inosine 5 '-monophosphate) dehydrogenase 2 (IMPDH2) autoantibodies in a subject comprising the steps of: (a) preparing a IMPDH2 protein; (b) preparing antibodies to the IMPDH2 protein; (c) reacting the antibodies to the IMPDH2 protein with a sample from the subject; and (d) detecting the presence of IMPDH2 autoantibodies in the subject sample. [0014] In a further embodiment, the present invention relates to a panel assay to detect Wolf-Hirschhorn syndrome candidate 2 (WHSC2) and (inosine 5 ^' -monophosphate) dehydrogenase 2 (IMPDH2) autoantibodies in a subject comprising the steps of: a first assay for detecting the level of WHSC2 specific autoantibodies in the subject comprising: (a) preparing a WHSC2 protein; (b) reacting the WHSC2 protein with a sample from the subject; and (c) detecting the WHSC2 autoantibodies in the sample; and a second assay for detecting the level of IMPDH2 specific autoantibodies in the subject comprising: (a) preparing a IMPDl 12 protein; (b) reacting the IMPDH2 protein with a sample from the subject; and (c) detecting the IMPDH2 autoantibodies in the sample.

[0015] Other aspects of the invention are found throughout the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a graphical depiction of antibodies to WHSC2 in sera from subjects with HCC and asymptomatic healthy individuals ( Example 1 ).

[0017] Figure 2 is a graphical depiction of antibodies to IMPDH2 in sera from subjects with HCC and asymptomatic healthy individuals (Example 1 ).

[0018] Figure 3 is a scatterplot of WHSC2 and IMPDH2 ELISA results on HCC and normal sera (Example 2).

[0019] Figure 4 is a graphical representation of WHSC2 and IMPDH2 ELISA results on HCC and normal sera (Example 2).

[0020] Figure 5 is a graphical representation of WHSC2 and IMPDH2 ELISA results on HCC and normal sera (Example 3).

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates generally to the discovery of autoantibodies to Wolf-Hirschhorn syndrome candidate 2 (WHSC2) and (inosine 5 '-monophosphate) dehydrogenase 2 (IMPDH2) as biomarkers of hepatocellular carcinoma (HCC). More specifically, the present invention relates to the detection and quantitation of WSHC2 and IMPDH2 specific autoantibodies for diagnosing, monitoring or evaluating the status of HCC in a subject. [0022] In the description that follows, a number of terms used in the field of molecular biology, immunology and medicine are extensively utilized. The following non- limiting definitions provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms.

[0023] When the terms "one," "a," or "an" are used in this disclosure, they mean "at least one" or "one or more," unless otherwise indicated.

[0024] The term "antibody" refers to an immunoglobulin molecule that is capable of binding an epitope or antigenic determinant. The term "antibody" includes whole antibodies and antigen-binding fragments thereof, including single-chain antibodies.

Accordingly, the term "antibody" includes human antigen binding antibody and antibody fragments, including, but not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. The antibodies may be from any animal origin such as for example mammals including human, murine, rabbit, goat, guinea pig. camel, horse and the like.

[0025] The term "antigen" refers to a molecule that is capable of eliciting an immune response. The term "antigen" includes any molecule that is capable of being bound by an antibody. Additionally, the term "antigen" may refer to a proteinaceous or non- proteinaceous antigen. The antigen may be a whole protein or a portion thereof.

[0026] The term "autoantibody" refers to an immunoglobulin directed against self-protein, carbohydrate, nucleic acid or other molecule present in the human body. More specifically, the term "autoantibody" refers to an antibody that is capable of binding to a self antigen or fragment thereof.

[0027] The term "peptide" as used herein refers to a sequence of two or more amino acid residues joined by peptide bonds. A peptide is distinguished from a native protein because its sequence is generally less than the native protein sequence. The peptide may be isolated from a protein digest or synthetically prepared. The amino acid sequence of the peptide may be identical to the native protein sequence or modified. Modification may be by enzymatic conversion of one or more particular amino acid residues into another amino acid residue. In addition, modification may be effected by replacing, deleting or inserting one or more amino acid residues during peptide synthesis. [0028] The term "subject" as used herein refers to an animal, including, but not limited to, an ovine, bovine, ruminant, lagomorph, porcine, equine, canine, feline, rodent or primate, e.g. a human. Typically, the terms "subject" and "patient" are used interchangeably herein in reference, for example, to a mammalian subject, particularly a human subject.

[0029] The term "sample" as used herein refers to biological samples obtained from animals (e.g. humans) and refers to a biological material or compositions found therein, including, but not limited to, bone marrow, blood, serum, cells, plasma, interstitial fluid, urine, cerebrospinal fluid, nucleic acid, DNA, tissue and purified or filtered forms thereof. Samples for detecting circulating autoantibodies include, for example, blood, serum and plasma. In one embodiment, for convenience, the sample is plasma or serum, although saliva has been reported to be a suitable biological sample for measuring the presence of anti-microbial antigens, HIV. or autoantibodies (anti-transglutaminase, etc) as described herein.

