PROTEIN MARKERS FOR DETECTING LIVER CANCER AND METHOD FOR IDENTIFYING THE MARKERS THEREOF

Abstract

The present invention relates to the diagnosis of liver cancer. It discloses the use of protein ERBB3 and protein IGFBP2 in the diagnosis of liver cancer. It relates to a method for diagnosis of liver cancer from a liquid sample, derived from an individual by measuring ERBB3 protein and IGFBP2 protein in the sample. Measurement of ERBB3 protein and IGFBP2 protein can, e.g., be used in the early detection or diagnosis of liver cancer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a division of U.S. patent application Ser. No. 12/831,447, filed on Jul. 7, 2010, titled Protein Markers for Detecting Liver Cancer and Method for Identifying the Markers Thereof, listing Sen-Yung Hsieh as inventor.

FIELD OF THE INVENTION

[0002] The present invention relates to protein markers for detecting liver cancer, also called hepatoma, in plasma or serum and a method for detecting liver cancer thereof. The present invention also relates to a method for identifying a novel marker in plasma/ serum for detecting liver cancer. In particular, the present invention relates to protein markers expressed in tissue interstitial fluid and a method for identifying novel marker in a tissue interstitial fluid for detecting liver cancer. Especially, the present invention relates to plasma/serums ERBB3 and IGFBP2 protein markers used for detecting liver cancer precisely.

BACKGROUND OF THE INVENTION

[0003] Cancer remains a major public health challenge despite progress in detection and therapy. Whole blood, serum, plasma, or nipple aspirate fluid are the most widely used sources of sample in clinical routine. Conventionally, researchers try to find valuable markers from plasma/serum to detect liver cancer. However, up to 90 percentage of the plasma/serum are composed by 6 constant serum proteins, and 99 percentage are composed by about 20 constant proteins. The metabolic and the physiological conditions could be represented in whole blood, serum or plasma. Some specific proteins with diagnosis values are secreted into whole blood, serum or plasma, but they always present in a trace amount and are hard to be found. Therefore, an urgent clinical need exists to improve the method to identify biomarkers for the diagnosis of liver cancer from plasma/serum.

[0004] Some researchers tried to find tumor markers from hepatocellular carcinoma (hereinafter may be referred to as "liver cancer", "hepatoma" or "HCC") tissue or cell culture media. However, neither tumor tissues nor cell culture media of hepatocellular carcinoma has been proved to be an adequate source for identifying new serum markers for hepatoma. In contrast, the tissue interstitial fluid is the media between tumor cells and the circulation, and tumor interstitial fluid represents the microenvironment that tumor cells inhabit. Tumor markers shed into circulation may also be generated by interaction of tumor cells with its microenvironment. It is, therefore, tempting to examine whether tumor interstitial fluid is the source for discovery of serum biomarkers.

[0005] So far, some markers, including alpha-fetoprotein (AFP), alpha-fetoprotein lectin fraction-L3 fraction, PIVKA-II, AFU and GPC3, have conventionally been employed for liver cancer diagnosis. However, results obtained from detecting by the foregoing tumor markers often show false-positive or false-negative, so that their functions of detection are limited clinically. Despite the large and ever growing list of candidate protein markers in the field of liver cancer, to date clinical/diagnostic utility of these molecules is not known. In order to be of clinical utility, a new diagnostic marker as a single marker should be at least as good as the best single marker known in the art. Or, a new marker should lead to a progress in diagnostic sensitivity and/or specificity either if used alone or in combination with one or more other markers, respectively.

[0006] Therefore, there is a keen need in the art to develop a new tumor marker for clinical diagnosis and increase the precision of diagnosis. It was the task of the present invention to investigate whether a new marker can be identified which may aid in liver cancer diagnosis. Surprisingly, it has been found that use of the marker ERBB3 or IGFBP2 can at least partially overcome the problems known from the state of the art.

SUMMARY OF THE INVENTION

[0007] The present invention therefore relates to a novel protein marker ERBB3 for detecting liver cancer.

[0008] The present invention therefore relates to a novel protein marker IGFBP2 for detecting liver cancer.

[0009] The present invention therefore relates to a method for the detection of liver cancer comprising the steps of a) providing a liquid sample obtained from an individual, b) contacting said sample with a specific binding agent for ERBB3 or IGFBP2 under conditions appropriate for formation of a complex between said binding agent and ERBB3 or IGFBP2, and c) correlating the amount of complex formed in (b) to the detection of liver cancer.

[0010] The present invention also relates to a method for identifying a marker for detecting liver tumor in plasma/serum, comprising: [0011] obtaining fresh tissues of liver cancer and non-cancer liver tissues from patients with liver cancer, cutting the tissues and washing by PBS solution twice, culturing the cut tissues in an incubator for 10 minutes, and then precipitating by centrifugation at 1000-2000 rpm/min for 2-5 minutes to obtain cell pellets and removing the contaminations; [0012] re-suspending the cell pellets in PBS solution, culturing the suspended cells in PBS solution in the incubator for 60 minutes, precipitating by centrifugation at 1000-2000 rpm/min for 2-5 minutes to remove cell pellets and obtain a crude tissue interstitial fluid; [0013] centrifugating the crude tissue interstitial fluids by centrifugation at 5000-15000 rpm/min for 15-30 minutes to remove undissolved cell matrix and obtain a pure tissue interstitial fluid; [0014] comparing the difference of the protein components between the tissue interstitial fluids obtained from the liver cancer tissues and non-cancer liver tissues by proteomic methods, then identifying the relatively high-content proteins in tissue fluids of the liver cancer cells, and listing those relatively high-content proteins as candidate biomarkers for hepatoma detection; [0015] detecting the candidate biomarkers in serum by ELISA and measuring the concentrations of the candidate markers, and [0016] analyzing the concentrations difference by student t-test analysis and Receiver Operating Characteristic curve (ROC curve) to check the function of the candidate biomarkers.

