Plasma biomarker tool for the diagnosis of liver cancer comprising liver carboxylesterase 1 and liver cancer screening method

Abstract

The present invention relates to a plasma biomarker for diagnosing hepatocellular carcinoma (HCC), in particular to the discovery of a protein in plasma using 2-D fluorescence differential gel electrophoresis (2-D DIGE), immunoprecipitation and Nano-liquid chromatography mass spectrometry (Nano-LC-MS/MS) system that was unknown on the basis of conventional techniques. By demonstrating the presence of liver carboxylesterase 1 (hCE1) in human plasma and confirming that its secretion level is higher in patients with HCC than in healthy volunteers, this invention may be used as a screening method to diagnose HCC at an early stage.

Claims

.[.1. A screening method for hepatocellular carcinoma (HCC), comprising: collecting human blood, and detecting the presence of human liver carboxylesterase 1(hCE1) protein in plasma of the human blood as a plasma biomarker for HCC diagnosis; wherein the level of hCE1 protein is increased more in the plasma of patients with HCC than in the plasma of healthy patients..].

.[.2. The method of claim 1, wherein the level of hCE1 protein is increased, on average, 2-5 fold more in the plasma of patients with HCC compared to the plasma of healthy patients..].

.[.3. The method of claim 1, wherein the presence of the hCE1 protein is detected by an anti-hCE1 antibody..].

.Iadd.4. A method for diagnosing hepatocellular carcinoma (HCC) in a subject, the method comprising the steps of: a) collecting a sample of human blood from the subject; b) contacting a portion of the blood sample with an antibody having specific binding affinity for human liver carboxylesterase 1 (hCE1), thereby forming a complex between the antibody and hCE1, the antibody having a detectable label; c) separating the complex formed in said contacting step (b) from labeled antibody not comprising the complex; d) quantifying a signal from the detectable label of the antibody comprising the complex formed in said contacting step (b), the signal being proportional to an amount of hCE1 in the blood sample, whereby the amount of hCE1 in the sample is calculated; e) comparing the amount of hCE1 calculated in said quantifying step (d) to a hCE1 reference amount; and f) providing a diagnosis of HCC in the subject if the amount of hCE1 in the sample calculated in said quantifying step (d) is greater than the hCE1 reference amount; wherein the hCE1 reference amount is an amount of hCE1 in a blood sample from a subject not having HCC. .Iaddend.

.Iadd.5. The method of claim 4, wherein the blood sample comprises plasma. .Iaddend.

.Iadd.6. The method of claim 4, wherein the blood sample comprises serum. .Iaddend.

.Iadd.7. The method of claim 4, further comprising the step of separating human liver carboxylesterase 1 (hCE1) protein from other remaining proteins in the blood sample, wherein said separating hCE1 protein step occurs between steps (a) and (b). .Iaddend.

.Iadd.8. The method of claim 7, wherein the hCE1 protein is separated from other remaining proteins in the blood sample by immunoprecipitation. .Iaddend.

.Iadd.9. The method of claim 7, wherein the step of separating hCE1 protein from other remaining proteins in the blood sample comprises the following steps: i) contacting a portion of the sample from the subject with an antibody having specific binding affinity for hCE1, thereby forming a complex between the antibody and hCE1; and ii) precipitating the complex formed in said contacting step (i); iii) separating the precipitated complex from the supernatant of the sample, the supernatant comprising the other remaining proteins and antibody not comprising the complex; wherein the hCE1 protein is separated from the other remaining proteins in the blood sample. .Iaddend.

.Iadd.10. The method of claim 9, wherein the antibody is linked to a magnetic molecule. .Iaddend.

.Iadd.11. The method of claim 4, wherein the hCE1 reference amount is an amount of hCE1 in the blood of a healthy subject. .Iaddend.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a photograph showing a 2-D DIGE image of fractionated glycoproteins from non-tumorous liver tissue from a patient with HCC; the position of hCE1 is indicated.

(2) FIG. 2 is a photograph showing a 2-D DIGE image of fractionated glycoproteins from tumorous liver tissue from a patient with HCC; the position of hCE1 is indicated.

(3) FIG. 3 is a photograph showing the differences in hCE1 expression levels in paired samples of non-tumorous and tumorous liver tissue from additional ten patients with HCC.

(4) N: Non-tumorous liver tissue

(5) T: Tumorous liver tissue

(6) FIG. 4 is a photograph of an immunohistochemically stained, paraffin-embedded tissue array constructed from paired non-tumorous and tumorous liver sections from 47 patients with HCC showing the level of hCE1 expression.

(7) FIG. 5 is a graph depicting the frequency distribution of staining scores determined by immunohistochemical staining in paired non-tumorous and tumorous liver sections of patients with HCC. The results indicate that the level of hCE1 expression is specifically reduced in the tumorous liver sections compared to the non-tumorous liver sections.

