Diagnostic Value of Serum SMP30 and Anti-SMP30 Antibody in Hepatocellular Carcinoma

Diagnostic Value of Serum SMP30 and Anti-SMP30 Antibody in Hepatocellular Carcinoma Abstract Objective To evaluate the clinical value of senescence marker protein 30 (SMP30) and anti-SMP30 antibody in serum. Methods We used enzyme-linked immunosorbent assay (ELISA) to analytically validate serum levels of SMP30 and anti-SMP30 antibody in 143 patients with hepatocellular carcinoma (HCC), compared with those levels in serum from 137 patients with chronic hepatitis (CH), 51 with liver cirrhosis (LC), and 165 healthy control individuals. Results The positivity rate of SMP30 in the HCC group (8.39%) was significantly higher than that rate in the CH group (.73%) and in the healthy control group (1.21%). The positivity rate for anti-SMP30 antibody in patients with HCC was 25.87%, that in the CH group was 4.38%, and that in the LC group was 3.92%. Conclusion Anti-SMP30 antibody levels can be used as a biomarker for diagnosing HCC; marked results have been observed for patients with alpha-fetoprotein (AFP) negativity, in particular. hepatocellular carcinoma (HCC), senescence marker protein 30 (SMP30), serum, anti-SMP30 antibody, chronic hepatitis (CH), liver cirrhosis (LC) It has been reported1,2 that approximately 80% of liver cancer worldwide is primary liver cancer (PLC). Also, the hepatocellular carcinoma (HCC) histological subtype is highly common in China; this subtype ranks first among all malignant tumors in Guangxi province. Despite the improvement in diagnosing and treatment of HCC, 5-year overall survival for the patients is only 3% to 5%.3 This finding is partly attributed to the lack of a reliable method for early diagnosis (to our knowledge). So far, alpha-fetoprotein (AFP) has been used as the most common serum biomarker for diagnosis of HCC. However, its sensitivity is only 25% to 65% at the standard cutoff of 25 ng per mL, especially when diagnosing in the early stages of HCC.4 Therefore, it is important to develop serological markers for effectively diagnosing HCC at early stages and to monitor aggressiveness, responsiveness to treatments, tumor recurrence, and patient survival rate. It has been shown that senescence marker protein 30 (SMP30), otherwise known as regucalcin (RGN), a calcium-binding protein discovered by Fujita et al in 1992,5 plays a crucial role in protective effects against apoptosis and oxidative stress.6 Using the serologic identification by recombinant expression cloning (SEREX) approach, SMP30 was identified in the Guangxi HCC complementary DNA (cDNA) expression library as being an HCC-associated antigen.7 The results of immunohistochemical analysis showed that the levels of SMP30 expression in HCC paracancerous tissues were higher than those in the corresponding HCC.8 The low expression of SMP30 protein in HCC is closely related to the survival prognosis for patients.9 The aforementioned studies have shown that expressions of SMP30 are important in the progression of liver cancer. However, whether the concentration of SMP30 in serum can be used as a diagnostic indicator of liver cancer has not been reported, to our knowledge. Numerous studies10,11 have shown that the tumor itself can induce the immune response of the body to produce antibody related to the antitumor antigen. This response can be tested even when the tumor antigen expression is low. Therefore, the antibody related to the antitumor antigen is understood as the symbol of tumor diagnosis.12 Previously, Western blotting (WB) has been used to confirm the presence of SMP30 and anti-SMP30 antibody in the serum of patients with HCC, which suggests that detection of SMP30 and anti-SMP30 antibody levels may be understood as auxiliary indices for HCC diagnosis.13 However, as a serological diagnostic