[0017] The candidate biomarkers were further used to detect serum samples obtained from liver cancer patients and non-liver cancer patients by ELISA and ROC curve. When area under curve values (AUC values) of the candidate markers in serums are greater than 90%, the protein is classified as suitable markers for hepatoma detection.

[0018] Comparing with the conventional method of detecting liver cancer, it is hard to find a suitable marker from serum for detecting liver cancer by the conventional methods. The present invention provides a novel ERBB3 protein and a novel IGFBP2 protein as markers for liver cancer detection. ERBB3 protein and IGFBP2 protein are found from tissue interstitial fluids and have been proven their powerful functions in identifying liver cancers. Detection by the concentrations of ERBB3 protein and IGFBP2 protein in patients' serum/plasma or whole blood could increase the sensitivity of liver cancer diagnosis.

[0019] As a skilled artisan will appreciate, any such diagnosis is made in vitro. The patient sample is discarded afterwards. The patient sample is merely used for the in vitro diagnostic method of the invention and the material of the patient sample is not transferred back into the patient's body. Typically, the sample is a liquid sample.

[0020] A specific binding agent preferably is an antibody reactive with ERBB3 or IGFBP2. The term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody. Any antibody fragment retaining the above criteria of a specific binding agent can also be used.

[0021] In a preferred embodiment the method according to the present invention is practiced with serum as liquid sample material.

[0022] In a further preferred embodiment the method according to the present invention is practiced with plasma as liquid sample material.

[0023] In a further preferred embodiment the method according to the present invention is practiced with whole blood as liquid sample material.

[0024] In a further preferred embodiment the method according to the present invention is practiced with tissue interstitial fluid of liver as liquid sample material.

[0025] Whereas application of routine proteomics methods to tissue interstitial fluid obtained from tissue samples, leads to the identification of many potential marker candidates for the tissue selected, the inventors of the present invention have been able to surprisingly detect both or one of ERBB3 and IGFBP2 in a bodily fluid sample. Even more surprising they have been able to demonstrate that the presence of ERBB3 and IGFBP2 in such liquid sample obtained from an individual can be correlated to the diagnosis of liver cancer.

[0026] Antibodies to ERBB3 and IGFBP2 with great advantages can be used in established procedures, e.g., to detect liver cancer cells in situ, in biopsies, or in immunohistological procedures.

[0027] Preferably, an antibody to ERBB3 is used in a qualitative (ERBB3 present or absent) or quantitative (ERBB3 amount is determined) immunoassay.

[0028] Preferably, an antibody to IGFBP2 is used in a qualitative (IGFBP2 present or absent) or quantitative (IGFBP2 amount is determined) immunoassay.

[0029] Measuring the level of protein ERBB3 or IGFBP2 has proven very advantageous in the field of liver cancer. Therefore, in a further preferred embodiment, the present invention relates to use of protein ERBB3 or/and IGFBP2 as a marker molecule in the diagnosis of liver cancer from a liquid sample obtained from an individual.

[0030] The details of one or more embodiments of the technology are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the technology will be apparent from the description and drawings, and from the claims. All cited patents, and patent applications and references (including references to public sequence database entries) are incorporated by reference in their entireties for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a scheme showing steps performing in accordance with the present invention.

[0032] FIG. 2A is a picture showing a two-dimensional gel electrophoresis (2-DE) of Example 1 in accordance with the present invention.

[0033] FIG. 2B is a picture showing a two-dimensional differential fluorescence gel electrophoresis (2-D DICE) condition of Example 1 in accordance with the present invention.

[0034] FIG. 3 is a picture showing results of normal tissue interstitial fluid and tumor tissue interstitial fluid on a biochip.

[0035] FIG. 4 is a chart showing different concentrations of ERBB3 proteins obtained from different tissues.

[0036] FIG. 5A is a chart showing ROC curve of serum ERBB3 discriminating hepatoma from non-hepatoma controls in the discovery group in accordance with the present invention.

[0037] FIG. 5B is a chart showing ROC curve of serum ERBB3 discriminating hepatoma from non-hepatoma controls in the validation group in accordance with the present invention.

[0038] FIG. 5c is a chart showing ROC curve of serum ERBB3 discriminating hepatoma from non-hepatoma controls in the discovery and validation groups in accordance with the present invention.

[0039] FIG. 6 is a chart showing different IGFBP2 concentrations in Example 1 obtained from different tissues.

[0040] FIG. 7A is a chart showing ROC curve of serum IGFBP2 discriminating hepatoma from non-hepatoma controls in the discovery group in accordance with the present invention.

[0041] FIG. 7B is a chart showing ROC curve of serum IGFBP2 discriminating hepatoma from non-hepatoma controls in the validation group in accordance with the present invention.

[0042] FIG. 7C is a chart showing ROC curve of serum IGFBP2 discriminating hepatoma from non-hepatoma controls in the discovery and validation groups in accordance with the present invention.

[0043] FIG. 8A is a chart showing ROC curve of serum ERBB3, AFP, or combined ERBB3 and AFP in discriminating hepatoma from non-hepatoma controls in one of the present embodiment in accordance with the present invention.

[0044] FIG. 8B is a chart showing ROC curve of serum IGFBP2, AFP, or combined IGFBP2 and AFP in discriminating hepatoma from non-hepatoma controls in one of the present embodiment in accordance with the present invention.

[0045] FIG. 8C is a chart showing ROC curve of serum IGFBP2, ERBB3, or combined IGFBP2 and ERBB3 in discriminating hepatoma from non-hepatoma controls in one of the present embodiment in accordance with the present invention.