[0030] The term "controls" or "control samples" refers to samples, such as serum samples, taken from subjects who have tested negative for the determinant (e.g., autoantibodies) in question. For example, a control for testing for the presence of HCC might be taken from a subject who does not have HCC, which might be a healthy individual or one with another carcinoma.

[0031 ] The term "diagnosis" as used herein is not meant to imply an absolute diagnosis of a disease such as HCC. which is normally determined by performing invasive diagnostic procedures, such as a colonoscopy. Instead, it is meant to refer to the determination that a subject is more likely to have the disease than not. As such, detecting the presence of characteristic diagnostic markers can be used in conjunction with other diagnostic tests to help differentiate from other disease states, confirm a diagnosis, or monitor treatment. In addition, the term "diagnosis" can mean that a subject who is not known to have a disease, whether or not a more invasive diagnostic procedure has been performed, is more likely not to have that disease, and/or more likely than not to develop the disease in the future. Accordingly, a marker or panel of markers is "diagnostic" if its detection (singly or by composite) is positively correlated with the presence of, or the likelihood of developing, a disease. [0032] The phrase "amino acid substitution" as used herein refers to the replacement or conversion of an amino acid residue in a sequence with another molecule. The molecule may be a natural or non-natural amino acid, an organic molecule or other chemical moiety. In replacement, one amino acid residue in a sequence being synthesized may be replaced with another amino acid residue or molecule. For example, replacing leucine with isoleucine, which would be a conservative substitution wherein one

hydrophobic amino acid residue is replaced with another hydrophobic amino acid residue.

[0033] The term "peptide" as used herein refers to a sequence of two or more amino acid residues joined by peptide bonds. A peptide is distinguished from a native protein because its sequence is generally less than the native protein sequence. The peptide may be isolated from a protein digest or synthetically prepared. The amino acid sequence of the peptide may be identical to the native protein sequence or modified. Modification may be by enzymatic conversion of one or more particular amino acid residues into another amino acid residue. In addition, modification may be effected by replacing, deleting or inserting one or more amino acid residues during peptide synthesis.

Biomarkers of the Invention

[0034] The present invention is based in part on the discovery of highly specific autoantibody biomarkers for HCC detection. In general terms, biomarkers are biological characteristics that serve as indicators of normal and disease processes occurring in the body. Certain autoantibodies serve as biomarkers in that their presence correlates with the onset or decline of a bodily process, such as HCC. Such autoantibodies can be found in fluids and tissues throughout the body. When using autoantibodies as biomarkers, the presence or amount of biomarker peptide in a biological sample from a subject can be used to identify the presence, severity, or type of disease.

[0035] The biomarkers of the present invention can be used to assess HCC status in a subject. "HCC status" in this context subsumes, inter alia, the presence or absence of disease, the risk of developing disease, the stage of the disease, and the effectiveness of treatment of disease. Based on this status, further procedures may be indicated, including additional diagnostic tests or therapeutic procedures or regimens, such as endoscopy, biopsy, surgery, chemotherapy, immunotherapy, and radiation therapy. More particularly, the biomarkers of the invention are capable of identifying HCC with high specificity and thus can be used to qualify HCC status or risk in a subject.

[0036] The biomarkers according to the present invention were identified using commercially available microarrays containing approximately 8000 human recombinant proteins (Human ProtoarrayR v4.0, Invitrogen, Carlsbad, CA). More specifically, candidate biomarkers were identified from microarray data analysis using a cell-free ELISA system from Ambergen, Inc. (Boston, MA). However, any suitable methods known by those of ordinary skill in the art can be used to delect one or more the biomarkers described herein. These methods include, without limitation, mass spectrometry, fluorescence, cell-free ELISA, surface plasmon resonance, ellipsometry and atomic force microscopy.

Wolf-Hirschhorn Syndrome Candidate 2 (WHSC2)

[0037] The Wolf-Hirschhorn Syndrome Candidate 2 (WHSC2) gene is expressed ubiquitously with higher levels in fetal than in adult tissues. The WHSC2 gene encodes a protein having an amino acid sequence according to SEQ ID NO: l [UniProt/Swiss-Prot: Q9H3P2; 57277 Da (GeneCards database); Ensembl ENSGOOOOO 185049]. The encoded protein is believed to be capable of reacting with HLA-A2-restricted and tumor-specific cytotoxic T lymphocytes, and has also been shown to be a member of the negative elongation factor (NELF) protein complex. (GeneCards database). The WHSC2 gene is located at position 4pl6.3 on Chromosome 4 : NC_000004.1 1 , NT_006051.18; . The NCBI protein reference sequence is NI )05654.3,. WHSC2 DNA sequence is further described in record NCBI Reference Sequence: NG_009232.1 of NCBFs database.

