WO2007053659A2 - Method of screening for hepatocellular carcinoma - Google Patents

Abstract

A method of screening for hepatocellular carcinoma (HCC) comprising detecting in a sample the expression of three or more HCC biomarkers selected from the group consisting of Gpc3, Mdk, Serpinl1, PeglO, and Qp-c, or detecting in a sample the expression of Serpinl1 or Qp-c, as well as a method for monitoring the progression or regression of HCC in a subject, a method for screening a compound for anti-HCC activity, and an array and kit useful for detecting HCC.

Description

METHOD OF SCREENING FOR HEPATOCELLULAR CARCINOMA

BACKGROUND OF THE INVENTION

[0001] Hepatocellular carcinoma (HCC), endemic in Asia and Africa with a rising incidence in the western countries, is one of the most common and aggressive cancers worldwide. It has been the third most deadly cancer in the world and the second most deadly cancer in China since the 1990s. Globally, the five year survival rate of HCC is less than 5% and approximately 598,000 HCC patients die each year. The high mortality is mainly attributed to the inability to reliably diagnose HCC patients at an early stage. In fact, most of the symptomatic HCC patients are diagnosed at an advanced stage, thus precluding their chance for surgical intervention. In contrast, studies have shown that HCC patients who are diagnosed at an early stage and receive curative resection have a significantly improved survival time. Thus, early detection and resection have been generally recognized as the most important factors to achieve long term survival for HCC patients.

[0002] Since its detection in the serum of HCC patients in 1970s, alpha-fetoprotein

(AFP) has been the only reliable serological biomarker for HCC. AFP levels are commonly used in conjunction with ultrasonography to diagnose HCC patients. However, elevated serum AFP is observed only in about 60-70% of HCC patients and to a lesser extent (33- 65%) in patients with small HCC tumors. Moreover, nonspecific elevation of serum AFP has been found in 15-58% of patients with chronic hepatitis and 11-47% patients with liver cirrhosis. Thus, the use of AFP as a serum biomarker for HCC allows the identification of only a small subset of HCC patients at an early stage.

[0003] There remains a need for new methods of screening for HCC, especially methods that can detect AFP-negative and small-tumor HCC cases.

BRIEF SUMMARY OF THE INVENTION

[0004] The invention provides a method of screening for hepatocellular carcinoma (HCC) comprising detecting in a sample the expression of three or more HCC biomarkers selected from the group consisting of Gpc3, Mdk, Serpinll, Peg 10, and Qp-c. The invention also provides a method of screening for HCC comprising detecting in a sample the expression of Serpinll or Qp-c. [0005] The invention further provides a method of monitoring the progression or regression of HCC in a subject comprising (a) measuring the expression level of three or more HCC biomarkers in a first sample obtained from the subject at a first point in time, (b) measuring the expression level of three or more HCC biomarkers in a second sample obtained from the subject at a second point in time, and (c) comparing the expression levels of the HCC biomarkers of the first and second samples, wherein the HCC biomarkers are selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c. The invention additionally provides a method of monitoring the progression or regression of HCC in a subject, comprising (a) measuring the expression level of Qp-c or Serpinll in a first sample obtained from the subject at a first point in time, (b) measuring the expression level of Qp-c or Serpinll in a second sample obtained from the subject at a second point in time, and (c) comparing the expression levels of Qp-c or Serpinll of the first and second samples. [0006] The invention provides a method of screening a compound for anti-HCC activity comprising (a) contacting an HCC cell with a test compound, (b) measuring the expression level of three or more of the HCC biomarkers selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c in the HCC cell, and (c) comparing the expression level of the HCC biomarkers with a control. In a related aspect, the method of screening a compound comprises (a) contacting an HCC cell with a test compound, (b) measuring the expression level of Serpinll or Qp-c in the HCC cell, and (c) comparing the expression level of the HCC biomarkers with a control.

[0007] An array also is provided by the invention, which comprises (a) a substrate and (b) three or more different addressable elements that each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C. m a related aspect, the array comprises (a) a substrate and (b) two or more different addressable elements that each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, wherein at least one of the addressable elements comprises a polynucleotide that binds to the mRNA transcript of Qp-c or Serpinll, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C, wherein at least one of the addressable elements comprises a polypeptide that binds to QP-C or SERPINIl. IH' IU Ii / iι,..ιι ai . i i:::: :::o ,.. ,

[0008] A kit suitable for detecting HCC also is provided herein. The kit comprises three or more different probes that each comprise (a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, or (b) a polypeptide that binds to a polypeptide selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C. In a related aspect, the kit comprises two or more different probes that each comprise (a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp- c, wherein at least one of the probes comprises a polynucleotide that binds to the mRNA transcript of Qp-c or Serpinll, or (b) a polypeptide that binds to a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C, wherein at least one of the probes comprises a polypeptide that binds to QP-C or SERPINIl .

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0009] Figure 1 is a graph of the fold-increase in mRNA levels of each biomarker plotted against test samples categorized as AFP- tumor samples, AFP+ tumor samples, and non- tumor samples. Each data point represents an individual case (average of triplicate measurements +/- SD). Circular data points represent HCC tumor samples, triangular data points represent HCC non-tumor samples, and square data points represent samples from cirrhosis patients. Solid data points correspond to cases that were used both in the present analysis and in prior microarray studies used to discover the biomarkers of interest, whereas open data points correspond to cases that were used only in the present analysis. [0010] Figure 2 is a graph illustrating the relative increase in mRNA levels of Gpc3, Mdk, Serpinll, PeglO, and Qp-c in AFP-high (>300 ng/ml) and AFP-normal (<20 ng/ml) HCCs, as determined by qRT-PCR. The levels are expressed as fold-change after normalization to 18S ribosomal gene and then to a pool of 7 disease-free normal liver samples. Median values are indicated. Nonparametric Mann-Witney test was used to calculate the p values.

[0011] Figure 3 is a graph of the probability of correct classification with 10-fold cross validation (CV) of each sample on the basis of the five-gene set according to the PAM algorithm plotted against the individual samples classified. N refers to classification as non- tumor sample, and T refers to classification as tumor sample. In the graph, squares represent individual HCC samples, and diamonds represent individual non-tumor cases. [0012] Figures 4A-D further illustrate the classification of HCC samples on the basis of Gpc3, Mdk, Serpinll, PeglO, and Qp-c biomarkers by PAM analysis. Figure 4 A is a graph of the misclassification error rate based on 10-fold crossvalidation plotted against the number of biomarkers used in the classification. Figure 4B is an illustration of the shrunken centroid classification for the five-gene dataset, wherein "N" is non-tumor samples and "T" is tumor samples. Figure 4C is a graph of the misclassification error rate for individual classes (non- tumor and tumor) plotted against the number of biomarkers used in the classification, wherein "N" is non-tumor samples and "T" is tumor samples. Figure 4D is a graph of the probability of correct classification with 10-fold cross validation (CV) of each sample on the basis of the five-gene set according to the PAM algorithm plotted against the individual samples classified. N refers to classification as non-tumor sample, and T refers to classification as tumor sample. In the graph, squares represent individual HCC samples, and diamonds represent individual non-tumor cases.