[0046] FIG. 8D is a chart showing ROC curve of serum ERBB3, IGFBP2, AFP, or combined ERBB3, IGFBP2, and AFP in discriminating hepatoma from non-hepatoma controls in one of the present embodiment in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0047] The following examples illustrate the invention without limiting its scope.

[0048] The present invention relates to a method for the detection of liver cancer, comprising the steps of:

[0049] a) providing a liquid sample obtained from an individual,

[0050] b) contacting said sample with an antibody specific for at least one of ERBB3 protein(SEQ ID NO:2) and IGFBP2 protein(SEQ ID NO: 4) under conditions appropriate for formation of a complex between said antibody and at least one of said proteins, and

[0051] c) correlating an amount of the complex formed in (b) to the detection of liver cancer.

[0052] With reference to FIG. 1, the method for obtaining the novel ERBB3 and IGFBP2 protein markers for hepatoma detection comprises the steps of:

[0053] Step 1 (11) Obtaining liver cancer tissues and non-cancer liver tissues from individuals respectively:

[0054] cutting the obtained liver cancer tissues and the non-cancer liver tissues into 1×1×3 mm³ pellets,

[0055] washing the above pellets by PBS solution twice,

[0056] incubating the cell pellets by PBS solution at 37° C., 10% CO₂ incubator for 10 minutes,

[0057] centrifuging the cultured cell pellets at 1000 to 2000 rpm/min for 2 to 5 minutes to remove the contaminations on liver cancer tissues and non-cancer liver tissues,

[0058] Step 2 (12) Separating tissues and tissue interstitial fluid by low speed centrifugation:

[0059] culturing the cell pellets by PBS at 37° C., 10% CO₂ incubator for 60 minutes,

[0060] centrifuging the cultured broth at 1000-2000 rpm/min for 2-5 minutes to separate tissues and tissue interstitial fluid, and avoiding cell crack,

[0061] Step 3 (13) Removing the dissolved matrix by high speed centrifugation,

[0062] centrifuging the cultured broth by 5000-15000 rpm/min for 15-30 minutes to increase the purification and the sensitivity of the tissue interstitial fluid,

[0063] Step 4 (14) Finding candidate biomarkers for hepatoma detection:

[0064] comparing protein pattern obtained from liver cancer tissues and non-cancer liver tissues to select possible protein markers, said protein patterns may be performed such as 2-DE or antibody arrays,

[0065] identifying and listing the candidate biomarkers which are present in relatively high concentration and are highly different in the protein pattern of the liver cancer and non-cancer liver tissues.

[0066] Step 5 (15) Selecting candidate biomarkers for hepatoma detection proteins:

[0067] analyzing the candidate biomarkers by ELISA method and checking the concentrations of each candidate biomarker in cancer tissues and non-cancer tissues,

[0068] analyzing the concentrations obtained from the above sub-step by student t-test to identify the concentration of the biomarker with significant difference, and selecting the concentration of the biomarker with p value<0.01,

[0069] further analyzing the concentration with significant difference by ROC curve method and selecting the candidate biomarker with AUC value>90% as the biomarker for hepatoma detection.

[0070] In a preferred embodiment, the above selected markers were further analyzed by applying in the serum samples obtained from another liver cancer group and non-liver cancer group. The method may be performed by ELISA method and ROC curve method for getting their AUC values. When the AUC values>90%, the selected marker was confirmed to be a suitable marker for liver cancer detection.

[0071] As used herein, the term "non-liver cancer" refers to a patient that may have cirrhosis without liver cancer, chronic hepatitis or healthy individuals without liver cancer.

[0072] As used herein, "antibody" or "specific binding agent" includes immunoglobulin molecules and immunologically active determinants of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen. Structurally, the simplest naturally occurring antibody (e.g., IgG) comprises four polypeptide chains, two copies of a heavy (H) chain and two of a light (L) chain, all covalently linked by disulfide bonds. Specificity of binding in the large and diverse set of antibodies is found in the variable (V) determinant of the H and L chains; regions of the molecules that are primarily structural are constant (C) in this set. Antibody includes polyclonal antibodies, monoclonal antibodies, whole immunoglobulins, and antigen binding fragments of the immunoglobulins.

[0073] In the diagnostic and prognostic assays of the invention, the antibody can be a polyclonal antibody or a monoclonal antibody and in a preferred embodiment is a labeled antibody.

[0074] In this exemplary method a Receiver Operating Characteristic curve (ROC curve) is generated. An ROC curve is a plot of test sensitivity (plotted on the y axis) versus its False Positive Rate (or 1--specificity) (plotted on the x axis). Each point on the graph is generated by using a different cut point. The set of data points generated from the different cut points is the empirical ROC curve. Lines are used to connect the points from all the possible cut points. The resulting curve illustrates how sensitivity and the FPR vary together. ROC is a standard statistical method used in the evaluation of a biomarker in disease diagnosis. This analysis determines the ability of a test to discriminate diseased cases from normal cases. The value of the area under the ROC curve is a measure of test accuracy.

EXAMPLE 1

Markers Selection

[0075] 1-1 Sample Collection and Preparation

[0076] Step 1: Liver cancer tissues and non-cancer liver tissues were respectively collected from 10 patients with hepatoma received surgical resection of liver tumors. The contaminations on the liver cancer tissues and non-cancer liver tissues were removed by low speed centrifugation.

[0077] In a preferred embodiment of the present invention, liver cancer tissues and non-cancer liver tissues were obtained by surgical operation. The sizes of the tissues were cut as 1×1×3 mm, and then the cut tissues were cultured by PBS solution in an incubator at 37° C. and 10% CO₂ condition for 10 minutes. Then the culture broth was centrifuged at 1000-2000 rpm/min for 2-5 minutes for removing the contaminations on the tissues.

[0078] Step 2: Tissues and tissue interstitial fluid were separated by low speed centrifugation to obtain tissue interstitial fluid.