IMP (inosine 5 '-monophosphate) dehydrogenase 2 (IMPDH2)

[0038] The IMP (inosine 5 ' -monophosphate) dehydrogenase 2 (IMPDH2) gene encodes a rate-limiting enzyme in the de novo guanine nucleotide biosynthesis, and is involved in maintaining cellular guanine deoxy- and ribonucleotide pools needed for D A and RNA synthesis. The IMPDH2 gene encodes a protein having an amino acid sequence according to SEQ ID NO:2 [UniProt/Swiss-Prot: P12268; 55805 Da (GeneCards database); Ensembl ENSGOOOOO 178035]. The encoded protein catalyzes the NAD-dependent oxidation of inosine-5 '-monophosphate into xanthine-5 '-monophosphate, which is then converted into guanosine-5' -monophosphate. The IMPDH2 gene is listed as DNA sequence: NC_000003.1 1 , NT_022517.18; NM_000884.2; and protein sequence NP_000875.2.

IMPDH2 is further described in RefseqGene record NG_012091.1 of NCBFs database.

[0039] The WSHC2 and IMDPH2 proteins that are useful in the present invention may also be variants of the wild-type protein. Unless otherwise indicated, the terms

"WSHC2" and "IMDPH2 ^" refer both to native proteins as well as variants thereof. As used herein, a WSI IC2 or IMDPH2 variant is a protein that comprises an amino acid sequence having one or more amino acid substitutions, deletions, and/or additions (such as internal additions and/or fusion proteins) as compared to the amino acid sequence of a native protein, but which nonetheless retains immunological activity. Such functionally or immunologically equivalent variants may occur as natural biological variations (e.g., polypeptide allelic variants, polypeptide orthologs, and polypeptide splice variants), or they may be prepared using known techniques, for example by chemical synthesis or modification, or mutagenesis (e.g., site-directed or random mutagenesis). Thus, for example, an amino acid may be replaced by another which preserves the physicochemical character of the WSHC2 or IMDPH2 protein or its epitope(s), e.g. in terms of charge density,

hydrophilicity/hydrophobicity, size and configuration and hence preserves the

immunological structure. "Addition" variants may include N- or C-terminal fusions as well as intrasequence insertions of single or multiple amino acids. Deletions may be

intrasequence or may be truncations from the N- or C-termini.

[0040] The disclosure also features a variety of WSHC2 and I .VI PD 112 proteins, polypeptides and chemical analogs that are suitable for use in the present invention as a WSCH or IMPDH2 antigen for the detection of WSHC2 and IMPDH2 specific

autoantibodies. In various exemplary aspects of the invention, the WSHC2 and IMPDH2 antigen comprise an amino acid sequence that is at least 65% (e.g., at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95% at least 97% or 100%) identical to WSUC2 or IMDPH2. In some embodiments, the antigens can consist of, or consist essentially of, an amino acid sequence that is at least 65% (e.g., at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95% at least 97% or 100%) identical to WSHC2 or I M DPI! 2.

[0041] Percent identity between two amino acid sequences can be determined using a variety of algorithms and computer programs including, but not limited to, Clustal W (The European Bioinformatics Institute (EMBL-EBI), BLAST-Protein (National Center for Biotechnology Information (NCBI), United States National Institutes of Health), and PSAlign (University of Texas A&M; Sze et al. (2006) Journal of Computational Biology 13:309-319).

[0042] The disclosure also features WSHC2 and IMDPH2 antigens comprising an amino acid sequence identical to the amino acid sequence of WSHC2 or IMDPH2, but with not more than 20 (e.g., not more than 19, not more than 18, not more than 17, not more than 16. not more than 15, not more than 14, not more than 13, not more than 12, not more than 1 1 , not more than 10, not more than nine, not more than eight, not more than seven, not more than six, not more than five, not more than four, not more than three, not more than two, or not more than 1 ) substitutions. The substitutions can be, e.g., conservative or non- conservative (as described below). In some embodiments, an antigen can consist of, or consist essentially of, an amino acid sequence identical to the amino acid sequence of WSHC2 or 1MDPH2. but with not more than 20 substitutions.

[0043] Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The non-polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non- conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

[0044] The WSHC2 and IMPDH2 proteins of the present invention may also comprise a protein derivative, which is a chemically or biologically modified protein, including protein post-translation modification, such as acylation (i.e., acetylation or formylation), biotinylation, carboxylation, deamination, glutathionylation, glycosylation, lipidation (i.e., farnesylation, gernylgeranylation, prenylation, myristoylation,

palmitoylation, or stearoylation), methylation, phosphorylation, sulphation, fucosylation, and ubiquitination. Unless otherwise indicated, the term " WSHC2 protein" and "IMPDH2 protein" refers both to native proteins, and variants and derivatives thereof. A WSHC2 or IMPDH2 derivative may be modified in a manner that is different in the type, number, or location of the post- translation modification groups naturally attached to the polypeptide. For example, a WSHC2 or IMPDH2 derivative may have the number and/or type of glycosylation altered compared to the native protein. The resulting derivative may therefore comprise a greater or a lesser number of N-linked glycosylation sites than the native protein.

[0045] The WSHC2 and IMPDH2 polypeptides may also be modified by the covalent attachment of one or more polymers. Typically, the polymer selected is water- soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer may be of any molecular weight and may be branched or unbranched. The polymer each typically has an average molecular weight of between about 1 kDa to about 100 kDa.