[0013] Figures 5A-D illustrate serum midkine (MDK) levels and serum alpha-fetoprotein (AFP) levels in HCC patients. Figure 5A is a graph of serum MDK levels in healthy volunteers (n=26) and HCC patients (n=65) stratified by serum AFP and tumor size. Figure 5B is a graph of serum AFP levels as a function of median tumor size. Figure 5C is a graph of serum MDK levels as a function of median tumor size. Figure 5D is a graph of the relative serum MDK levels of hospital controls (n=20), cirrhosis (n=49), and HCC patients (n=32) stratified by serum AFP or Child-Pugh score.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The term "Gpc3" as used herein refers to a gene that encodes the glypican-3 protein (GPC3) and encompasses human Gpc3 as well as orthologs and naturally occurring variants thereof. Similarly, the term "glypican-3" or "GPC3" as used herein refers to the glypican-3 protein and encompasses human GPC3 protein as well as orthologs and naturally occurring variants thereof. Human Gpc3 is referenced by GenBank Accession no. NM_002391. The nucleic acid sequence of the coding region (e.g., mRNA) of human Gpc3 is provided herein as SEQ ID NO: 1. Human GPC3 is referenced by GenBank Accession no. NP_002382. The amino acid sequence of human GPC3 is provided herein as SEQ ID NO: 2. Orthologs of human Gpc3 (GPC3) are known in the art.

[0015] The term "Mdk" as used herein refers to a gene that encodes midkine (MDK) (also named neurite growth promoting factor 2) and encompasses human Mdk as well as orthologs and naturally occurring variants thereof. Similarly, the term "midkine" or "MDK" as used herein refers to the midkine protein and encompasses human MDK protein as well as orthologs and naturally occurring variants thereof. Human Mdk is referenced by GenBank Accession No. NM_002391. The nucleic acid sequence of the coding region (e.g., mRNA) of human Mdk is provided herein as SEQ ID NO: 3. Human MDK is referenced by GenBank Accession no. NP_002382. The amino acid sequence of human MDK is provided herein as SEQ ID NO: 4. Orthologs of human Mdk (MDK) are known in the art. [0016] The term "PeglO" as used herein refers to a gene that encodes paternally expressed 10 protein (PEGlO) and encompasses human PeglO as well as orthologs and naturally occurring variants thereof. Similarly, the term "paternally expressed 10" or "PEGlO" as used herein refers to the paternally expressed 10 protein and encompasses human PEGlO protein as well as orthologs and naturally occurring variants thereof. Human PeglO is referenced by GenBank Accession no. XM_496907. The nucleic acid sequence of the coding region (e.g., mRNA) of human PeglO is provided herein as SEQ ID NO: 5. Human PEGlO is referenced by GenBank Accession no. XP_496907. The amino acid sequence of PEGlO is provided herein as SEQ ID NO: 6. Orthologs of human PeglO (PEG 10) are known in the art.

[0017] The term "Serpinll" as used herein refers to a gene that encodes serpin peptidase inhibitor, clade I, member 1 (SERPINIl) (also named serine/cysteine proteinase inhibitor 12 and neuroserpin member 1) and encompasses human Serpinll as well as orthologs and naturally occurring variants thereof. Similarly, the term "serpin peptidase inhibitor, clade I, member 1" or "SERPINH" as used herein refers to the serpin peptidase inhibitor, clade I, member 1 protein and encompasses human SERPINIl protein as well as orthologs and naturally occurring variants thereof. Human Serpinll is referenced by GenBank Accession no. NM_005025. The nucleic acid sequence of the coding region (e.g., mRNA) of human Serpinll is provided herein as SEQ ID NO: 7. Human SERPINIl is referenced by GenBank Accession no. NP_005016. The amino acid sequence of human SERPINIl is provided herein as SEQ ID. NO: 8. Orthologs of human Serpinll (SERPINIl) are known in the art. [0018] The term "Qp-c" as used herein refers to a gene that encodes low molecular mass ubiquinone-binding protein (9.5kD) (QP-C) and encompasses human Qp-c as well as orthologs and naturally occuring variants thereof. Similarly, the term "low molecular mass ubiquinone-binding protein" or "QP-C" as used herein refers to the low molecular mass ubiquinone-binding protein and encompasses human QP-C protein as well as orthologs and naturally occurring variants thereof. Human Qp-c is referenced by GenBank Accession no. NM_014402. The nucleic acid sequence of the coding region (e.g., mRNA) of human Qp-c is provided herein as SEQ ID NO: 9. Human QP-C is referenced by GenBank Accession no. NP_055217. The amino acid sequence of human QP-C is provided herein as SEQ ID NO: 10. Orthologs of human Qp-c (QP-C) are known in the art.

[0019] The invention provides a method of screening for HCC comprising detecting in a sample the expression of three or more HCC biomarkers selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c. Desirably, the three or more HCC biomarkers comprise Gpc3, Mdk, and PeglO. While the method of the invention can be practiced by detecting the expression of three or more of the above HCC biomarkers in a sample, the detection of more than three of the above HCC biomarkers can increase the sensitivity or accuracy of the screen. Thus, it is preferred that the expression of four or more of the above HCC biomarkers are detected. More preferably, the method comprises detecting the expression of all five of the above HCC biomarkers.

[0020] In a related aspect, the invention provides a method of screening for HCC comprising detecting the expression of Serpinll or Qp-c. Preferably, the method comprises detecting Serpinll and Qp-c, desirably in combination with the detection of one or more, two or more, or all three of the other HCC biomarkers described above. [0021] Overexpression of one or more of the HCC biomarkers is indicative of HCC, while normal or below-normal expression levels of the biomarkers suggests that no HCC is present. Overexpression of the HCC biomarkers can be determined by comparing the expression of the biomarkers in a test sample with a control (e.g., a positive or negative control). A control can be provided, for example, by measuring the expression of the same biomarkers in a sample known to be HCC negative (negative control) or known to be HCC positive (positive control), or by a previously determined standard for the biomarkers prepared by any suitable method (e.g., an expression profile of biomarkers generated from a pool of HCC negative (normal) samples or a pool of HCC positive samples). When comparing the expression of the biomarkers to a negative control, overexpression in the sample can be defined as any level of expression greater than the level of expression of the control (e.g., 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or even greater expression as compared to the negative control).