[0079] The cutting tissues were collected and further cultured by PBS solutions in an incubator at 37° C., 10% CO₂ condition for 60 minutes. Then the culture broths were centrifuged at 1000-2000 rpm/min for 2-5 minutes for removing cells to obtain a crude tissue interstitial fluid.

[0080] Step 3: The crude tissue interstitial fluid were centrifuged again to remove undissolved matrix by high speed centrifugation to obtain a pure tissue interstitial fluid.

[0081] To obtain pure tissue interstitial fluids respectively from liver cancer tissues and non-cancer liver tissues, the crude tissue interstitial fluids were centrifuged at 5000-15000 rpm/min for 15-30 minutes to remove the undissolved matrix and obtain a pure tissue interstitial fluid.

[0082] Step 4: Candidate biomarkers for hepatoma detection were selected.

[0083] The electropherograms of FIGS. 2A and 2B illustrate the unique 2-DE and 2-D DIGE patterns of the diseased (liver cancer) and normal (non-cancer liver) tissues. The representative images of two-dimensional gel electrophoresis (2-DE) (FIG. 2A) and two-dimensional differential fluorescence gel electrophoresis (2-D DIGE) (FIG. 2B). 2-DE was performed on immobilized pH 4-7 gradient strips, followed by the second-dimensional separation on 10-16% gradient polyacrylamide gels. For 2-DE, the separated proteins were stained with SYPRO Ruby. Images were captured, and relative volumes for each protein were normalized, matched across gels and determined with the aide of software analysis. For 2-D DIGE, equal amount of protein lysate from TIF (hepatoma interstitial fluid) and NIF (non-hepatoma interstitial fluid) were labeled with Cy3 and Cy5 dyes respectively, and vice versa, using the minimal labeling procedures. TIF and NIF were mixed together and then separated by 2-dimensional gel electrophoresis. Proteins with significant differentiation between TIF and NIF were selected for protein identification using mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight/ time-of-flight mass spectrometry, MALDI-TOF/TOF MS). Results showed that the concentrations of ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3) protein and IGFBP2 (Insulin-like growth factor binding protein 2) protein in the tissue interstitial fluid of the liver cancer tissues were higher than them appeared in the tissue interstitial fluid of non-cancer liver tissues.

[0084] Furthermore, the tissue interstitial fluids obtained from liver cancer tissues and non-cancer liver tissues were also analyzed by Antibody Array (Human Cytokine Antibody Array G Series 2000, RayBiotech Inc.) method, comprising:

[0085] A. 100 mg tissue interstitial fluids were dropped on each reaction well of the array chip for reaction at room temperature for 2 hours.

[0086] B. The reaction wells were washed by washing solution for 5 times, and the blocking buffer was mixed well with antibodies which had linked with biotin.

[0087] C. The blocking buffer containing antibodies linked with biotin were added into each reaction wells at room temperature for 2 hours.

[0088] D. The blocking buffer containing antibodies linked with biotin were removed. Then the reaction wells were washed by washing solution for 5 times, and a diluted Cy3-conjugated streptavidin which was included in the kit were added for reacting in dark at room temperature for 2 hours.

[0089] E. The Cy3-conjugated streptavidin were removed and the reaction wells were washed again by washing solution for 5 times and dried in dark at room temperature.

[0090] F. With reference to FIG. 3, the results showed on the protein array were read by Confocal Scanner Chip Reader. The strength of fluorescence showed on the protein array was further analyzed by Gene Pix Pro 4.1 software. Results showed that both concentrations of ERBB3 protein and IGFBP2 protein in the tissue interstitial fluids of the cancer tissues were higher than the non-cancer tissues. ERBB3 protein and IGFBP2 protein were selected as candidate biomarkers for hepatoma detection.

[0091] Complete amino acid sequence of ERBB3 protein was shown as SEQ ID NO. 1, and nature amino acid sequence in human serum was shown as SEQ ID NO: 2. Sequence of SEQ ID NO: 2 is same as the sequence from the 20 to 643 amino acid sequence of SEQ ID NO: 1. Complete amino acid sequence of IGFBP2 was shown as SEQ ID NO: 3, and nature amino acid sequence in human serum was shown as SEQ ID NO: 4. Sequence of SEQ ID NO: 4 is same as the sequence from the 40 to 328 amino acid sequence of SEQ ID NO: 3.

[0092] Step 5: The candidate biomarkers were used for hepatoma detection

[0093] A. Detect the concentrations of ERBB3 protein and IGFBP2 protein in serum:

[0094] To measure the concentration of ERBB3 protein and IGFBP2 protein correctly, ELISA methods comprised human ErbB3 kit (DY348) and human IGFBP2 kit (DY674) (R&D Systems Europe, Ltd) were used. Human ErbB3 protein and human IGFBP2 proteins which were produced by genetic engineer technology were used as standard.

[0095] Antibody for detecting ERBB3 protein in ELISA assay:

[0096] 1. Capture antibody: an antibody which could bind to SEQ ID NO: 2 (R&D Systems, MAB 3481).

[0097] 2. Detection antibody: a biotinylated monoclone antibody which could bind to SEQ ID NO: 2 (R&D Systems, BAM348).

[0098] ELISA antibody for IGFBP2 proteins:

[0099] 1. Capture antibody: an antibody which could bind to SEQ ID NO: 4 (R&D Systems, MAB6741).

[0100] 2. Detection antibody: a biotinylated antibody, goat IgG, which could bind to SEQ ID NO: 4 (R&D Systems, BAF674).