index of HCC, the specificity and sensitivity of those methods for HCC diagnosis still requires greater nontumor serological verification. Based on the aforementiond analyses, we have examined the levels of serum SMP30 together with anti-SMP30 antibody in 495 serum specimens from 143 patients with HCC, 51 patients with liver cirrhosis (LC), 137 patients with chronic hepatitis (CH), and 165 healthy control individuals to validate the diagnostic value. Materials and Methods Patients and Specimen Obtaining Procedures The BL21 (DE3) pLysS-pColdIII-SMP30-pTf16 recombinant genetic-engineering strain was constructed by our research team and stored at −80°C.14 This study included 331 patients, of whom 143 with HCC were recruited by the Affiliated Tumor Hospital of Guangxi Medical University, China. A total of 137 patients with CH and 51 with LC were followed up at the first Affiliated Hospital of the same university. The diagnoses of CH and LC were carried out by abdominal ultrasound scanning and laboratory testing. Patients with HCC were diagnosed by histology and laboratory testing, including computed tomography (CT) scanning, abdominal ultrasound scanning, and magnetic resonance imaging (MRI). The clinical stages were classified according to the International Union against Cancer TNM classification.15 Neither chemotherapy nor surgical treatments had been given to the patients. Physical examinations and laboratory tests were performed at the time that blood specimens were obtained. The normal human serum (NHS) specimens were obtained from 165 healthy college students, aged 17 through 23, who had tested negative for hepatitis B surface antigen (HBsAg) and whose liver function and abdominal ultrasound test results revealed no abnormalities (Table 1). The investigation procedures were conducted in compliance with the guidelines of the Ethical Committee of Guangxi Medical University. Oral informed consent was obtained from all participating patients and healthy donors. The serum specimens were divided into aliquots and stored at −80°C until they were analyzed. Table 1. Test Results From Specimens, by Donor Characteristics Variable HCC (n = 143) CH (n = 137) LC (n = 51) NHS (n = 165) Age, y Mean (SD) 50.06 (11.7) 41.28 (11.05) 45.47 (12.01) 19.13 (1.56)  Range 15–81 15–69 23–70 17–23 Sex  Male 123 120 42 95  Female 20 17 9 70 ALT  <40 U/L 70 29 18 165  ≥ 40 U/L 73 108 33 NA AFP  <5 ng/mL 60 77 27 NA  ≥ 25 ng/mL 83 60 24 NA  HBsAg 121 131 28 NA BCLC  A 46 NA NA NA  B 42 NA NA NA  C 55 NA NA NA Size of Tumor (Diameter)  <3 cm 20 NA NA NA  ≥ 3 cm 123 NA NA NA Variable HCC (n = 143) CH (n = 137) LC (n = 51) NHS (n = 165) Age, y Mean (SD) 50.06 (11.7) 41.28 (11.05) 45.47 (12.01) 19.13 (1.56)  Range 15–81 15–69 23–70 17–23 Sex  Male 123 120 42 95  Female 20 17 9 70 ALT  <40 U/L 70 29 18 165  ≥ 40 U/L 73 108 33 NA AFP  <5 ng/mL 60 77 27 NA  ≥ 25 ng/mL 83 60 24 NA  HBsAg 121 131 28 NA BCLC  A 46 NA NA NA  B 42 NA NA NA  C 55 NA NA NA Size of Tumor (Diameter)  <3 cm 20 NA NA NA  ≥ 3 cm 123 NA NA NA HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis; NHS, normal human serum; ALT, alanine transaminase; NA, nonapplicable; AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer View Large Table 1. Test Results From Specimens, by Donor Characteristics Variable HCC (n = 143) CH (n = 137) LC (n = 51) NHS (n = 165) Age, y Mean (SD) 50.06 (11.7) 41.28 (11.05) 45.47 (12.01) 19.13 (1.56)  Range 15–81 15–69 23–70 17–23 Sex  Male 123 120 42 95  Female 20 17 9 70 ALT  <40 U/L 70 29 18 165  ≥ 40 U/L 73 108 33 NA AFP  <5 ng/mL 60 77 27 NA  ≥ 25 ng/mL 83 60 24 NA  HBsAg 121 131 28 NA BCLC  A 46 NA NA NA  B 42 NA NA NA  C 55 NA NA NA Size of Tumor (Diameter)  <3 cm 20 NA NA NA  ≥ 3 cm 123 NA NA NA Variable HCC (n = 143) CH (n = 137) LC (n = 51) NHS (n = 165) Age, y Mean (SD) 50.06 (11.7) 41.28 (11.05) 