[0022] The sample, as referred to herein, can be any suitable sample. Suitable samples include samples from a subject or host. The sample can be a liquid or fluid sample, such as a _{, 11}

sample of body fluid (e.g., blood, plasma, interstitial fluid, bile, lymph, milk, semen, saliva, urine, etc.), or a solid sample, such as a tissue sample (e.g., liver tissue or tumor tissue sample), which can be processed prior to use. A sample also may include a cell or cell line created under experimental conditions, which is not directly isolated from a subject or host, or a product produced in cell culture by normal, non-tumor, or transformed cells (e.g., via recombinant DNA technology).

[0023] As used herein, a subject or a host may be an animal, such as a mammal including a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject can be suspected of having HCC, diagnosed with HCC, of an unknown HCC status, or a control subject that is confirmed not to have HCC. Diagnostic methods for HCC and the clinical delineation of HCC diagnoses are known to those of ordinary skill in the art. [0024] The term "detect" as used herein with respect to the expression of HCC biomarkers means to determine the presence or absence of detectable expression of a biomarker. Thus, detection encompasses, but is not limited to, measuring or quantifying the expression level of a biomarker by any method. Preferably, the method involves detecting or measuring the expression of the biomarkers in such as way as to facilitate the comparison of expression levels between samples.

[0025] The expression of HCC biomarkers can be detected or measured by any suitable method. For example, the HCC biomarkers can be detected or measured on the basis of mRNA or protein levels. Suitable methods of detecting or measuring mRNA include, for example, Northern Blotting, reverse-transcription PCR (RT-PCR), and real-time RT-PCR. Such methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989. Of these methods, realtime RT-PCR is used. In real-time PCR, which is described in Bustin, J. MoI. Endocrinology 25: 169-193 (2000), PCRs are carried out in the presence of a labled (e.g., fluorogenic) oligonucleotide probe that hybridizes to the amplicons. The probes can be double-labeled, for example, with a reporter fluorochrome and a quencher fluorochrome. When the probe anneals to the complementary sequence of the amplicon during PCR, the Taq polymerase, which possesses 5' nuclease activity, cleaves the probe such that the quencher fluorochrome is displaced from the reporter fluorochrome, thereby allowing the latter to emit fluorescence. The resulting increase in emission, which is directly proportional to the level of amplicons, is monitored by a spectrophotometer. The cycle of amplification at which a particular level of fluorescence is detected by the spectrophotometer is called the threshold cycle, C_T- It is this value that is used to compare levels of amplicons. Probes suitable for detecting mRNA levels of the HCC biomarkers are commercially available and/or can be prepared by routine methods, such as methods discussed elsewhere herein.

[0026] Suitable methods of detecting protein levels in a sample include Western Blotting, radioimmunoassay, and Enzyme-Linked Immunosorbent Assay (ELISA). Such methods are described inNakamura et al., Handbook of Experimental Immunology, 4^th ed., WoI. 1, Chapter 27, Blackwell Scientific Publ., Oxford, 1987. When detecting proteins in a sample ■ using an immunoassay, the sample is typically contacted with antibodies or antibody fragments (e.g., F(ab)₂' fragments, single chain antibody variable region fragment (ScFv) chains, and the like) that specifically bind the target protein (e.g., the HCC biomarker protein). Antibodies and other polypeptides suitable for detecting the HCC biomarkers in conjunction with immunoassays are commercially available and/or can be prepared by routine methods, such as methods discussed elsewhere herein (e.g., Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Publishers, Cold Spring Harbor, NY, 1988). [0027] The immune complexes formed upon incubating the sample with the antibody are subsequently detected by any suitable method. In general, the detection of immune complexes is well-known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art. U.S. Patents concerning the use of such labels include U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. [0028] For example, the antibody used to form the immune complexes can, itself, be linked to a detectable label, thereby allowing the presence of or the amount of the primary immune complexes to be determined. Alternatively, the first added component that becomes bound within the primary immune complexes can be detected by means of a second binding ligand that has binding affinity for the first antibody. In these cases, the second binding ligand is, itself, often an antibody, which can be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected. IK iι,,,,. H ," iι,,,ιι αι- . :;

[0029] Other methods include the detection of primary immune complexes by a two- step approach. A second binding ligand, such as an antibody, that has binding affinity for the first antibody can be used to form secondary immune complexes, as described above. After washing, the secondary immune complexes can be contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. A number of other assays are contemplated; however, the invention is not limited as to which method is used. [0030] The method of screening for HCC can be used for any purpose. For example, the method of screening for HCC can be used to assist in the detection or diagnosis of HCC, or in conjunction with a method of monitoring the progression or regression of disease in a subject. In this respect, the invention provides a method of monitoring the progression or regression of HCC in a subject, comprising (a) measuring the expression level of three or more HCC biomarkers in a first sample obtained from the subject at a first point in time, (b) measuring the expression level of three or more HCC biomarkers in a second sample obtained from the subject at a second point in time, and (c) comparing the expression levels of the HCC biomarkers of the first and second samples, wherein the HCC biomarkers are selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c. Desirably, the three or more HCC biomarkers comprise Gpc3, Mdk, and PeglO. Preferably, the method comprises measuring and comparing the expression of four or more of the biomarkers, or all five of the biomarkers.

[0031] The invention also provides a method of monitoring the progression or regression of HCC in a subject comprising (a) measuring the expression level of Qp-c or Serpinll in a first sample obtained from the subject at a first point in time, (b) measuring the expression level of Qp-c or Serpinll in a second sample obtained from the subject at a second point in time, and (c) comparing the expression levels of Qp-c or Serpinll of the first and second samples. Preferably, the method comprises measuring and comparing the expression levels of both Qp-c and Serpinll, desirably in combination with one or more, two or more, or all three of the other biomarkers (e.g., Gpc3, Mdk, and PeglO).

[0032] Comparison of the expression of the biomarkers (e.g., step (c) of the method of monitoring the progression or regression of HCC) can be performed by directly comparing the level of expression of a biomarker of the first sample with the level of expression of the biomarker in the second sample. Alternatively, or in addition, the expression levels of a biomarker in the first and second samples can be indirectly compared to each other by comparing the expression level of each sample to a control. A control can be provided, for example, by measuring the expression of the same biomarkers in the sample of a subject known to be free of HCC or a non-cancerous sample from a subject that has HCC (negative control), or in the sample of a subject known to have HCC or a known HCC sample (positive control). Alternatively, the control can be provided by a previously determined standard for the biomarkers prepared by any suitable method (e.g., an expression profile of biomarkers generated from a pool of non-HCC samples (negative expression profile) or known HCC samples (positive expression profile)).