[0101] Steps for operation:

[0102] (a) Both capture antibodies were diluted to the concentration of 4 mg/ml, and added 100 μl to each reaction well at room temperature for reacting overnight;

[0103] (b) The obtained serum were diluted (the average dilution rate 10-100×), and 100 μl diluted serum were added into each reaction well at room temperature for 2 hours;

[0104] (c) The diluted serum were removed and the reaction wells were washed by wash solution, then 2 mg/ml of 100 μl biotinylated detection antibodies were added into each reaction wells at room temperature for 2 hours;

[0105] (d) The reaction wells were washed again, and strptavidin-HRP which was included in the kit was added and reacted in dark at room temperature for 20 minutes;

[0106] (e) The reaction wells were washed again, the subtracts which was also included in the kit were added for reaction at room temperature for 20 minutes;

[0107] (f) The data were read by microplate reader at 450 nm and 540 nm and corrected by 540 nm absorption as background value. After correction, the true absorption values were obtained. Then, the concentration of ERBB3 proteins and IGFBP2 proteins were evaluated by comparing with the concentration of standard samples.

[0108] Serum samples were collected from 113 liver cancer patients and 111 non-liver cancer patients (including 47 cirrhosis patients, 64 chronic hepatitis B) underwent the concentration of ERBB3 and IGFBP2 in serum samples for liver cancer detection.

[0109] With reference to FIGS. 4 to 6, the concentrations of serum ERBB3 protein and serum IGFBP2 protein in 113 liver cancer patients and 111 non-liver cancer patients were analyzed by student t-test to understand the difference. Results showed that false-positive values were less than 1/100 and p<0.01 were preliminarily selected for further analysis. The concentrations of serum ERBB3 proteins and serum IGFBP2 proteins, which showed a significant difference, were following analyzed by ROC curve analysis.

[0110] B. ROC Curve Analysis

[0111] To further understand whether ERBB3 protein and IGFBP2 protein were suitable for being markers for liver cancer detection, we provided another two group samples for each candidate markers for further check.

[0112] For IGFBP2 protein check experiment, there were 57 liver cancer patients and 35 non-liver cancer patients in Group I (taken as discovery group), and there were 56 liver cancer patients and 36 non-liver cancer patients in Group II (taken as validation group).

[0113] For ERBB3 protein check experiment, there were 56 liver cancer patients and 32 non-liver cancer but with hepatitis B patients in Group I (taken as discovery group), and there were 57 liver cancer patients and 32 non-liver cancer but with hepatitis B patients in Group II (taken as validation group). Results were shown in table 1 and table 2, respectively.

TABLE-US-00001 TABLE 1 Concentrations of IGFBP2 protein in serum Group I (Discovery Group) Group II (validation Group) Liver Non-liver Liver Non-liver cancer cancer cancer cancer patient patient patient patient (ng/ml) (ng/ml) (ng/ml) (ng/ml) 1 43.48484 37.16512915 1 101.4429 29.72011915 2 42.84814 27.8824984 2 67.78099 34.66665435 3 94.947 32.18498435 3 74.59434 29.72011915 4 51.20713 26.6626694 4 72.53931 26.6626694 5 94.23048 20.6265424 5 49.26238 20.02870635 6 102.898 36.538935 6 347.0552 25.4470416 7 113.2013 26.6626694 7 898.2424 26.6626694 8 66.43093 30.95045115 8 69.13526 38.42066835 9 47.32708 23.0283896 9 73.22327 36.538935 10 63.07414 31.5671926 10 61.07267 23.0283896 11 78.73277 12.352415 11 72.53931 19.4319206 12 175.1997 27.8824984 12 59.08066 32.8038264 13 99.99205 23.0283896 13 1059.827 40.3118544 14 32.80383 27.8824984 14 158.0795 23.0283896 15 29.72012 21.8253654 15 202.1783 29.72011915 16 73.22327 29.1065286 16 31.56719 31.5671926 17 118.4302 33.42371875 17 54.46938 42.21249315 18 150.8841 32.18498435 18 70.49373 21.22542875 19 61.73878 32.8038264 19 47.32708 25.4470416 20 58.41875 19.4319206 20 72.53931 35.91379115 21 176.8544 15.28382875 21 103.6272 21.22542875 22 84.30948 37.7923736 22 57.0981 21.22542875 23 46.04213 30.95045115 23 108.024 24.84080315 24 143.7737 14.6954454 24 49.90958 21.8253654 25 35.91379 36.538935 25 69.81397 21.22542875 26 37.16513 19.4319206 26 57.7579 28.49398835 27 116.183 21.22542875 27 35.91379 13.5218296 28 82.909 21.22542875 28 150.0898 26.05433035 29 792.7261 21.22542875 29 131.3431 22.42635235 30 76.65883 19.4319206 30 112.4586 30.95045115 31 115.436 67.7809926 31 142.9889 15.8732624 32 89.24425 14.10811235 32 283.8218 13.5218296 33 100.717 12.93659715 33 75.96962 30.33476 34 286.6897 17.64786515 34 38.42067 39.0500134 35 84.30948 15.8732624 35 162.1137 42.84814 36 43.48484 36 785.7038 15.28382875 37 77.34909 37 196.1848 38 75.96962 38 63.07414 39 80.12064 39 55.78164 40 47.32708 40 181.0094 41 55.12499 41 95.66457 42 42.21249 42 35.2897 43 37.79237 43 59.08066 44 31.56719 44 36.53894 45 35.2897 45 35.2897 46 32.18498 46 36.53894 47 147.7134 47 30.33476 48 49.26238 48 63.07414 49 38.42067 49 52.50888 50 40.31185 50 46.04213 51 65.75747 51 162.1137 52 70.49373 52 283.8218 53 42.21249 53 71.8564 54 55.78164 54 31.56719 55 61.73878 55 32.18498 56 42.84814 56 42.21249 57 42.21249