45.47 (12.01) 19.13 (1.56)  Range 15–81 15–69 23–70 17–23 Sex  Male 123 120 42 95  Female 20 17 9 70 ALT  <40 U/L 70 29 18 165  ≥ 40 U/L 73 108 33 NA AFP  <5 ng/mL 60 77 27 NA  ≥ 25 ng/mL 83 60 24 NA  HBsAg 121 131 28 NA BCLC  A 46 NA NA NA  B 42 NA NA NA  C 55 NA NA NA Size of Tumor (Diameter)  <3 cm 20 NA NA NA  ≥ 3 cm 123 NA NA NA HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis; NHS, normal human serum; ALT, alanine transaminase; NA, nonapplicable; AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer View Large Preparation of Soluble Recombinant HIS-SMP30 Protein We prepared soluble recombinant HIS-SMP30 protein in our laboratory. In brief, the BL21(DE3)pLysS-pColdIII-SMP30-pTf16 recombinant genetic-engineering strain was cultured and induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) to express HIS-SMP30; the fusion protein is 165 amino acids near the C-terminal. The supernatant of bacteria with sonication was harvested and loaded into the nitrilotriacetic acid (Ni-NTA) affinity column, and then purified HIS-SMP30 genetic-engineering protein was obtained. We performed verification testing of the target protein by WB in which the primary antibody was the murine-origin anti-SMP30 monoclonal antibody and the secondary antibody was goat anti-mouse horseradish peroxidase–conjugated immunoglobulin G (IgG-HRP). The aliquots of target protein were frozen and stored at −80°C for further use. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for Serum Anti-SMP30 Antibody We used 2.5 μg per mL purified HIS-SMP30 protein to coat the ELISA plate at 4°C overnight. The wells were then emptied and washed 3 times with phosphate-buffered saline with Tween 20 (PBST) and with 0.5% bovine serum albumin (BSA) closure at 37°C for 1 hour. We then added serum diluted (1:1024) with PBST at 37°C for 1 hour; all serum specimens underwent this process twice. After washing, we added biotin goat anti–human IgG antibody (Boster Biological Engineering) to the wells and then incubated the resulting product for 1 hour at 37°C, followed by washing. The horseradish peroxidase (HRP)–avidin was added for 40 minutes at 37°C, the plates were washed, and 100 μL of tetramethyl benzidine (TMB) substrate solution was added. The TMB color development was conducted for 15 minutes and then 2 M of sulfuric acid was used to terminate the reaction. The optical densities were read at 450 nm using a microplate reader. The optical density (OD) values of all the results were deducted from the blank control values, and the result was determined by the X¯+3SD of the NHS group as the cutoff value. Double Antibody Sandwich ELISA for Serum SMP30 ELISA plates were prepared by coating them with 100 μL per well of rabbit anti–human SMP30 polyclonal antibody at 4°C overnight. The wells were emptied, followed by washing 3 times with PBST, and with 0.5% BSA closure at 37°C for 1 hour. All serum specimens were diluted to 1:5 with PBST and loaded with 100 μL of diluted serum per well. The plates were incubated for 1 hour at 37°C and washed 3 times. The plates were incubated with 100 μL per well of mouse anti–human SMP30 monoclonal antibody for 1 hour at 37°C. The plates were once again washed 3 times and then incubated with 100 μL per well of goat anti-mouse IG-HRP solution in phosphate-buffered saline (PBS). After all the aformentioned treatments, the plates were further incubated for 1 more hour at 37°C plus washing. We then added 100 μL of TMB substrate solution. The TMB color development was conducted for 15 minutes, and then 2 M of sulfuric acid was used to terminate the reaction. We used a microplate reader to read the OD values at 450 nm. The OD values of all the results were deducted from the blank control results, and the result was determined by the X¯+3SD of the NHS group as the cutoff value. Statistical