[0033] A difference in the expression level of one or more HCC biomarkers as between the first and second samples indicates a change in the status of the HCC, wherein increasing expression levels between an earlier point in time and a later point in time suggests progression of the disease and a decrease in the expression levels between an earlier point in time and a later point in time suggests a regression of the disease. No difference in the expression levels of the biomarkers suggests stasis of the disease.

[0034] Other aspects of the method of monitoring the progression or regression of HCC in a subject are as previously described herein with respect to the method of screening for HCC.

[0035] The methods of screening for HCC or monitoring the status (e.g., progression or regression) of HCC in a host can be useful for many applications, such as for prognosticating the course of the disease, estabilishing toxic limits of a drug, developing dosing regimens, or monitoring the effectiveness of a particular treatment against HCC.

[0036] The methods described herein also can be used to screen compounds for potential anti-HCC activity. Candidate compounds may be screened for potential activity against HCC by detecting changes in the expression of one or more of the HCC biomarkers described herein prior to and after contacting HCC cells or tissues with the candidate compound. For instance, such a method can comprise (a) contacting an HCC cell with a test compound, (b) measuring the expression level of three or more HCC biomarkers selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c in the HCC cell, and (c) comparing the expression level of the HCC biomarkers with a control. Desirably, the three or more HCC biomarkers comprise Gpc3, Mdk, and PeglO. Preferably, the method comprises measuring the expression level of four or more, or even all five of the HCC biomarkers. [0037] In a related aspect, the method of screening a compound can comprise (a) contacting an HCC cell with a test compound, (b) measuring the expression level oiSerpinll or Qp-c in the HCC cell, and (c) comparing the expression level of the HCC biomarkers with a control. Preferably, the method comprises measuring and comparing the expression levels of both Serpinll and Qp-c, desirably in combination with one or more, two or more, or all three of the other HCC biomarkers (e.g., Gpc3, Mdk, and PeglO). [0038] A control can be provided, for example, by measuring the expression of the biomarkers in the HCC cell prior to contacting the cell with the test compound, or by measuring the expression of the biomarkers in a cell of the same type as the cell contacted with the test compound. Alternatively, the control can be provided by a previously determined standard for the biomarkers prepared by any suitable method (e.g., an expression profile of the biomarkers generated from a pool of HCC cells).

[0039] Alterations in the expression of one or more of the HCC biomarkers in the HCC cell tested before and after contact with a test compound indicate potential efficacy of the test compound against HCC and concomitant identification of candidate compounds for therapeutic use in HCC. In particular, a decrease in the expression of one or more of the above HCC biomarkers as compared to a negative control (e.g., HCC-positive cell in the absence of the test compound) indicates potential pharmaceutical efficacy. [0040] Any HCC cell can be used in conjunction with the method of screening a compound for anti-HCC activity. The cell can be a cell of a sample obtained from a host or subject, as previously defined herein, or the cell can be a cell created under experimental conditions, which is not directly isolated from a subject or host. The cell can be employed in the method as an isolated cell or cell culture, or as part of a tissue or other sample as previously described herein. The cell also can be in a subject or host, such as an animal, preferably a mammal. Examples of suitable animals are described elsewhere herein. [0041] The test compound can be any compound that one desires to test, without limitation. In general, candidate or test compounds are identified from large libraries of both natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. As those skilled in the field will understand, the precise source of test extracts or compounds is not critical to the method.

[0042] Other aspects of the method of screening a compound for anti-HCC activity are as previously described herein with respect to the other methods of the invention. IK iI H ," iι.,.ιι ;riι . " i::: :::iι a

[0043] Any of the methods described herein can further comprise, in addition to detecting or measuring the expression of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, detecting or measuring the expression of other HCC biomarkers known in the art. As illustrated by the Examples, expression of the HCC biomarkers described herein is not correlated with tumor size or AFP levels. Thus, in preferred aspects of the invention, the methods provided herein are sensitive to AFP-negative HCC and can, therefore, be used to detect or monitor the progression or regression of AFP-negative HCC and early-stage (small-tumor) HCC, which is often undetectable by conventional AFP-screening. As used herein the term "AFP-negative HCC" is used to refer to HCC cases in which AFP levels are not elevated as compared to the AFP levels of a non-HCC patient (i.e., AFP-normal). Also, the method of screening compounds for anti-HCC activity can be used to screen specifically for activity against AFP- negative HCC (e.g., by testing compounds in an AFP-negative HCC cell). Accordingly, the methods described herein can be used to compliment conventional AFP-based screening ■ techniques. Thus, the methods described herein advantageously can further comprise detecting or measuring the expression of AFP as an additional biomarker. AFP expression can be detected or measured according to the methods described herein and other methods known in the art. Other suitable HCC biomarkers that can be used in conjunction with the methods described herein are also known in the art.

[0044] The invention also provides an array that can be used to detect or measure the expression of the HCC biomarkers described herein in accordance with the foregoing methods, or for any other purpose. The array comprises (a) a substrate and (b) three or more different addressable elements that each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C. Desirably, the three or more different addressable elements each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, and PeglO, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, or PEGlO. In a related aspect, the array comprises (a) a substrate and (b) two or more different addressable elements that each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, wherein at least one of the addressable elements comprises a polynucleotide that binds to the mRNA transcript of Qp-c or Serpinll, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C, wherein at least one of the addressable elements comprises a polypeptide that binds to QP-C or SERPINIl . Preferably, the array comprises four or more, or even five of the foregoing addressable elements, thereby comprising a probe for each of the five HCC biomarkers.

[0045] As used herein, the term "addressable element" means an element that is attached to the substrate at a predetermined position and specifically binds a known target molecule, such that when target-binding is detected (e.g., by fluorescent labeling), information regarding the identity of the bound molecule is provided on the basis of the location of the element on the substrate. Addressable elements are "different" for the purposes of the present invention if they do not bind to the same target molecule. The addressable element typically comprises a polynucleotide or polypeptide depending on whether the target molecule is a polynucleotide or polypeptide, respectively. Any given element can comprise more than one copy of the polynucleotide or polypeptide, or even more than one different polynucleotide or polypeptide, provided that the polynucleotides or polypeptides selectively bind the same target molecule. The addressable element also can comprise a detectable label, suitable examples of which are well known in the art.