TABLE-US-00002 TABLE 2 Concentrations of ERBB3 protein in serum Group I (Discovery Group) Group II (Validation Group) Liver Non-liver Liver Non-liver cancer cancer cancer cancer patient patient patient patient (ng/ml) (ng/ml) (ng/ml) (ng/ml) 1 819.626 136.066 1 768.546 1106.586 2 666.386 187.146 2 1023.946 289.306 3 1177.186 238.226 3 2505.266 289.306 4 2403.106 187.146 4 1381.506 340.386 5 1279.346 289.306 5 1892.306 187.146 6 921.786 136.066 6 1483.666 646.866 7 1483.666 289.306 7 1126.106 391.466 8 1534.746 62.346 8 921.786 62.346 9 870.706 442.546 9 2096.626 289.306 10 819.626 544.706 10 2249.866 136.066 11 1279.346 902.266 11 768.546 187.146 12 1075.026 187.146 12 870.706 62.346 13 1687.986 595.786 13 870.706 136.066 14 7306.786 136.066 14 1177.186 62.346 15 1687.986 271.84 15 972.866 238.226 16 819.626 271.84 16 1790.146 339.2 17 1228.266 372.88 17 1228.266 137.12 18 666.386 406.56 18 1841.226 305.52 19 1177.186 406.56 19 1177.186 69.76 20 2096.626 137.12 20 1075.026 69.76 21 972.866 810.72 21 1432.586 574.96 22 541.586 204.48 22 1279.346 103.44 23 1330.426 642.32 23 921.786 204.48 24 870.706 305.52 24 1841.226 36.08 25 1687.986 372.88 25 1126.106 473.92 26 768.546 271.84 26 1126.106 204.48 27 870.706 541.28 27 1177.186 238.16 28 972.866 137.12 28 1177.186 473.92 29 1432.586 204.48 29 1330.426 170.8 30 666.386 58.16 30 666.386 291.68 31 870.706 204.11 31 768.546 116.54 32 921.786 466.82 32 921.786 320.87 33 1381.506 33 666.386 34 1228.266 34 768.546 35 768.546 35 819.626 36 8685.946 36 921.786 37 1330.426 37 1023.946 38 717.466 38 870.706 39 1075.026 39 1177.186 40 1177.186 40 5825.466 41 1636.906 41 1177.186 42 819.626 42 1330.426 43 1177.186 43 768.546 44 717.466 44 1177.186 45 1841.226 45 972.866 46 972.866 46 972.866 47 972.866 47 768.546 48 1023.946 48 1075.026 49 819.626 49 819.626 50 1652.72 50 305.52 51 608.64 51 4650.24 52 507.6 52 1955.84 53 2730.48 53 1046.48 54 878.08 54 574.96 55 676 55 2797.84 56 1248.56 56 204.48 57 271.84

[0114] The concentrations data shown in table 1 and table 2 were further described as follow:

[0115] (1) Results showed that ERBB3 proteins was a proper biomarker for liver cancer (hepatoma) detection [0116] (i) With reference to FIG. 4, the ERBB3 protein concentration in serum in 113 liver cancer patients were higher than 47 cirrhosis without liver cancer patients (p<0.0001, student's t-test), and also higher than 64 chronic hepatitis B patients (p<0.0001, student's t-test). [0117] (ii) With reference to FIG. 5A, the AUC values of Group I which had 56 liver cancer patients and 32 non-liver cancer but with hepatitis B patients was 97.7%. [0118] (iii) With reference to FIG. 5B, the AUC values of Group II which had 57 liver cancer patients and 32 non-liver cancer but with hepatitis B patients was 96.1%. [0119] (iv) With reference to FIG. 5c, analyzing by the cut-off value of Youden index, the sensitivity value for detecting liver cancer was 89.3% after combining Group I and Group II patient samples, and the specificity value was 99.7%. Similarly, by analyzing AFP in serum, the AUC values of the liver cancer patients and non-liver cancer patients were 91.5% after combining the two groups, but the AUC values of ERBB3 proteins only were 96.8%.

[0120] The above results showed that analyzed by the concentration of ERBB3 protein in serum is more sensitive than analyzed by AFP in serum. Therefore, it is powerful to use ERBB3 protein as a biomarker for detecting liver cancer.

[0121] (2) Results showed that IGFBP2 proteins was a proper biomarker for liver cancer (hepatoma) detection [0122] (i) With reference to FIG. 6, the concentration of IGFBP2 protein in serum in 113 liver cancer patients were higher than 47 cirrhosis without liver cancer patients (p<0.001, student's t-test), and also higher than 64 chronic hepatitis B patients (p<0.001, student's t-test). [0123] (ii) With reference to FIG. 7A, the AUC value of the serum in Group I which had 57 liver cancer patients and 35 hepatitis B without liver cancer patients were 96.4%. [0124] (iii) With reference to FIG. 7B, the AUC value of the serum in Group II which had 56 liver cancer patients and 36 hepatitis B without liver cancer patients were 96.24%. [0125] (iv) With reference to FIG. 7C, the AUC value was 96.2% after combing Group I and Group II patient samples. However, by detecting AFP in serum, the AUC values of the liver cancer patients and non-liver cancer patients were 71.5% after combining the two Groups. Therefore, detecting by the concentration of IGFBP2 in serum to identify liver cancer patients and non-liver cancer with hepatitis B patients were more sensitive and specificities than detecting by AFP values.

[0126] The above results showed that analyzed by the concentration of IGFBP2 protein in serum is more sensitive than analyzed by AFP values in serum. Therefore, it is powerful to use IGFPB2 protein as a biomarker for detecting liver cancer.