[0046] Suitable water-soluble polymers or mixtures thereof include, but are not limited to, polyalkylene glycol (such as mono-(Cl -C„) alkoxy-, aryloxy-polyethylene glycol, poly(N-vinyl pyrrol idone) polyethylene glycol, propylene glycol homopolymers, or polypropylene oxide/ethylene oxide copolymers), carbohydrate-based polymers (such as dextran or cellulose), polyoxyethylated polyols, and polyvinyl alcohols. Also encompassed by the present invention are bifunctional crosslinking molecules which can be used to prepare covalently attached WSHC2 and 1MPDI 12 polypeptide multimers.

[0047] In general, chemical derivatization may be performed under a suitable condition by reacting a protein with an activated polymer molecule. Methods for preparing chemical derivatives of polypeptides will generally comprise the steps of: (a) reacting the polypeptide with the activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions whereby a WSHC2 or IMPDH2 protein becomes attached to one or more polymer molecules, and (b) obtaining the reaction products. The optimal reaction conditions may vary depending upon the WSHC2 or IMPDH2 protein selected and chemical reagents used, and are generally determined experimentally. The PEGylation of a polypeptide may be carried out using any f the PEGylation reactions known in the art, including, but not limited to, acylation, alkylation, or Michael addition. [0048] As described in the Examples section further below, several new autoantibody biomarkers have been identified as useful for diagnosing, monitoring and/or assessing the status of HCC in a subject. In particular, it was discovered that these biomarkers are capable of identifying HCC with a predictive success of at least 28% or greater, either alone or in combination. Further, at least two of the biomarkers identified demonstrated both high specificity and no overlap in sera in which they were detected. The biomarkers of the present invention represent known genes, the sequences of which are available through public databases known to those of ski ll in the art. Furthermore, in the present invention, the recombinant WSHC2 and IMDPH2 proteins are produced using standard molecular biology protocols that are well known to those skilled in the art.

[0049] The Examples that follow below are intended to be illustrative of the methods used to detect autoantibodies specific to WSHC2 and IMPDH2. They are not meant to limit or construe the invention in any way.

Methods of Detection

[0050] Various systems and methods may be employed in detecting the HCC biomarkers disclosed herein. In one embodiment, a sample (e.g., whole blood, plasma or serum) is assayed for the presence of autoantibodies to WSHC2 and IMPDH2. In other words, a blood sample may be drawn, and a blood product, e.g., whole blood, plasma or serum, may be tested. Further, the autoantibodies may be detected using specific capture agents, e.g., antibody proteins. In certain embodiments, the method employed may provide a quantitative evaluation of the presence of one or more of the above-described autoantibodies to WSHC2 and IMPDH2 in a subject sample.

[0051 ] Various detection platforms may be employed in such methods, including antibody arrays, labeled bead assays, ELISA and RIA formats, binding of labeled antibodies in suspension/solution and detection by flow cytometry, mass spectroscopy, and the like. A variety of different assays can be utilized to quantitate HCC marker protein levels, including both methods that detect gene transcript and protein levels. Many of such methods are known to one of skill in the art, including ELISA, protein arrays, eTag system, bead based system, antibody-based systems, nucleic acid and/or small molecule systems, tag or other array based systems. Examples of such methods are set forth in the art, including, inter alia, chip-based capillary electrophoresis: Colyer et al. ( 1997) J Chromatogr A. 781 ( 1 -2): 271 -6 ; mass spectroscopy: Petricoin et al. (2002) Lancet 359: 572-77; eTag systems: Chan-Hui et al. (2004) Clinical Immunology 1 1 1 : 162-174; microparticle-enhanced nephelometric immunoassay: Monagne et al. (1992) Eur J Clin Chem Clin Biochem. 30(4):21722; and the like, each of which are herein incorporated by reference.,

[0052] There are many different types of immunoassays suitable for use in the present invention. Any of the well known immunoassays may be adapted to detect the level of WSHC2 and IMPDH2 specific autoantibodies in a sample which react with the WSHC or IMPDH2 antigen, such as, e.g., enzyme linked immunoabsorbent assay (ELISA), fluorescent immunosorbent assay (FIA), chemical linked immunosorbent assay (CLIA), radioimmuno assay (RIA), chemiluminescent immunoassay(CIA), immunoblotting, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and

Immunoelectrophoresis assays, etc. For a review of the different immunoassays which maybe used, see: The Immunoassay Handbook, David Wild, ed., 3rd edition, Stockton Press, New York, 2005. A competitive immunoassay with solid phase separation or an

immunometric assay for antibody testing is particularly suitable for use in the present invention. See, The Immunoassay Handbook, chapter 2.