[0046] The array can comprise addressable elements that bind to HCC biomarkers other than the biomarkers described herein. Suitable HCC biomarkers are known in the art. For example, the array can advantageously comprise an addressable element that binds to AFP or its mRNA transcript. It will be appreciated, however, that an array capable of detecting a vast number of targets (e.g., mRNA or polypeptide targets), such as arrays designed for comprehensive expression profiling of a cell line (e.g., gene profiling) or the like, are not economical or convenient for use as a diagnostic tool or screen for any particular condition like HCC. Thus, to facilitate the convenient use of the array as a diagnostic tool or screen, for example, in conjunction with the methods described herein, the array preferably comprises a limited number of addressable elements. In this regard, the array desirably comprises no more than about 100 or fewer addressable elements, such as about 75 or fewer addressable elements, or even about 50 or fewer addressable elements. Of course, even smaller arrays can comprise about 25 or fewer addressable elements, such as about 15 or fewer addressable elements, or even about 10 or fewer addressable elements (e.g., about 7 or fewer or even 5 or fewer addressable elements). [0047] The substrate can be any rigid or semi-rigid support to which polynucleotides or polypeptides can be covalently or non-covalently attached. Suitable substrates include membranes, filters, chips, slides, wafers, fibers, beads, gels, capillaries, plates, polymers, microparticles, and the like. Materials that are suitable for substrates include, for example, nylon, glass, ceramic, plastic, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, and the like. [0048] The polynucleotides or polypeptides of the addressable elements (hereinafter referred to as "probes") can be attached to the substrate in a pre-determined 1- or 2- dimensional arrangement, such that the pattern of hybridization or binding to a probe is easily correlated with the expression of particular HCC biomarkers. Because the probes are located at specified locations on the substrate, the hybridization or binding patterns and intensities create a unique expression profile, which can be interpreted in terms of expression levels of particular biomarkers and can be correlated with the presence or absence of HCC, or the progression, regression, or stasis of HCC.

[0049] Polynucleotide and polypeptide probes can be generated by any suitable method known in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2" Ed, Cold Spring Harbor Press, Cold Spring Harbor, N. Y., 1989). For example, polynucleotide probes that specifically bind to the mRNA transcripts of the biomarkers described herein can be created using the nucleic acid sequences of mRNA targets themselves (e.g., SEQ ID NOs: 1, 3, 5, 7, and 9, or fragments thereof) by routine techniques (e.g., PCR or synthesis). As used herein, the term "fragment" means a contiguous part or portion of a polynucleotide sequence comprising about 10 or more nucleotides, preferably . about 15 or more nucleotides, more preferably about 20 or more nucleotides (e.g., about 30 or more or even about 50 or more nucleotides). By way of further illustration, a polynucleotide probe that binds to the mRNA transcript of Gpc3 can be provided by a polynucleotide comprising a nucleic acid sequence that is complementary to SEQ ID NO: 1 or a fragment thereof, or sufficiently complementary to SEQ ID NO: 1 or fragment thereof that it will selectively bind to SEQ ID NO: 1. The same is true with respect to the other biomarkers described herein. The exact nature of the polynucleotide probe is not critical to the invention; any probe that will selectively bind the mRNA target can be used. Typically, the polynucleotide probes will comprise 10 or more nucleic acids (e.g., 20 or more, 50 or more, or 100 or more nucleic acids). In order to confer sufficient specificity, will have a sequence identity to a compliment of the target sequence (e.g., SEQ ID NOs: 1, 3, 5, 7, and 9, or

corresponding fragment thereof) of about 90% or more, preferably about 95% or more (e.g., about 98% or more or about 99% or more) as determined, for example, using the well-known Basic Local Alignment Search Tool (BLAST) algorithm (available through the National Center for Biotechnology Information (NCBI), Bethesda, MD).

[0050] Similarly, polypeptide probes that bind to the protein biomarkers described herein can be created using the amino acid sequences of the protein biomarker targets (e.g., SEQ ID NOs: 2, 4, 6, 8, and 10, or fragments thereof) using routine techniques. As used herein, the term fragment means a contiguous part or portion of any of a polypeptide sequence comprising about 5 or more amino acids, preferably about 10 or more amino acids, more preferably about 15 or more amino acids (e.g., about 20 or more amino acids or even about 30 or more or 50 or more amino acids). For example, antibodies to the protein biomarkers can be generated in a mammal using routine techniques, which antibodies can be harvested to serve as probes for the protein biomarkers. The exact nature of the polypeptide probe is not critical to the invention; any probe that will selectively bind to the protein biomarker target can be used. Preferred polypeptide probes include antibodies and antibody fragments antibodies or antibody fragments (e.g., F(ab)₂' fragments, single chain antibody variable region fragment (ScFv) chains, and the like). Antibodies suitable for detecting the HCC biomarkers can be prepared by routine methods, and are commercially available. See, for instance, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Publishers, Cold Spring Harbor, NY, 1988.

[0051] The array can comprise other elements common to polynucleotide and polypeptide arrays. For instance, the array also can include one or more elements that serve as a control, standard, or reference molecule, such as a housekeeping gene or portion thereof (e. g. , PBGD, GAPDH), to assist in the normalization of expression levels or the determination of nucleic acid quality and binding characteristics, reagent quality and effectiveness, hybridization success, analysis thresholds and success, etc. These other common aspects of the arrays or the addressable elements, as well as methods for constructing and using arrays, including generating, labeling, and attaching suitable probes to the substrate, consistent with the invention are well-known in the art. Other aspects of the array are as previously described herein with respect to the methods of the invention. [0052] The invention also provides a kit suitable for detecting HCC. The kit can comprise three or more different probes that each comprise (a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Ii ii,,,;. if

n ..-^{■ 11}r¹ ic; :::n :: o

Qp-c, or (b) a polypeptide that binds to a polypeptide selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C. Desirably, the three or more different probes each comprise (a) a polynucleotide that binds to an niRNA transcript selected from the group consisting of Gpc3, Mdk, andPeglO, or (b) a polypeptide that binds to a polypeptide selected from the group consisting of GPC3, MDK, and PEGlO. In a related aspect, the kit can comprise two or more different probes that each comprise (a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, wherein at least one of the probes comprises a polynucleotide that binds to the mRNA transcript of Qp-c or Serpinll, or (b) a polypeptide that binds to a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C, wherein at least one of the probes comprises a polypeptide that binds to QP-C or SERPINIl . The kit preferably comprises four or more, or even five of the above- described probes thereby comprising a probe for each of the five biomarkers described herein.

[0053] The probes used in the kit are as previously described herein with respect to the array. Indeed, the probes of the kit can be provided in the form of an array. The kit also can comprise polynucleotide or polypeptide probes for other HCC biomarkers known in the art, such as a probe for AFP or its mRNA transcript. However, to facilitate convenient use in a method of detecting HCC or screening for anti-HCC activity, such as any of the methods described herein, the probe set is preferably limited to a reasonable number of probes. Thus, the kit preferably comprises about 100 or fewer different probes, such as about 50 or fewer different probes, or even about 25 or fewer different probes (e.g., about 15 or fewer, about 10 or fewer, or even about 7 or fewer different probes).