[0127] Combination detection of AFP, ERBB3 protein and IGFBP2 protein in serum to increase the sensitivity and specificity of liver cancer detection [0128] (i) The AUC values of AFP, ERBB3 and IGFBP2 were 84.3%, 96.8% and 96.1%, respectively. [0129] (ii) With reference to FIGS. 8A and 8B, the AUC values of AFP+ERBB3 and AFP+IGFBP2 were 96.9% and 94.5%, respectively. With reference to FIG. 8C, the AUC value of ERBB3+IGFBP2 was 98.5%. Furthermore, with reference to FIG. 8D, the AUC value of AFP+ERBB3+IGFBP2 was 99.1%. Therefore, to increase the sensitivity and specificity to almost 100%, it is very useful to combine AFP value, ERBB3 value and IGFBP2 value for diagnosis of hepatoma.

[0130] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0131] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.

Sequence CWU 1

411342PRTHomo sapiens 1Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu1 5 10 15Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 20 25 30Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile65 70 75 80Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 85 90 95Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 100 105 110Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 115 120 125His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 130 135 140Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr145 150 155 160Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 165 170 175Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 180 185 190Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 195 200 205Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn 210 215 220Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp225 230 235 240Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val 245 250 255Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu 260 265 270Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala 275 280 285Ser Cys Pro His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala 290 295 300Cys Pro Pro Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys305 310 315 320Glu Pro Cys Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser 325 330 335Gly Ser Arg Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val 340 345 350Asn Cys Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 355 360 365Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 370 375 380Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln385 390 395 400Ser Trp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 405 410 415Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 420 425 430Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 435 440 445Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr 450 455 460His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu465 470 475 480Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu 485 490 495Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro 500 505 510Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val 515 520 525Cys Val Thr His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala 530 535 540His Glu Ala Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu545 550 555 560Gly Thr Ala Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys 565 570 575Ala His Phe Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly 580 585 590Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn 595 600 605Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro 610 615 620Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr625 630 635 640His Leu Thr Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe 645 650 655Met Met Leu Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln 660 665 670Asn Lys Arg Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu 675 680 685Pro Leu Asp Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe 690 695 700Lys Glu Thr Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly Val Phe705 710 715 720Gly Thr Val His Lys Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys 725 730 735Ile Pro Val Cys Ile Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser 740 745 750Phe Gln Ala Val Thr Asp His Met Leu Ala Ile Gly Ser Leu Asp His 755 760 765Ala His Ile Val Arg Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln 770 775 780Leu Val Thr Gln Tyr Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg785 790 795 800Gln His Arg Gly Ala Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val 805 810 815Gln Ile Ala Lys Gly Met Tyr Tyr Leu Glu Glu His Gly Met Val His 820 825 830Arg Asn Leu Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val 835 840 845Gln Val Ala Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys 850 855 860Gln Leu Leu Tyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu865 870 875 880Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser 885 890 895Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr 900 905 910Ala Gly Leu Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu 915 920 925Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met 930 935 940Val Lys Cys Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu945 950 955 960Leu Ala Asn Glu Phe Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu 965 970 975Val Ile Lys Arg Glu Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro 980 985 990His Gly Leu Thr Asn Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu 995 1000 1005Leu Asp Leu Asp Leu Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr 1010 1015 1020Thr Thr Leu Gly Ser Ala Leu Ser Leu Pro Val Gly Thr Leu Asn Arg1025 1030 1035 1040Pro Arg Gly Ser Gln Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro 1045 1050 1055Met Asn Gln Gly Asn Leu Gly Glu Ser Cys Gln Glu Ser Ala Val Ser 1060 1065 1070Gly Ser Ser Glu Arg Cys Pro Arg Pro Val Ser Leu His Pro Met Pro 1075 1080 1085Arg Gly Cys Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr Gly Ser 1090 1095 1100Glu Ala Glu Leu Gln Glu Lys Val Ser Met Cys Arg Ser Arg Ser Arg1105 1110 1115 1120Ser Arg Ser Pro Arg Pro Arg Gly Asp Ser Ala Tyr His Ser Gln Arg 1125 1130 1135His Ser Leu Leu Thr Pro Val Thr Pro Leu Ser Pro Pro Gly Leu Glu 1140 1145 1150Glu Glu Asp Val Asn Gly Tyr Val Met Pro Asp Thr His Leu Lys Gly 1155 1160 1165Thr Pro Ser Ser Arg Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser 1170 1175 1180Val Leu Gly Thr Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met1185 1190 1195 1200Asn Arg Arg Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser 1205 1210 1215Leu Glu Glu Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser 1220 1225 1230Ala Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile 1235 1240 1245Met Pro Thr Ala Gly Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn 1250 1255 1260Arg Gln Arg Asp Gly Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly1265 1270 1275 1280Ala Cys Pro Ala Ser Glu Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln 1285 1290 1295Gly Pro Gly His Gln Ala Pro His Val His Tyr Ala Arg Leu Lys Thr 1300 1305 1310Leu Arg Ser Leu Glu Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp Tyr 1315 1320 1325Trp His Ser Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr 1330 1335 13402624PRTHomo sapiens 2Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr Leu Asn Gly1 5 10 15Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr Leu Tyr Lys 20 25 30Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu Ile Val Leu 35 40 45Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile Arg Glu Val 50 55 60Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu65 70 75 80Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe 85 90 95Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu 100 105 110Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val 115 120 125Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp 130 135 140Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp Asn145 150 155 160Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly Arg Cys Trp 165 170 175Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala 180 185 190Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys 195 200 205His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys 210 215 220Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys225 230 235 240Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn 245 250 255Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys Pro 260 265 270His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala Cys Pro Pro 275 280 285Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys Met Cys Glu Pro Cys 290 295 300Gly Gly Leu Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg305 310 315 320Phe Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr 325 330 335Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp 340 345 350Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu Asn Val Phe 355 360 365Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln Ser Trp Pro 370 375 380Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr Thr Ile Gly385 390 395 400Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile Met Lys Asn 405 410 415Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala 420 425 430Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser 435 440 445Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp 450 455 460Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu Gly Lys Val465 470 475 480Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro Gly 485 490 495Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val Cys Val Thr 500 505 510His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu Phe Ala His Glu Ala 515 520 525Glu Cys Phe Ser Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala 530 535 540Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe545 550 555 560Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly 565 570 575Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg 580 585 590Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu Gln 595 600 605Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr His Leu Thr 610 615 6203328PRTHomo sapiens 3Met Leu Pro Arg Val Gly Cys Pro Ala Leu Pro Leu Pro Pro Pro Pro1 5 10 15Leu Leu Pro Leu Leu Pro Leu Leu Leu Leu Leu Leu Gly Ala Ser Gly 20 25 30Gly Gly Gly Gly Ala Arg Ala Glu Val Leu Phe Arg Cys Pro Pro Cys 35 40 45Thr Pro Glu Arg Leu Ala Ala Cys Gly Pro Pro Arg Val Ala Pro Pro 50 55 60Ala Ala Val Ala Ala Val Ala Gly Gly Ala Arg Met Pro Cys Ala Glu65 70 75 80Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Ser Val Cys Ala Arg Leu 85 90 95Glu Gly Glu Ala Cys Gly Val Tyr Thr Pro Arg Cys Gly Gln Gly Leu 100 105 110Arg Cys Tyr Pro His Pro Gly Ser Glu Leu Pro Leu Gln Ala Leu Val 115 120 125Met Gly Glu Gly Thr Cys Glu Lys Arg Arg Asp Ala Glu Tyr Gly Ala 130 135 140Ser Pro Glu Gln Val Ala Asp Asn Gly Asp Asp His Ser Glu Gly Gly145 150 155 160Leu Val Glu Asn His Val Asp Ser Thr Met Asn Met Leu Gly Gly Gly 165 170 175Gly Ser Ala Gly Arg Lys Pro Leu Lys Ser Gly Met Lys Glu Leu Ala 180 185 190Val Phe Arg Glu Lys Val Thr Glu Gln His Arg Gln Met Gly Lys Gly 195 200 205Gly Lys His His Leu Gly Leu Glu Glu Pro Lys Lys Leu Arg Pro Pro 210 215 220Pro Ala Arg Thr Pro Cys Gln Gln Glu Leu Asp Gln Val Leu Glu Arg225 230 235 240Ile Ser Thr Met Arg Leu Pro Asp Glu Arg Gly Pro Leu Glu His Leu 245 250 255Tyr Ser Leu His Ile Pro Asn Cys Asp Lys His Gly Leu Tyr Asn Leu 260 265 270Lys Gln Cys Lys Met Ser Leu Asn Gly Gln Arg Gly Glu Cys Trp Cys 275 280 285Val Asn Pro Asn Thr Gly Lys Leu Ile Gln Gly Ala Pro Thr Ile Arg 290 295 300Gly Asp Pro Glu Cys His Leu Phe Tyr Asn Glu Gln Gln Glu Ala Arg305 310 315 320Gly Val Asp Thr Gln Arg Met Gln3254289PRTHomo sapiens 4Glu Val Leu Phe Arg Cys Pro Pro Cys Thr Pro Glu Arg Leu Ala Ala1 5 10 15Cys Gly Pro Pro Arg Val Ala Pro Pro Ala Ala Val Ala Ala Val Ala 20 25 30Gly Gly Ala Arg Met Pro Cys Ala Glu Leu Val Arg Glu Pro Gly Cys 35 40 45Gly Cys Cys Ser Val Cys Ala Arg Leu Glu Gly Glu Ala Cys Gly Val 50 55 60Tyr Thr Pro Arg Cys Gly Gln Gly Leu Arg Cys Tyr Pro His Pro Gly65 70 75 80Ser Glu Leu Pro Leu Gln Ala Leu Val Met Gly Glu Gly Thr Cys Glu 85 90 95Lys Arg Arg Asp Ala Glu Tyr Gly Ala Ser Pro Glu Gln Val Ala Asp 100 105 110Asn Gly Asp Asp His Ser Glu Gly Gly Leu Val Glu Asn His Val Asp 115 120 125Ser Thr Met Asn Met Leu Gly Gly Gly Gly Ser Ala Gly Arg Lys Pro 130 135 140Leu Lys Ser Gly Met Lys Glu Leu Ala Val Phe Arg Glu Lys Val Thr145 150 155 160Glu Gln His Arg Gln Met Gly Lys Gly Gly Lys His His Leu Gly Leu 165 170