[0053] In one exemplary embodiment of the invention, the diagnostic assay is an immunometric assay for detecting the level of WSHC2 and IMPDH2 specific autoantibodies in a sample. In the immunometric assay, a WSHC2 and/or 1MPDH2 antigen is immobilized on a solid support directly or indirectly through a capture agent, such as anti-WSHC2 and/or anti-IMPDH2 antibodies. An aliquot of a sample, such as a serum sample, from a subject is added to the solid support and allowed to incubate with the WSHC2 and/or IMPDH2 antigen on the solid phase. A secondary antibody that recognizes a constant region in the autoantibodies present in the sample which have reacted with the WSHC2 and/or IMPDH2 antigen is added. When the subject is a human, this secondary antibody is an anti-human immunoglobulin. The secondary antibody which is specific for IgA, IgG, or IgM heavy chain constant regions may be employed. After separating the solid support from the liquid phase, the support phase is examined for a detectable signal. The presence of the signal on the solid support indicates that autoantibodies to the native WSHC2 and/or IMPDH2 protein present in the sample have bound to the WSHC2 and/or IMPDH2 antigen on the solid support. Increased optical density or radiolabeled signal when compared to the control samples from normal subjects correlates with a diagnosis of HCC in a subject.

[0054] Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray (e.g., microtiter plates), test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, glass or silicon chips, sheep (or other animal) red blood cells, duracytes and others. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional molecule which has the ability to attract and immobilize the capture reagent. The additional molecule can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the WSHC2 and/or 1MPDH2 antigen through a specific binding reaction. The molecule enables the indirect binding of the WSHC2 and/or IMPDH2 antigen to a solid support material before the performance of the assay or during the performance of the assay.

[0055] The signal producing system is made up of one or more components, at least one of which is a label, which generate a detectable signal that relates to the amount of bound and/or unbound label, i.e., the amount of label bound or unbound to the WSHC2 and/or IMPDH2 antigen. The label is a molecule that produces or which may be induced to produce a signal. Examples of labels include fluorescers, enzymes, chemiluminescers, photosensitizers or suspendable particles. The signal is detected and may be measured by detecting enzyme activity, luminescence or light absorbance. Radiolabels may also be used and levels of radioactivity detected and measured using a scintillation counter.

[0056] Examples of enzymes which may be used to label the anti-human immunoglobulin include β-D-galactosidase, horseradish peroxidase, alkaline phosphatase, and glucose-6-phosphate dehydrogenase ("G6PDH")- Examples of fluorescers which may be used to label the anti-human immunoglobul in include fluorescein, isothiocyanate, rhodamines, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, and Alexa Fluor® dyes (that is, sulfonated courmarin, rhodamine, xanthene, and cyanine dyes). Chemiluminescers include e.g., isoluminol. For example, the anti-human

immunoglobulin may be enzyme labeled with either horseradish peroxidase or alkaline phosphatase.

[0057] Enzymes may be covalently linked to WSHC2 and/or IMPDH2 antigen reactive antibodies for use in the methods of the present invention using well known methods. There are many well known conjugation methods. For example, alkaline phosphatase and horseradish peroxidase may be conjugated to antibodies using

glutaraldehyde. Horseradish peroxidase may also be conjugated using the periodate method. Commercial kits for enzyme conjugating antibodies are widely available. Enzyme conjugated anti-human and anti-mouse immunoglobulin specific antibodies are available from multiple commercial sources.

[0058] Biotin labeled antibodies may be used as an alternative to enzyme linked antibodies. In such cases, bound antibody would be detected using commercially available streptavidin horseradish peroxidase detection systems.

[0059] Enzyme labeled antibodies produce different signal sources, depending on the substrate. Signal generation involves the addition of substrate to the reaction mixture. Common peroxidase substrates include A B I S (2,2'-azinobis (ethylbenzothiazoline-6- sulfonate)), OPD (0-phenylenediamine) and TMB (3,3', 5,5'-tetramethylbenzidine). These substrates require the presence of hydrogen peroxide. p-Nitrophenyl phospate is a commonly used alkaline phosphatase substrate. During an incubation period, the enzyme gradually converts a proportion of the substrate to its end product. At the end of the incubation period, a stopping reagent is added which stops enzyme activity. Signal strength is determined by measuring optical density, usually via spectrophotometer.

[0060] Alkaline phosphatase labeled antibodies may also be measured by fluorometry. Thus in the immunoassays of the present invention, the substrate 4- methylumbelliferyl phosphate (4-UMP) may be used. Alkaline phosphatase dephos- phorylated 4-UMP to form 4-melhy 1 u mbe 11 i ferone (4-MU), the fluorophore. Incident light is at 365 nm and emitted light is at 448 nm. [0061] The amount of color, fluorescence, luminescence, or radioactivity present in the reaction (depending on the signal producing system used) is proportionate to the amount of autoantibodies in a sample which react with the WSHC2 and/or IMPDH2 antigen. Quantification of optical density may be performed using spectrophotometric or

fluorometric methods, including flow cytometers. Quantification of radiolabel signal may be performed using scintillation counting.

[0062] In another exemplary embodiment, the assay is a competitive

immunoassay, which employs one or more WSHC2 and/or IMPDH2 specific antibodies that binds to the same epitopes as the WSHC2 and/or IMP H2 specific autoantibodies. In the assay, the WSHC2 and/or IMPDH2 specific antibodies and the WSHC2 and/or IMPDH2 specific autoantibodies in a sample compete for binding to the WS1IC2 and/or IMPDH2 antigen. Typically, a constant amount of a labeled antibody which is known to bind to WSHC2 and/or IMPDH2 antigen is incubated with different concentrations of a sample from a subject. The WSHC2 and/or IMPDH2 specifc antibodies may be monoclonal or polyclonal.