Analysis We adapted SPSS statistical software, version 17.0 (IBM) and GraphPad Prism software (GraphPad Software, Inc) for analyses. We conducted receiver operating characteristic (ROC) analyses to assess sensitivity, specificity, and area under the curve (AUC) with 95% confidence intervals (CIs). The sensitivity and specificity of each biomarker were calculated separately from the concentrations in 496 clinical serum specimens. We used the Pearson χ2 bilateral detection method to test the relationship between the serum test results from the HCC group, various clinical indices, and the positive rate for each group. Test results were considered significant when the P value was less than .05. Results Expression and Identification of the Interest Protein We used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis on the identified genetic-engineering protein HIS-SMP30. The results (Figure 1A) showed that the target product could be well separated and purified under NTA-100 conditions. A protein fragment of approximately 21 kDa can appear in the purified product; this finding is consistent with the previous results.14 To verify whether the expressed protein is the protein of interest, anti-SMP30 antibody has been used simultaneously in carrying out WB experiments. The results of WB have shown that the purified protein can be combined with anti-SMP30 antibody (Figure 1B). The HIS-SMP30 antigenic protein fragment that can be combined with the anti-SMP30 antibody has been expressed efficiently. Figure 1 View largeDownload slide Results of testing of genetic-engineering protein HIS-SMP30 and anti-SMP30 antibody. A, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) testing on genetic engineering protein HIS-SMP30. B, Western blotting. SMP30 indicates senescence marker protein 30. Figure 1 View largeDownload slide Results of testing of genetic-engineering protein HIS-SMP30 and anti-SMP30 antibody. A, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) testing on genetic engineering protein HIS-SMP30. B, Western blotting. SMP30 indicates senescence marker protein 30. The results of WB confirmed that the extracted protein and the protein were effectively bound with anti-SMP30 antibody. SMP30 protein meets the experimental requirements of ELISA and can be used to detect the presence of antibody in serum. Positive Rates of SMP30 and Anti-SMP30 Antibody in Serum Specimens ELISA has been used to detect the absorbance of SMP30 and anti-SMP30 antibody in the serum of patients with HCC, CH, and LC, as well as the healthy control group. According to X¯+3SD of absorbance in the healthy control group, the cutoff values of SMP30 and anti-SMP30 antibody in serum are defined as 0.340 and 0.049, respectively, and serum specimens that tested positive have been divided. The positive rate of SMP30 in HCC group was 8.39%, and the positive rates in CH group and healthy control group were 0.55% and 1.21%, respectively. The positive rate of SMP30 in LC group was negative (Figure 2A). The rate of positivity for anti-SMP30 antibody in patients with HCC was 25.87% and that of the CH group was 4.38%. In the LC group, the rate of positivity for anti-SMP30 antibody was 3.92%; no positive results were detected in the healthy group (Figure 2B). Figure 2 View largeDownload slide Senecence marker protein 30 (SMP30) and anti-SMP30 results in patients with various conditions and in healthy control individuals. A, Results for SMP30 in hepatocellular carcinoma (HCC), chronic hepatitis (CH), liver cirrhosis (LC), and healthy control groups. B, Results for anti-SMP30 in HCC, CH, LC, and healthy control groups. OD indicates optical density; NHS, normal human serum. Figure 2 View largeDownload slide Senecence marker protein 30 (SMP30) and anti-SMP30 results in patients with various conditions and in healthy