(8) FIG. 6 is a table showing the tryptic peptides of hCE1 identified by Nano-LC-MS/MS system in the plasma of healthy volunteers and patients with HCC, and used to validate the presence of hCE1 in human plasma.

(9) FIG. 7 is a representative Nano-LC-MS/MS chromatograph of the common peptides shown in FIG. 6.

(10) FIG. 8 is a photograph showing the differences in hCE1 levels between representative three healthy volunteers and three patients with HCC, determined using Western blot analysis after immunoprecipitation.

(11) Np: plasma of healthy volunteer

(12) Tp: plasma of patient with HCC

(13) FIG. 9 is a graph comparing the hCE1 levels in the plasma of eight healthy volunteers and eight patients with HCC.

BEST MODE FOR CARRYING OUT THE INVENTION

(14) The present invention is described in detail in the following embodiments. The embodiments are only included to describe the present invention, however, the scope of the present invention is not limited to the embodiments.

(15) Embodiment 1: Collection of Clinical Tissues and Plasma

(16) The non-tumorous and tumorous liver tissue from patients with HCC were obtained along with pathological individual information from the Department of Pathology, Yonsei University College of Medicine, Seoul, Korea. The following tests were used to assess the appropriateness of plasma from healthy volunteers to serve as a negative control: HIV-1 and HIV-2 antibodies derived from HIV, which is a liver-cancer indicating standard test element; HIV-1 antigen; hepatitis B surface antigen; hepatitis B core antigen; hepatitis C virus; T-Cell Leukemia Virus (HTLV-I/II) antigen; and Treponema pallidum. Plasma was obtained according to a standardized sample separation method formally adopted by the Human Proteome Organization (HUPO).

(17) Each blood sample (4 ml) from man or woman was maintained in a tube containing di-potassium ethylenediamine tetraacetic acid (K.sub.2EDTA) for 30 minutes at room temperature, followed by centrifugation for 15 minutes at 2,400×g. A supernatant (plasma) was retained. Samples of non-tumorous, tumorous liver tissue, and plasma were stored at −70° C. until ready for use. Liver tissues and plasma were acquired according to approved procedures of the Institutional Review Board (IRB) at Yonsei University College of Medicine with informed consent of the patients.

(18) Embodiment 2: Separation of Glycoproteins in Non-Tumorous and Tumorous Liver Tissue

(19) A 100 mg sample of each tissue was homogenized in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, pH 7.4) at 4° C. Glycoproteins were separated by lectin-affinity chromatography using a mixture of five different agarose-bound lectins: concanavalin A, wheat germ agglutinin, Jacalin, Sambucus nigra, and Aleuria aurantia. The lectin mixture, with binding specificities for glycoproteins with different sugar compositions, was packed into a 2 ml PD-10 column (Pierce). Glycoprotein-containing samples were applied to the column in binding buffer (20 mM Tris, 1 mM MnCl.sub.2, 1 mM CaCl.sub.2, 0.15 M NaCl, pH 7.4) and allowed to interact with the column for 30 minutes.

(20) The bound glycoproteins were eluting with a buffer (0.2 M methyl-D-mannopyroside, 0.2 M methyl-D-glucopyroside, 0.2 M N-acetylglucosamine, 0.2 M galactose, 0.1 M lactose, 0.1 M fucose, 20 mM Tris, 0.5 M NaCl, pH 7.0) that displaces glycoproteins on the basis of sugar composition. The eluted glycoproteins were concentrated using a 5-kDa membrane filter, and precipitated with 50% trichloroacetic acid and 100% ice-cold acetone. The precipitate was dissolved in 2-D lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, pH 8.5), and the resulting protein solution was adjusted to pH 8.5. The concentration of the protein was measured using a 2D Quant kit (GE Healthcare).

(21) Embodiment 3: 2-Dimensional Fluorescence Differential Gel Electrophoresis (2-D DIGE)

(22) Ten paired samples (five from non-tumorous liver tissue and five from tumorous liver tissue) from five patients were used to prepare analytic samples (50 μg each) for 2-D DIGE. Analytic samples and a corresponding pooled standard sample were labeled individually with the fluorescent dyes, Cy3, Cy5 and Cy2 (400 pmol; GE Healthcare) in the dark for 30 minutes.

(23) The reaction was stopped by adding 1 μl 10 mM lysine, and the three labeled samples were mixed, adjusted to a final volume of 450 μl by adding sample buffer (6 M urea, 2 M thiourea, 4% CHAPS, 60 mM dithiothreitol (DTT), 30 mM Tris, pH 8.5) and rehydrated by incubating with a 2% IPG 3-10 NL buffer solution for 16 hours at room temperature.

(24) Isoelectric focusing (IEF) was performed using the Immobiline DryStrip pH 3-10 NL under optimized conditions (up to 95,000 V) on the MultiPhor II electrophoresis system (GE Healthcare), followed by a one-step reduction and alkylation in which the DryStrip was incubated for 25 minutes in a tributylphosphine (TBP) buffer solution (6 M urea, 2% sodium dodecyl sulfate [SDS], 30 mM Tris, 20% glycerol, 2.5% acrylamide solution, 5 mM TBP).