[0063] As described herein above, the WSHC2 and/or IMPDH2 specific antibody may be labeled with a fluorescer, enzyme, chemiluminescer, photosensitizer, suspendable particles, or radioisotope. After incubation, bound labeled antibodies are separated from free antibodies. Depending on the signal producing system used and if necessary, an appropriate substrate with which the labeled antibody reacts is added and allowed to incubate. The signal generated by the sample is then measured. A decrease in optical density or radioactivity from before and after addition of the serum sample or between experimental and control samples, is indicative that autoantibodies in the sample have bound to the WSHC2 and/or IMPDH2 antigen. Decreased optical density or radiolabeled signal when compared to control samples from normal subjects correlates with a diagnosis or indication of a likelihood of HCC in a subject.

[0064] In an alternative exemplary embodiment of the competitive immunoassay, an indirect method using two antibodies is provided. The first antibody is a WSHC2 and/or IMPDH2 antigen specific antibody as described in the preceding paragraph with the exception that it is not labeled. The first antibody is incubated with different concentrations of a sample from a subject. A constant amount of a second antibody is then added to the mixture of the sample and the first antibody. The second antibody recognizes constant regions of the heavy chains of the first antibody. For example, the second antibody may be an antibody which recognizes constant regions of the heavy chains of mouse immunoglobulin which has reacted with the WSHC2 and/or IMPDH2 antigen (anti-mouse immunoglobulin). The second antibody may be labeled with a fluorophore, chemilophore or radioisotope, as described above. Free labeled second antibody is separated from bound antibody. If an enzyme-labeled antibody is used, an appropriate substrate with which the enzyme label reacts is added and allowed to incubate. A decrease in optical density or radioactivity from before and after addition of the serum sample in comparison with control samples is indicative that autoantibodies in the serum sample have bound to the WSHC2 and/or IMPDH2 antigen. Decreased optical density or radioactivity when compared to control samples from normal subject correlates with a diagnosis or indication of likelihood of HCC in a subject.

[0065] In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in U.S. Pat. Nos. 5,885,530, 4,981 , 785, 6, 159,750, and 5,358,691 , each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a prognosis based on the presence or absence of a series of proteins corresponding to autoimmune or chronic inflammatory disease markers is utilized. In some embodiments, the WSHC2 and/or IMPDH2 specific autoantibody level may be used together with other biological markers as a panel for HCC diagnosis. The panel allows for the simultaneous analysis of multiple markers correlating with liver disease, including HCC. For example, a panel may include markers identified as correlating with HBV. HCV or cirrhosis in a subject that is/are likely or not likely to respond to a given treatment. Depending on the subject, panels may be analyzed alone or in combination in order to provide the best possible diagnosis and prognosis. Markers for inclusion on a panel are selected by screening for their predictive value using any suitable method, including but not limited to, those described in the illustrative examples below.

[0066] Since the diagnostic assay of the present invention is used for autoantibody detections, it should be understood that the antibodies can be directed to any WSHC2 and/or IMPDH2 epitope(s). Accordingly, there are no need to characterize any specific WSHC2 and/or IMPDH2 epitope to practice the present invention. Likewise, the autoantibodies may be of any variety as described. Data Analysis

[0067] In the present invention, a computer-based analysis program may also be used to translate the raw data generated by the detection assay into data of predictive value for a clinician. The clinician can readily access the predictive data using any suitable means. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

[0068] The present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information provides, medical personal, and subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy or a serum or urine sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication system). Once received by the profiling service, the sample is processed and a profile is produced, specific for the diagnostic or prognostic information desired for the subject.

[0069] The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of a liver disease such as HCC to respond to a specific therapy) for the subject, along with recommendations for particular treatment options. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor. [0070] In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

[007 1 ] In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or severity of disease

Panel Assays

[0072] In addition to measuring the presence of WSCH2 and IMPDH2 specific autoantibodies either alone or in combination, the present invention also contemplates "panel assays" measuring WSCH2 and IMPDH2 autoantibodies along with one or more other analytes. Such panel assays may be provided as a kit containing two or more separate assay platforms or they may be combined on a single platform such as an immunoassay strip or ELISA plate

Kits

[0073] Finally, kits for use in practicing the subject invention are also provided. The subject kits at least include an HCC evaluation system as discussed above. For example, certain embodiments of the subject invention include kits that have a plurality of capture agents for HCC biomarkers attached to the surface of a solid support. Further, a kit may comprise the computer-readable medium described above.

[0074] The kits may further include one or more additional components necessary for carrying out a binding assay, such as binding buffers, and the like. As such, the kits may include one or more containers such as vials or bottles, with each container containing a separate component for the assay, and reagents for carrying out an array assay such as a nucleic acid hybridization assay or the like. The kits may also include a reagent for preparing the sample, and a syringe for drawing blood from a subject, as well as suitable negative and positive controls.