control individuals. A, Results for SMP30 in hepatocellular carcinoma (HCC), chronic hepatitis (CH), liver cirrhosis (LC), and healthy control groups. B, Results for anti-SMP30 in HCC, CH, LC, and healthy control groups. OD indicates optical density; NHS, normal human serum. Diagnostic Value of SMP30 and Anti-SMP30 Antibody in Patients With HCC We measured SMP30 and anti-SMP30 antibodies in serologic specimens from the cohort with HCC and the control cohort and report that the levels of SMP30 and anti-SMP30 antibody are noticeably higher in the HCC group than in other groups. We used ROC curves to analyze the positive test results from the HCC group (Figure 3). The AUC of SMP30 is 0.461, indicating that there is a low degree of diagnostic value, with 8.39% sensitivity and 99.15% specificity. Although SMP30 antigen yields poor diagnostic performance in patients with HCC, its corresponding anti-SMP30 antibody has a high diagnostic value, yielding AUC of 0.814, with 25.87% sensitivity and 97.73% specificity (Table 2). Table 2. AUC, Sensitivity, and Specificity of Different Variables and Variable Combinations Variable AUC (95% CI) Sensitivity Specificity P Value SMP30 0.461 (0.400-0.522) 8.39% 99.15% .17 Anti-SMP30 antibody 0.814 (0.773-0.856) 25.87% 97.73% <.001 AFP 0.731 (0.679-0.783) 58.04% 70.25% <.001 SMP30 + anti-SMP30 antibody 0.816 (0.775-0.858) 34.27% 96.88% <.001 SMP30 + AFP 0.737 (0.682-0.791) 61.54% 69.41% <.001 Anti-SMP30 antibody + AFP 0.856 (0.819-0.893) 69.23% 67.99% <.001 Variable AUC (95% CI) Sensitivity Specificity P Value SMP30 0.461 (0.400-0.522) 8.39% 99.15% .17 Anti-SMP30 antibody 0.814 (0.773-0.856) 25.87% 97.73% <.001 AFP 0.731 (0.679-0.783) 58.04% 70.25% <.001 SMP30 + anti-SMP30 antibody 0.816 (0.775-0.858) 34.27% 96.88% <.001 SMP30 + AFP 0.737 (0.682-0.791) 61.54% 69.41% <.001 Anti-SMP30 antibody + AFP 0.856 (0.819-0.893) 69.23% 67.99% <.001 AUC, area under the curve; SMP30, senescence marker protein 30; AFP, alpha-fetoprotein. View Large Table 2. AUC, Sensitivity, and Specificity of Different Variables and Variable Combinations Variable AUC (95% CI) Sensitivity Specificity P Value SMP30 0.461 (0.400-0.522) 8.39% 99.15% .17 Anti-SMP30 antibody 0.814 (0.773-0.856) 25.87% 97.73% <.001 AFP 0.731 (0.679-0.783) 58.04% 70.25% <.001 SMP30 + anti-SMP30 antibody 0.816 (0.775-0.858) 34.27% 96.88% <.001 SMP30 + AFP 0.737 (0.682-0.791) 61.54% 69.41% <.001 Anti-SMP30 antibody + AFP 0.856 (0.819-0.893) 69.23% 67.99% <.001 Variable AUC (95% CI) Sensitivity Specificity P Value SMP30 0.461 (0.400-0.522) 8.39% 99.15% .17 Anti-SMP30 antibody 0.814 (0.773-0.856) 25.87% 97.73% <.001 AFP 0.731 (0.679-0.783) 58.04% 70.25% <.001 SMP30 + anti-SMP30 antibody 0.816 (0.775-0.858) 34.27% 96.88% <.001 SMP30 + AFP 0.737 (0.682-0.791) 61.54% 69.41% <.001 Anti-SMP30 antibody + AFP 0.856 (0.819-0.893) 69.23% 67.99% <.001 AUC, area under the curve; SMP30, senescence marker protein 30; AFP, alpha-fetoprotein. View Large Figure 3 View largeDownload slide Receiver operating characteristic (ROC) curve measurement of positive results for senecence marker protein 30 (SMP30) in patients with hepatocellular carcimona (HCC). OD indicates optical density; HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis; NHS, normal human serum; AUC, area under the curve; AFP, alpha-fetoprotein. Figure 3 View largeDownload slide Receiver operating characteristic (ROC) curve measurement of positive results for senecence marker protein 30 (SMP30) in patients with hepatocellular carcimona (HCC). OD indicates optical density; HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis; NHS, normal human serum; AUC, area under the curve; AFP, alpha-fetoprotein. The result of Pearson χ2 bilateral detection of various indices of case individuals with HCC is shown in Table 3. The P values of the groups are all