[0054] The kit also can comprise an appropriate buffer, suitable controls or standards as described elsewhere herein, and written or electronic instructions. Other aspects of the kit are as previously described with respect to the methods or the array of this invention. [0055] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0056] The following example demonstrates that expression of Gpc3, Mdk, Serpinll,

PeglO, and Qp-c is detectably increased in HCC tissues as compared to normal tissue samples, without correlation to AFP-status or tumor size. if"¹ ιι,,.ιr ii ,.^■ 'ui ■„,.» II. J o / " II": :::iι ... .

[0057] qRT-PCR was performed on 36 non-cancerous liver tissue samples and 58 HCC tumor tissue samples, of which 21 HCC tumor samples and 31 non-cancerous liver tissues were of sources used in prior microarray studies used to discover the biomarkers of interest. Characteristics of the tissue samples and patient sources are provided in Table 1. [0058] Of the 58 HCC samples, 50 samples were categorized as AFP-negative (<20 ng/ml of serum AFP; range 1-18 ng/ml, 6 ng/ml ave.) and 8 samples were categorized as high-AFP (>300 ng/ml of serum AFP; range 329-4620 ng/ml, 1758 ng/ml ave.). 95% of the samples were derived from cirrhotic livers, and the average size of primary tumor lesions from the 58 HCC patients was 4.6 cm in diameter range 1.1-15.3 cm). [0059] Of the 36 non-cancerous liver tissue samples, 10 samples were non-cancerous specimens obtained from HCC patients randomly selected from the 58 patient sources of the HCC samples. The remaining 26 non-cancerous liver tissue samples were obtained from cirrhotic and other non-cancer patients (primary biliary cirrhosis (n=15); autoimmune hepatitis (n=7); alcoholic liver disease (n=4)). The 10 samples selected from HCC patients included 8 AFP-negative samples (5 ng/ml ave.; 1-8 ng/ml range) and 2 high-AFP samples (1061 ng/ml ave.; 329-1794 ng/ml range).

«" Ii,,,,. Ii ./ W%Ά II "

Table 1. Clinical Characteristics of 58 Patients used for real time PCR analysis

Clinical HCC samples Cirrhotic samples from non-cancer patients variable # range % # range %

Sex

Male 52 89.7 / 26.9

Female 6 10.4 18 69.2 unknown 1 3.9

Age (yr)

<50 20 33-49 34.5 8 19-48 30.8 >50 38 50-74 65.5 18 50-68 69.2

Cirrhosis* yes 55 94.8 26 100.0 no 3 5.2 0 0.0

Child-Pugh staging

A 52 94.5 0 0.0

B 3 5.5 13 50.0

C 0 0.0 10 38.5 unknown 0 0.0 3 11.5

AFP (ng/ml)

<20 50 1-18.3 86.2

>300 8 328.9- 13.8 4620

Tumor size <3 cm 20 1.13-2.93 34.5 3-5 cm 20 3.0-4.67 34.5 >5 cm 5.17-

18 15.33 31.0

TNM staging

I 25 43.1

Il 22 37.9

III 10 17.3

IV 1 1.7

' The cirrhosis status was determined by the histological evaluation. - „■^■ :::»

[0060] To perform qRT-PCR, 5 μg isolated total RNA was converted to cDNA. Thermal cycling conditions were as follows: 25⁰C for 10 min, 37⁰C for 120 min. Amplification was carried out in triplicate for each sample and each gene. The PCR thermal program consisted of 5O⁰C for 2 min, 95°C for 10 min and 40 two-step cycles of 95°C for 15 sec and 60⁰C for 1 min. Gene target sequences were: Gpc3: 5'-CTTCCTTGCAGAACTGGCCTATGAT-S' (SEQ ID NO: 11); PeglO: 5'-GCTCTCCCACCTCGAGGTCGCCAAG-S' (SEQ ID NO: 12); Mdk: 5'-AAGGCCAAAGCCAAGAAAGG GAAGG-3' (SEQ ID NO: 13); Serpinlh 5'- TATACCTGCCCAGGTTCACAGTGGA-3' (SEQ ID NO: 14); Qp-c: 5'-TTTCGCGTGG TGCCGCAGTTTGTAG-S' (SEQ ID NO: 15). Primers were constructed about 40 nucleotides upstream or downstream of these sequences. Probes were labeled with the reporter dye (6-carboxy-fluorescein, FAM) at the 5'-end and a non-fluorescent quencher (TAMRA) at the 3' end.

[0061] The final RT-PCR products were analyzed by agarose gel electrophoresis to ensure the unique amplification of targeted genes. Upon completion of qRT-PCR analysis, 5 ul of each reaction was analyzed by 3% agarose (NuSieve 3:1 agarose) gel electrophoresis. Representative two tumor samples are shown. Molecular weight markers (M) are indicated as base pair (bp). Human 18 S rRNA labeled with VIC™ reporter dye was used as an endogenous control for each sample. An RNA pool from 7 disease-free normal liver donors was used as a reference. Only AFP -negative HCC cases (<20 ng/ml of serum AFP) and high AFP HCC cases (>300 ng/ml of serum AFP) were analyzed by qRT-PCR. The relative mRNA expression levels were analyzed by the 2^"ΔΔCt method (see Livak et al., Methods, 25, 402-408 (2001)).

[0062] The relative mRNA abundance of each gene was obtained by normalizing to the 18S ribosomal gene and then to the pool of 7 disease-free normal liver tissues. A "normal" (non-cancer) threshold for each gene was established as the mean level of the gene in the 36 non-cancerous liver samples plus two standard deviations of the variance. Thus, an HCC sample was considered to have an elevated expression of a given gene when its lowest level in a triplicate measurement was above the "normal" threshold. [0063] The results are reported in Table 2 and Figure 1. The relative mean levels (expressed as a fold-increase as compared to the disease-free normal liver pool) of Gpc3, Mdk, Serpinll, PeglO, and Qp-c mRNA were 26.9, 23.8, 40.2, 6.2, 4.8 in the HCC tumor samples, and 0.2, 0.2, 1.4, 1.1, 0.8 in the non-cancerous tissue samples, respectively. The ¹ Il H / i|..,ιι an lu :: 3. -

difference between the HCC tumor samples and the non-cancerous samples was statistically significant (pO.OOOl ; nonparametric Mann Whitney tests) in all 5 genes. [0064] As illustrated by Figure 1, a majority of the HCC cases had an elevated expression of all 5 genes above the defined 'normal' threshold (horizontal dashed line in Figure 1). Moreover, the increased expression levels of the five genes in the HCC samples appeared to be independent of AFP status and tumor size, since most of the AFP-negative samples and samples obtained from smaller tumors(< 3 or 5 cm in diameter) had elevated expression of each gene. The vertical solid lines in Figure 1 demark tumor size stratifications and separate tumor from non-tumor specimens, as indicated at the top of the table. The dashed vertical line separates AFP-normal cases (left of the dashed line) from high AFP-cases (right of the dashed line.