[0075] In addition to a system, a subject kit may also include written instructions for use in the above-described method. The instructions may be printed on a substrate, such as paper or plastic. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging). In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, etc. The instructions may include software or algorithms for data visualization or data analysis as described above. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the Internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0076] The subject kit may also include a computer-readable medium containing the above-described instructions, or means for accessing such instructions such as means for obtaining the algorithms from a remote source, e.g. via the Internet.

[0077] In many embodiments of the subject kits, the components of the kit are packaged in a kit containment element to make a single, easily handled unit, where the kit containment element, e.g., box or analogous structure, may or may not be an airtight container, e.g., to further preserve the one or more biopolymeric arrays and reagents, if present, until use.

EXAMPLES

Example 1

Screening HCC Biomarker Candidates

[0078] Commercially available protein microarrays containing approximately 8000 human recombinant proteins (Human Protoarray® v4.0, Invitrogen, Carlsbad, CA) were obtained for screening purposes. A total of 86 sera samples from 49 subjects with HCC, 5 sera samples from subjects with inflammatory bowel disease (IBD), 6 sera samples from subjects with autoimmune hepatitis (AIH), 6 sera samples from subjects with primary sclerosing cholangitis (PSC), and 20 sera samples from asymptomatic healthy donors (ProMedDx LLC, Norton, MA) were individually screened on the microarrays according to the manufacturer's instructions. A total of 90 microarrays were utilized for the experiments.

[0079] Binding of autoantibodies to the microarrays was assessed by scanning the microarrays with a fluorescent microarray scanner. Image analysis of the microarrays and the derived data was obtained with GenePix Pro v6.1 data acquisition software (Molecular Devices Corp., Sunnyvale, CA). Data obtained from the microarrays was analyzed using the ProtoArray® Prospector v5.1 software (Invitrogen, Carlsbad, CA) and Immune Response Profiling (IRP) program. Standard techniques including M-statistics, Z-scores, were employed to detect differences between samples from subjects with HCC compared to samples from subjects without HCC.

[0080] Materials:

1) INVITROGEN Proteo Array Prospector Version 5.1

2) Three sets of sera were evaluated:

a) HCC Set 1 - Pre-validalion Set, N= 13

a. Specific sera ID: mix of various HCC sera from 4 different sources b) HCC Set 2, N= 10

a. Specific sera ID: Cape Cod (commercial vendor)

c) HCC Set 3, N= 85

a. Specific sera ID: 83 Japanese sera (all advanced HCC disease, prior to liver resection ), 2 Italian

d) HCC Set 4, N=51

a. Specific sera ID: from subjects with HCC [0081 ] Results: A list of eleven candidate proteins, or autoantigens, was developed based on the composite analysis of the array results with the various disease sera. The 1 1 candidate proteins were as follows: IMPDH2; CINP; WHSC2; GMNN; GSDMD; SEPT 9; REL A; TCAP; TYR03 EXT; TYR03 ΓΝΤ; NEK1 . [IMPDH2= inosine 5 ^* - monophosphate dehydrogenase 2 , CINP= cyclin-dependent kinase 2 interacting protein , WHSC2= Wolf-Hirschhorn syndrome candidate 2 , GMN = geminin, DNA replication inhibitor, GSDMD= gasdermin D , SEPT9= septin 9 , REL A= reticuloendotheliosis viral oncogene homolog A (avian), TCAP= titin-eap (telethonin), TYRC)3-EXT= tyrosine kinase 3, TYR()3-INT= tyrosine kinase 3, NEK 1 = NEver in mitosis gene a-related Kinase 1]

[0082] Full-length open reading frames (ORFs) of candidate autoantigens were cloned into a modified pETBIue-2 (EMD Biosciences, Inc. San Diego,CA) or a modified pCR®-Blunt II-TOPO® (Invitrogen, Carlsbad, CA) vector compatible with cell-free protein expression and containing the T7 TNA polymerase promoter, a Kozak (ribosome binding) sequence, a start codon, an N-terminal VSV-G epitope tag as well as a C-terminal HSV and polyhistidine epitope tag. Using a cell-free transcription/translation coupled rabbit reticulocyte lysate system, candidate autoantigens were expressed from the constructed clones. Next a cell-free ELISA system, termed the T2-ELISA (AmberGen Inc., Watertown, MA), which is based on the use of dual-epitope tagged cell-free expressed protein antigens, was used to screen the 1 1 candidate autoantigens (see WO 2007/002106; Lini et al., Analytical Biochem. 383( 1 ), Dec. 2008, 103- 1 15; Lim et al., Breast Cancer Res 2010 12:R78). In this system, a mouse anti-HSV antibody is first coated on to polystyrene microtiter plates. After appropriate washing and blocking, cell-free reaction solutions were added and incubated for 30 minutes. Following washing, diluted sera ( 1 : 1000) were added, incubated for 30 minutes, washed, and bound autoantibodies detected by addition of mouse anti -human IgG-horseradish peroxidase conjugate and incubation for an additional 30 minutes. After removal of conjugate and washing, 50 ul of chemiluminescent substrate was added, incubated for 15 minutes, and luminosity read on a luminescence plate reader.