greater than .05, which means that there is no statistical significance between SMP30 and anti-SMP30 antibody and the clinic parameters of the patients, including age, sex, pathologic grading, AFP value, HbsAg, alanine transaminase (ALT) and aspartate transaminase (AST) values, and tumor size. Table 3. Results of Pearson χ2 Bilateral Detection of Various Indices of Case Individuals With HCC Variable Value SMP30 Anti-SMP30 Antibody Positive Result/ Total No. in Cohort Positivity Rate (%) x2 P Value Positive Positivity Rate (%) x2 P Value Age, y ≥ 50 5/74 6.75 0.533 .46 21/74 28.37 0.501 .48 <50 7/69 10.14 16/69 23.19 Sex Male 11/123 8.94 0.024 .88 32/123 26.02 0.009 .92 Female 1/20 5.00 5/20 25.00 AFP <25 6/60 10.00 0.348 .56 16/60 26.67 0.034 .85 ≥ 25 6/83 7.22 21/83 25.30 <400 7/86 8.14 0.018 .89 20/86 23.26 0.771 .38 ≥ 400 5/57 8.77 17/57 29.82 ALT <40 6/70 8.52 0.006 .94 18/70 25.71 0.002 .97 ≥ 40 6/73 8.22 19/73 26.03 AST <40 5/60 8.33 0 .98 11/60 18.33 3.065 .08 ≥ 40 7/83 8.43 26/83 31.33 BCLC A 4/46 8.70 1.179 .55 11/46 23.91 0.482 .79 B 2/42 4.76 10/42 23.81 C 6/55 10.91 16/55 29.09 HBsAg + 2/22 9.09 0.017 .90 7/22 31.82 0.479 .49 − 10/121 8.26 30/121 24.79 Tumor size <3 cm 2/20 10.00 0.078 .78 6/20 30.00 0.206 .65 ≥ 3 cm 10/123 8.13 31/123 25.20 Variable Value SMP30 Anti-SMP30 Antibody Positive Result/ Total No. in Cohort Positivity Rate (%) x2 P Value Positive Positivity Rate (%) x2 P Value Age, y ≥ 50 5/74 6.75 0.533 .46 21/74 28.37 0.501 .48 <50 7/69 10.14 16/69 23.19 Sex Male 11/123 8.94 0.024 .88 32/123 26.02 0.009 .92 Female 1/20 5.00 5/20 25.00 AFP <25 6/60 10.00 0.348 .56 16/60 26.67 0.034 .85 ≥ 25 6/83 7.22 21/83 25.30 <400 7/86 8.14 0.018 .89 20/86 23.26 0.771 .38 ≥ 400 5/57 8.77 17/57 29.82 ALT <40 6/70 8.52 0.006 .94 18/70 25.71 0.002 .97 ≥ 40 6/73 8.22 19/73 26.03 AST <40 5/60 8.33 0 .98 11/60 18.33 3.065 .08 ≥ 40 7/83 8.43 26/83 31.33 BCLC A 4/46 8.70 1.179 .55 11/46 23.91 0.482 .79 B 2/42 4.76 10/42 23.81 C 6/55 10.91 16/55 29.09 HBsAg + 2/22 9.09 0.017 .90 7/22 31.82 0.479 .49 − 10/121 8.26 30/121 24.79 Tumor size <3 cm 2/20 10.00 0.078 .78 6/20 30.00 0.206 .65 ≥ 3 cm 10/123 8.13 31/123 25.20 SMP30, senescence marker protein 30; AFP, alpha-fetoprotein; ALT, alanine transaminase; AST, aspartate transaminase; BCLC, Barcelona Clinic Liver Cancer; HBsAg, hepatitis B surface antigen. View Large Table 3. Results of Pearson χ2 Bilateral Detection of Various Indices of Case Individuals With HCC Variable Value SMP30 Anti-SMP30 Antibody Positive Result/ Total No. in Cohort Positivity Rate (%) x2 P Value Positive Positivity Rate (%) x2 P Value Age, y ≥ 50 5/74 6.75 0.533 .46 21/74 28.37 0.501 .48 <50 7/69 10.14 16/69 23.19 Sex Male 11/123 8.94 0.024 .88 32/123 26.02 0.009 .92 Female 1/20 5.00 5/20 25.00 AFP <25 6/60 10.00 0.348 .56 16/60 26.67 0.034 .85 ≥ 25 6/83 7.22 21/83 25.30 <400 7/86 8.14 0.018 .89 20/86 23.26 0.771 .38 ≥ 400 5/57 8.77 17/57 29.82 ALT <40 6/70 8.52 0.006 .94 18/70 25.71 0.002 .97 ≥ 40 6/73 8.22 19/73 26.03 AST <40 5/60 8.33 0 .98 11/60 18.33 3.065 .08 ≥ 40 7/83 8.43 26/83 31.33 BCLC A 4/46 8.70 1.179 .55 11/46 23.91 0.482 .79 B 2/42 4.76 10/42 23.81 C 6/55 10.91 16/55 29.09 HBsAg + 2/22 9.09 0.017 .90 7/22 31.82 0.479 .49 − 10/121 8.26 30/121 24.79 Tumor size <3 cm 2/20 10.00 0.078 .78 6/20 30.00 0.206 .65 ≥ 3 cm 10/123 8.13 31/123 25.20 Variable Value SMP30 Anti-SMP30 Antibody Positive Result/ Total No. in Cohort Positivity Rate (%) x2 P Value Positive Positivity Rate (%) x2 P Value Age, y ≥ 50 5/74 6.75 0.533 .46 21/74 28.37 0.501 .48 <50 7/69 10.14 16/69 23.19 Sex Male 11/123 8.94 0.024 .88 32/123 26.02 0.009 .92 Female 1/20 5.00 5/20 25.00 AFP <25 6/60 10.00 0.348 .56 16/60 26.67 0.034 .85 ≥ 25 6/83 7.22 21/83 25.30 <400 7/86 8.14 0.018 .89 20/86 23.26 0.771 .38 ≥ 400 5/57 8.77 17/57 29.82 ALT <40 6/70 8.52 0.006 .94 18/70 25.71 