[0065] Furthermore, the expression levels of MDK and SERPINIl were significantly higher in AFP-high cases than in AFP-normal cases, while the level of QP-C was significantly lower in AFP-high cases as compared to AFP-normal cases (Figure 2). Only a small number of cases had an expression below the threshold. The expression levels of GPC3 and PEGlO also were higher in AFP-positive cases as compared to AFP-normal cases, although the differences were statistically insignificant. It should be noted that GPC3 and PEGlO are highly expressed in AFP-normal cases.

[0066] The results show that Gpc3, Mdk, Serpinll, PeglO, and Qp-c can be used as diagnostic biomarkers to discriminate HCC samples from non-tumor liver specimens, regardless of the AFP status and tumor size.

Table 2

HCC cases that are positive for the expression

Total

Range GPC3 PEG10 MDK SERPINI1 QP-C (n) % n n %

AFP AFP+ 329-4620 8 7 88 5 63 8 100 8 100 2 25

Status

Diameter >3-<5 3-4.7 20 18 90 13 60 14 70 15 75 13 65

(cm) >5 5.2-15 18 12 72 12 67 15 83 15 83 14 78

T early I 25 21 84 15 60 16 65 17 68 20 80

I M INΠlVΛl int. Il

Staging 22 19 86 17 77 16 73 16 73 17 77 adv. M-IV 11 9 82 6 55 11 100 9 82 6 55 _"

EXAMPLE 2

[0067] The following example demonstrates the probability of correctly classifying

HCC and non-HCC samples on the basis of Gpc3, Mdk, Serpinll, PeglO, and Qp-c expression.

[0068] The qRT-PCR expression data generated in Example 1 was used to determine the probability of correctly classifying these 58 HCC samples arid 36 non-tumor liver specimens by the Prediction Analysis of Microarrays (PAM) algorithm (Tibshirani et al., "Diagnosis of multiple cancer types by shrunken centroids of gene expression." Proc.Natl.Acad.Sci.U.S.A, 99: 6567-6572 (2002)). Multidimensional scaling analysis based on Euclidean distance was used to visualize the classification outcome. The positive predictive value (PPV) and negative predictive value (NPV) were calculated as: PPV=nll/(nll+n21) and NPV=n22/(nl2+n22), respectively, where nl 1= number of HCC predicted as HCC, nl2= number of HCC predicted as non-HCC, n21= number of non-HCC predicted as HCC, and n22=number of non-HCC predicted as non-HCC.

[0069] The results of the PAM analysis are presented in Figures 3 and 4. PAM analysis with 10-fold crossvalidation predicted 100% correct classification of non-tumor samples and 84% correct classification of HCC samples (Figure 3). Similar results were obtained when these samples were randomly partitioned into 50% training and 50% testing cases, a process which was repeated 5 times (Figure 4D). Consistently, only the same 9 HCC samples were misclassified with these two approaches, suggesting that misclassification did not occur by random chance. Figures 4A and 4C show a sharp increase in the error rate when fewer than three of the five biomarkers are used for classification, according to the PAM algorithm. Thus, this 5-gene signature provides high sensitivity (84%) and specificity (100%) in classifying HCC cases. The PPV and NPV were calculated as 100% and 80%, respectively. PAM analysis of the same cohort after removing the samples that were used in prior microarray studies to discover the biomarkers showed that the 5-gene signature could provide 73% sensitivity (21 HCC) and 100% specificity (31 non-HCC). [0070] Multi-dimensional scaling analysis showed that most of the HCC samples clustered separately from non-tumor samples .demonstrating measurable differences between tumor and non-tumor samples based on the expression of the five genes. It appeared that Gpc3, PeglO, and Mdk provided the most weight to discriminate HCC from non-tumor liver specimens (Figure 4B). . _, .,

>ι,..ι> .,,,;ii HsJ . -

EXAMPLE 3

[0071] The following example illustrates that elevated MDK levels in HCC patients is independent of AFP level or degree of cirrhosis.

[0072] We determined the level of serum MDK levels in an additional 64 HCC patients and 26 normal blood donors (controls) using an ELISA-based assay. The medians (25th and 75th percentiles) of serum MDK were 442.8 (288 and 666.5) pg/ml in the cases and 11.7 (3.8 and 42.3) pg/ml in the controls. A statistically significant difference (p<0.0001; two-tailed) was observed between cases and controls, as determined by unpaired t-test with Welch's correction (Figure 5A). No significant difference was observed in the cases when stratified by overall AFP (p=0.96) or tumor size (p=.22) as determined by one-way ANOVA. However, when using the median tumor size (5 cm diameter) as the cutoff, we found that larger tumors (>5 cm) had significantly higher serum AFP than smaller tumors (<5 cm) (p=0.02) (Figure 5B). In contrast, the difference in serum MDK between small and large tumors was insignificant (p>0.05) (Figure 5C). These results suggest that serum AFP level may reflect the degree of tumor burden, while serum MDK is independent of tumor size. [0073] To further address the diagnostic value of MDK, we performed ELISA analysis on a different set of HCC patients consisting of 32 HCC cases, 20 normal blood donors, and 49 cirrhosis patients. We found that HCC patients had a significantly higher serum MDK level than patients with cirrhosis or hospital controls (p<0.0001 and p<0.0002, respectively) regardless of the AFP level or Child-Pugh score (Figure 5D).

[0074] Thus, it is possible to diagnose AFP-normal and small-tumor HCC using serum samples in accordance with the invention.

[0075] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0076] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not _, » .

limited to,") unless otherwise noted. Wherever an open-ended term is used, the substitution of a closed-ended term (e.g., "consisting essentially of or "consisting of) is specifically contemplated. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0077] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

, ,, ,, ,CLAIM(S):

1. A method of screening for hepatocellular carcinoma (HCC) comprising detecting the expression of three or more HCC biomarkers in a sample, wherein the HCC biomarkers are selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c.