175Glu Glu Pro Lys Lys Leu Arg Pro Pro Pro Ala Arg Thr Pro Cys Gln 180 185 190Gln Glu Leu Asp Gln Val Leu Glu Arg Ile Ser Thr Met Arg Leu Pro 195 200 205Asp Glu Arg Gly Pro Leu Glu His Leu Tyr Ser Leu His Ile Pro Asn 210 215 220Cys Asp Lys His Gly Leu Tyr Asn Leu Lys Gln Cys Lys Met Ser Leu225 230 235 240Asn Gly Gln Arg Gly Glu Cys Trp Cys Val Asn Pro Asn Thr Gly Lys 245 250 255Leu Ile Gln Gly Ala Pro Thr Ile Arg Gly Asp Pro Glu Cys His Leu 260 265 270Phe Tyr Asn Glu Gln Gln Glu Ala Arg Gly Val Asp Thr Gln Arg Met 275 280 285Gln

Claims

1. A method for detection of liver cancer, comprising steps of: a) providing a liquid sample obtained from an individual, b) contacting said sample with an antibody specific for at least one of ERBB3 protein (SEQ ID NO:2) under conditions appropriate for formation of a complex between said antibody and at least one of said proteins, and c) correlating an amount of the complex formed in step d) to the detection of liver cancer.

2 and 3. (canceled)

4. The method according to claim 1 wherein the liquid sample is whole blood.

5-9. (canceled)

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