0.002 .97 ≥ 40 6/73 8.22 19/73 26.03 AST <40 5/60 8.33 0 .98 11/60 18.33 3.065 .08 ≥ 40 7/83 8.43 26/83 31.33 BCLC A 4/46 8.70 1.179 .55 11/46 23.91 0.482 .79 B 2/42 4.76 10/42 23.81 C 6/55 10.91 16/55 29.09 HBsAg + 2/22 9.09 0.017 .90 7/22 31.82 0.479 .49 − 10/121 8.26 30/121 24.79 Tumor size <3 cm 2/20 10.00 0.078 .78 6/20 30.00 0.206 .65 ≥ 3 cm 10/123 8.13 31/123 25.20 SMP30, senescence marker protein 30; AFP, alpha-fetoprotein; ALT, alanine transaminase; AST, aspartate transaminase; BCLC, Barcelona Clinic Liver Cancer; HBsAg, hepatitis B surface antigen. View Large Combined Detection of AFP and Anti-SMP30 Antibody in Patients With HCC Through the data analyses, we report 16 cases of AFP negativity in patients who tested positive for anti-SMP30 antibody in the HCC group. With AFP of 25ng per mL or greater as a positive value, the positive rate of AFP in the diagnosis of HCC is 58.0%. We have assessed whether combined detection of anti-SMP30 antibody and AFP can improve the sensitivity of HCC detection. As we expected, testing of anti-SMP30 antibody and AFP have effectively increased the diagnostic accuracy for HCC with AUC of 0.856, compared with either test by itself (Figure 3). The positive rate in HCC is 69.23% for combined detection of AFP and Anti-SMP30 antibody (Table 2). Our results have shown that the combined detection has clinical significance and that it can significantly improve the detection rate of liver cancer (P <.01). Discussion Stenner-Liewen et al7 have used the SEREX technique for screening of HCC antigens from the serum of patients with HCC by using the complementary DNA (cDNA) library of HCC tissues, located in Guangxi, China. One of the antigens screened is homologous with human messenger RNA (mRNA) for 165 amino acids near the C-terminal of SMP30. Serologic tests have demonstrated that HCC ranging from 3 through 5 cm has an autoantibody-positive reaction to SMP30. All 20 serum specimens from different kinds of malignant neoplasms and 4 from hepatocirrhosis yielded negative reactions. The results of our serum analyses have suggested that SMP30 is a new HCC-associated antigen and thus can be of great value in the diagnosis of HCC. In a previous study,13 the research group had obtained BMP-SMP30 recombinant fusion protein by using genetic-engineering technology. The BMP-SMP30 fusion protein has shown strong water solubility and antigenicity and can be used to detect serum antibody. Our results have shown a 39% positive rate for autoantibody to SMP30 in serum specimens from patients with HCC. However, the molecular weight of BMP-labeled protein is too large for its 45-kD size. As a result, large-tag protein may be affecting the combining capacity of BMP-SMP30 fusion protein and anti-SMP30 antibody. HIS-SMP30 recombinant protein, when expressed, is water-insoluble. To resolve this problem, a molecular chaperone coexpression system (BL21[DE3] pLysS- pColdIII-SMP30 -pTf16) has been applied to express HIS-tag SMP30-soluble protein successfully.14 The label of such a fusion protein is small and does not have a great impact on the exposure of the SMP30 functional epitopes. Despite this finding, anti-SMP30 antibody levels from 143 patients with HCC have been detected by indirect ELISA, with soluble HiS-SMP30 fusion protein as antigen. Our results have shown that the positive rate is 25.87%, lower than the positive rate of 39% detected in the previous study. There are 3 possible reasons. First, the serum specimens from patients with HCC were tested in different batches, which may have influenced the rate of positivity. Second, the time spent, degree of freshness, and time saved with HCC serum specimens are different, leading to the differences in cutoff values. Third, although the molecular chaperone can promote the soluble expression of SMP30, it is worth