2. The method of claim 1, wherein the three or more HCC biomarkers comprise Gpc3, Mdk and PeglO.

3. The method of claim 1 comprising detecting the expression of four or more of the HCC biomarkers.

4. The method of claim 1 comprising detecting the expression level oϊGpcS, Mdk, Serpinll, PeglO, and Qp-c.

5. A method of screening for HCC comprising detecting the expression of Serpinll or Qp-c in a sample.

6. The method of claim 5 comprising detecting the expression of Serpinll .

7. The method of claim 5 comprising detecting the expression of Qp-c.

8. The method of claim 5 comprising detecting the expression of both Serpinll and Qp-c.

9. The method of any of claims 5-8 further comprising detecting the expression of one or more HCC biomarkers selected from the group consisting of Gpc3, Mdk, and PeglO.

10. The method of any of claims 1 -9, wherein expression is detected by mRNA detection.

11. The method of any of claims 1 -9, wherein expression is detected by protein detection.

12. The method of any of claims 1-11, wherein the method of screening for HCC is a method of screening for AFP-negative HCC. ,

13. A method of monitoring the progression or regression of HCC in a subject, comprising

(a) measuring the expression level of three or more HCC biomarkers in a first sample obtained from the subject at a first point in time,

(b) measuring the expression level of three or more HCC biomarkers in a second sample obtained from the subject at a second point in time, and

(c) comparing the expression levels of the HCC biomarkers of the first and second samples, wherein the HCC biomarkers are selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c.

14. The method of claim 13, wherein the three or more HCC biomarkers comprise Gpc3, Mdk, and PeglO.

15. The method of claim 13 comprising measuring the expression level of four or more of the HCC biomarkers.

16. The method of claim 13 comprising measuring the expression level of Gpc3, Mdk, Serpinll, PeglO, and Qp-c.

17. A method of monitoring the progression or regression of HCC in a subj ect, comprising

(a) measuring the expression level of Qp-c or Serpinll in a first sample obtained from the subject at a first point in time,

(b) measuring the expression level of Qp-c or Serpinll in a second sample obtained from the subject at a second point in time, and

(c) comparing the expression levels of Qp-c or Serpinll of the first and second samples.

18. The method of claim 17 comprising measuring the expression level of Serpinll.

19. The method of claim 17 comprising measuring the expression level of Qp-c

20. The method of claim 17 comprising measuring the expression level of both Serpinll and Qp-c. _{,, , -}

21. The method of any of claims 17-20, further comprising measuring the expression level of one or more HCC biomarkers selected from the group consisting of Gpc3, Mdk, and PeglO.

22. The method of any of claims 13-21, wherein expression is measured by mRNA detection.

23. The method of any of claims 13-21, wherein expression is measured by protein detection.

24. The method of any of claims 13-23, wherein the method is a method for monitoring the progression or regression of AFP-negative HCC.

25. A method of screening a compound for anti-HCC activity comprising

(a) contacting an HCC cell with a test compound,

(b) measuring the expression level of three or more of the HCC biomarkers selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c in the HCC cell, and

(c) comparing the expression level of the HCC biomarkers with a control.

26. The method of claim 25, wherein the three or more HCC biomarkers comprise Gpc3, Mdk and PeglO.

27. The method of claim 25 comprising measuring the expression level of four or more of the HCC biomarkers.

28. The method of claim 25 comprising measuring the expression level oϊGpc3, Mdk, Serpinll, PeglO, and Qp-c.

29. A method of screening a compound for anti-HCC activity comprising

(a) contacting an HCC cell with a test compound,

(b) measuring the expression level of Serpinll or Qp-c in the HCC cell, and

(c) comparing the expression level of the HCC biomarkers with a control.

30. The method of claim 29 comprising measuring the expression level of Serpinll. , ,

31. The method of claim 29 comprising measuring the expression level of Qp-c.

32. The method of claim 29 comprising measuring the expression level of both Serpinll and Qp-c.

33. The method of any of claims 29-32, further comprising measuring the expression level of one or more HCC biomarkers selected from the group consisting of Gpc3, Mdk, and PeglO.

34. The method of any of claims 25-33, wherein expression is measured by mRNA detection.

35. The method of any of claims 25-33, wherein expression is measured by protein detection.

36. The method of any of claims 25-33, wherein the cell is an AFP-negative HCC cell.

37. An array comprising

(a) a substrate and

(b) three or more different addressable elements that each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C.

38. The array of claim 37, wherein the three or more different addressable elements each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, and PeglO, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, and PEGlO.

39. An array comprising

(a) a substrate and

(b) two or more different addressable elements that each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, wherein at least one of the addressable elements comprises a polynucleotide that binds to the mRNA transcript of Qp-c or Serpinll, or (ii) a polypeptide that binds to a protein selected from the group consisting of ,_{, , ,,}

II¹" '!"^■>^■

GPC3, MDK, SERPINIl, PEGlO, and QP-C, wherein at least one of the addressable elements comprises a polypeptide that binds to QP-C or SERPINIl .

40. The array of claim 37 or 39 comprising four or more different addressable elements that each comprise (i) a polynucleotide that binds to an niRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp- c, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C.

41. The array of claim 37 or 39 comprising five different addressable elements that each comprise (i) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, or (ii) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C.

42. The array of any of claims 37-41 , wherein the array comprises less than about 100 addressable elements.

43. The array of claim 42, wherein the array comprises less than about 50 addressable elements.

44. The array of claim 43, wherein the array comprises less than about 10 addressable elements.

45. A kit for detecting HCC comprising three or more different probes that each comprise

(a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, or

(b) a polypeptide that binds to a polypeptide selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C.

46. The kit of claim 45, wherein the three or more different probes each comprise

(a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, and PeglO, or

(b) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, and PEGlO.

47. A kit for detecting HCC comprising two or more different probes that each comprise

(a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, wherein at least one of the probes comprises a polynucleotide that binds to the mRNA transcript of Qp-c or Serpinll, or

(b) a polypeptide that binds to a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C, wherein at least one of the probes comprises a polypeptide that binds to QP-C or SERPINIl.

48. The kit of claim 45 or 47 comprising four or more different probes that each comprise

(a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, or

(b) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C.

49. The kit of claim 45 or 47 comprising five different probes that each comprise

(a) a polynucleotide that binds to an mRNA transcript selected from the group consisting of the mRNA transcripts of Gpc3, Mdk, Serpinll, PeglO, and Qp-c, or

(b) a polypeptide that binds a protein selected from the group consisting of GPC3, MDK, SERPINIl, PEGlO, and QP-C.

50. The kit of any of claims 45-49, wherein the kit comprises less than about 100 different probes.

51. The kit of claim 50, wherein the kit comprises less than about 50 different probes.

52. The kit of claim 51, wherein the kit comprises less than about 10 different probes.