[0083] The initial eleven candidate autoantigens were screened using the system described above. A second round of screening narrowed the candidates to three proteins (WHSC2, IMPDH2, and GMNN). A final round of screening narrowed the most promising candidate antigens to two proteins (WHSC2 and IM PDH2 ), which gave a signal on HCC sera of at least four standard deviations greater than the signal obtained on non-HCC sera. [0084] Of the total of 95 sera tested, a total of 7 sera (7/95 = 7.3%) were interpreted as positive using a cutoff set to achieve 100% specificity. As shown in the table below, four sera samples were detected by WHSC2, and three sera samples were detected by IMPDH2. There was no overlap of positives, as evidenced in Figure 1 and Table 1 below.

Table 1

Detection of WHSC2 and IMPDH2 in Subjects with HCC and Healthy Individuals

Detection of WHSC2 and IMPDH2 Autoantibodies by ELISA

[0085] For WHSC2- and IMPDH2-specific autoantibody determination, polystyrene microliter plates were coated with either WHSC2 or IMPDH2 antigen at a concentration of 1 ug/ml incubated overnight at 5° C. Plates were washed with ELISA wash buffer and 180ul of ELISA Blocking buffer was added. Plates were blocked for 1 hour and then washed with ELISA wash buffer. Plates then were dried overnight.

[0086] Autoantibody was detected by adding sera diluted 1 : 10 in ELISA dilution buffer. Following a 30 minute incubation, the plates were washed, and goat anti-human IgG-horseradish peroxidase conjugate was added. Following an additional 30 minute incubation, the plates were washed, and TMB substrate was added for 30 minutes. The reaction was stopped by adding ELISA stop buffer and the resulting color was read on a spectrophotometer at 495 nm. The results of the reading are summarized in Table 2 below and depicted in Figures 3 and 4. Table 2

[0087] The data obtained as shown in Examples 1 and 2 demonstrate that the two proteins, WHSC2 and IMPDH2. are candidate biomarkers for HCC. The experiments showed that combining the results of testing for both antigens gave a low sensitivity (7%), but 100% specificity. The second series of experiments using a new cohort of sera from subjects with HCC, showed 45.5% (10/22) of HCC specimens were interpreted as positive for W1ISC2 autoantibodies, compared to 0% of healthy controls (0/2), and 9.8% (5/51) of HCC specimens were interpreted as positive for IMPDH2 autoantibodies, compared to 0% of healthy controls (0/2).

Example 3

Detection of WHSC2 and IMPDH2 Autoantibodies by ELISA

[0088] An additional set of experiments was performed in which the 57 HCC specimens and 12 healthy controls were tested for WHSC2 and IMPDH antibodies using the same experimental methods. Using a cutoff set to the mean of the normals plus 3 standard deviations, 1 1/57 (19.2%) of the HCC specimens and 0 of the healthy controls were positive for WHSC2. Using a cutoff set to the mean of the normals plus 3 standard deviations, 7/57 (12%) of the HCC specimens and 0 of the healthy controls were positive for IMPDH2. Combining the results of the two assays resulted in 16/57 (28%) of the HCC and 0 of the healthy controls positive. These results are summarized in Table 3 below and depicted in Figure 5. Table 3

30 HCC 0.055 0.1 12

31 HCC 0.75 0.1 12

32 HCC 0.06 0.123

33 HCC 1.313 0.125

34 HCC 0.062 0.126

35 HCC 0.183 0.127

36 HCC 0.244 0.129

37 HCC 0.188 0.13

38 HCC 0.147 0.134

39 HCC 0.513 0.134

40 HCC 0.524 0.135

41 HCC 0.209 0.138

42 HCC 0.129 0.148

43 HCC 0.524 0.182

44 HCC 0.12 0.187

45 HCC 1.279 0.199

46 HCC 0.225 0.201

47 HCC 3.376 0.201

48 HCC 0.201 0.233

49 HCC 0.378 0.237

50 HCC 3.671 0.281

51 HCC 0.241 0.297

52 HCC 2.956 0.313

53 HCC 0.733 0.354

54 HCC 2.092 0.432

55 HCC 0.277 0.452

56 HCC 0.616 0.869

57 HCC 0.104 1.607

58 Healthy 0.049 0.037 control

59 Healthy 0.345 0.049 control

60 Healthy 0.1 12 0.052 control

61 Healthy 0.19 0.055

control

62 Healthy 0.605 0.055

control

63 Healthy 0.555 0.058

control

64 Healthy n/a 0.069

control

65 Healthy 0.132 0.079

control

66 Healthy 0.453 0.123

control

67 Healthy n/a 0.141

control

68 Healthy n/a 0.196

control

69 Healthy 0.288 0.197

control

[0089] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use embodiments of the compositions, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes (for carrying out the invention that are obvious to persons of skill in the art) are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.