(25) After reduction and alkylation, the isoelectrically focused proteins were separated in the second dimension by electrophoresis on 9%-16% gradient polyacrylamide gels using an Ettan Dalt-twelve electrophoresis system (GE Healthcare). Each gel was then scanned at wavelengths corresponding to Cy2, Cy3 and Cy5 using a Typhoon 9400 (GE Healthcare) scanner, and images of each gel were analyzed using Decyder 2-D analysis software (GE Healthcare).

(26) Embodiment 4: Identification of 2-D DIGE-Separated Proteins

(27) After images were analyzed, a spot corresponding to a significantly differentially expressed protein was excised from a Coomassie blue-stained gel, destained and digested with trypsin. The digested peptides were desalted using a Poros R2 and Oligo R3 resin mixture. The protein was analyzed using a 4800 MALDI-TOF-MS (Applied Biosystem) and the spectra were identified using a MASCOT database.

(28) Embodiment 5: Validation of HCC Biomarker Expression Levels in Tissues by Western Blot Analysis

(29) Differences in HCC biomarker protein concentration between non-tumorous and tumorous liver tissue from ten patients were verified by Western blotting analysis. An equal amount of protein (5 μg) from each sample was separated by SDS-PAGE on 10% gels.

(30) Proteins were then transferred to a nitrocellulose (NC) membrane, and blocked by incubating in TBS-T buffer (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6) containing 5% skim milk for 1 hour at roam temperature. Membranes were then incubated with primary anti-hCE1 antibody (CE1, Abcam; 1:10000 in TBS-T/5% skim milk) for 1 hour at room temperature. A mouse β-actin antibody (Santa Cruz) was used as a positive control.

(31) After washing with TBS-T/5% skim milk, membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Santa Cruz; 1:20000 dilution) for 1 hour at room temperature. Immunoreactive proteins were detected using ECL Plus Western blot reagents (GE Healthcare), and blots were scanned and analyzed using a Typhoon 9400 scanner.

(32) Embodiment 6: Validation of HCC Biomarker Expression Levels in Tissues Using an Immunohistochemical Staining Method

(33) Paraffin-embedded tissue arrays constructed from paired tissues from non-tumorous and tumorous liver sections of 47 patients with HCC were used for immunohistochemistry. The slides were deparaffinized with xylene and alcohol, hydrated, and then treated with 0.6% hydrogen peroxide. Anti-hCE1 antibody (1:100) was applied to the slide and incubated for 30 minutes. hCE1-bound antibody color was developed using ChemMate Envision Kit (DAKO) and colorized with hematoxylin (used as a contrast staining agent) for 30 seconds. The intensity of staining was scored as 1) “−”, for no staining; 2) “+”, for weak staining; and 3) “++”, for strong staining.

(34) Embodiment 7: Validation of HCC Biomarker Protein Secretion Level in Human Plasma by Immunoprecipitation and Western Blot Analysis

(35) Dynabead MyOne™ Tosylactivated (Invitrogen) magnetic beads were coated with anti-hCE1 antibody as described by the manufacturer. Briefly, magnetic beads (10 mg) and anti-hCE1 antibody (400 μg) were mixed and incubated for 16 hours in binding buffer (0.1 M sodium borate, 1 M ammonium sulfate, pH 9.5) at 37° C., and then blocked with TBS-T/5% skim milk for an additional 16 hours. hCE1 was immunoprecipitated from the plasma of healthy volunteers and patients with HCC by transferring antibody-coated beads (500 μg) to 1 ml tubes containing 8 mg plasma and incubating for 2 hours.

(36) Antibody-bound proteins were eluted by adjusting the pH of the TBS-T buffer to pH 2. Differences in hCE1 levels between the plasmas of healthy volunteers and patients with HCC were determined by Western blot analysis using a primary anti-hCE1 antibody (Abcam; 1:1000) and a secondary anti-rabbit IgG-HRP antibody (Santa Cruz; 1:5000), as described in Embodiment 5.

(37) Embodiment 8: Validation of hCE1 Protein in Human plasma by Nano-LC-MS/MS System

(38) A band corresponding to hCE1 based on Western blot analysis (embodiment 7) was excised from the gel, and reduced and alkylated by dithiothrietol(DTT)/iodoacetic acid (IAA) treatment. After digesting with trypsin to yield peptides, the identity of the gel-isolated protein as hCE1 was validated using Nano-LC-MS/MS system and a linear trap quadrupole (LTQ) detector.

INDUSTRIAL APPLICABILITY

(39) As described above, hCE1 was discovered as a new biomarker for HCC diagnosis. According to the present invention, hCE1 protein secretion level in plasma can function as an index that allows early diagnosis of HCC, and thereby contributes to increasing the survival rate among patients with HCC by enabling timely therapeutic intervention.