considering the effect of molecular chaperone on the antigenic site of SMP30. CH and LC are important risk factors for the occurrence of HCC. In our study, we further detected the level of serum anti-SMP30 antibody in 137 patients with CH and 51 patients with LC. However, 8 serum specimens tested positive in patients with CH and 2 cases of a positive result in patients with LC. The positivity rate for the presence of anti-SMP30 antibody in the CH and LC groups was significantly lower than that in the HCC group (P <.05). Tumor material itself can induce the immune response of the human body to produce antibody related to the antitumor antigen; however, the specific mechanism is unclear. Part of the HCC serum anti-SMP30 antibody is negative for 2 possible reasons. First, the results of previous studies have shown that anti-SMP30 antibody appears in the early stages of HCC. The positivity rate for tumors less than 3 cm in diameter was higher than that of patients with tumors of 5 cm in diameter, which suggests that the presence of anti-SMP30 antibody may be an early indicator of HCC. In our current work, most of our serum specimens were donated by patients with advanced HCC. That type of patient has a lower immune response to tumor antigens, so some of the serum can not detect anti-SMP30 antibody. Second, anti-SMP30 antibody content is low and cannot be detected by ELISA assay. In this work, we detected anti-SMP30 antibody in some AFP-negative serum from patients with HCC. Therefore, anti-SMP30 antibody has great value in the diagnosis of HCC. It suggests that anti-SMP30 antibody production is closely related to the process of liver-cell malignant transformation and further shows that SMP30 autoantibody can be used as a biomarker for aiding with diagnosis of HCC. We report that the expression of SMP30 decreases in HCC-infected tissues but increases in adjacent cancerous tissues.8 Further, cell proliferation assay and migration assay results have shown that SMP30 can effectively inhibit the proliferation and migration of HCC cells, suggesting that SMP30 may function as a tumor suppressor.16,17 Because of low expression of SMP30 in liver cancer tissues and high expression in adjacent cancer tissues, it is valuable to study whether the appearance of serum anti-SMP30 antibody in patients with HCC is related to the protective reaction for HCC in the early stages. To study the relationship between serum anti-SMP30 antibody production and SMP30 expression levels, we have developed a method of double-antibody sandwich ELISA to detect SMP30 levels in serum. The results have shown that only 12 cases test positive for SMP30 in 143 cases of HCC, the positive rate is 8.39%, and the positive rate of SMP30 is lower than the antibody levels. Autoantibody to SMP30 has been detected in some HCC serum; however, the corresponding SMP30 has not been detected. Therefore, the mechanism of anti-SMP30 antibody appearance needs to be further investigated. Possible reasons for the low concentration of SMP30 in serum are that SMP30 mainly exists in cells, and normal tissue is rarely secreted into the serum; SMP30 expression in HCC tissues is too low to release in serum; and SMP30 from early stage liver cancer is released into the blood but as a foreign protein, making it easy to be removed by the body. At present, serum AFP testing is most commonly used for the diagnosis of HCC. However, the sensitivity of AFP is only 25% to 65%, which suggests that approximately 40% of patients with HCC may test false negative.4 We report that the positive rate of combined detection of anti-SMP30 antibody and AFP has increased to 69.23%, higher than the positive rate of 58.04% yielded by AFP single testing; the difference is statistically significant (P <.01). The combination of 2 biomarkers can improve the sensitivity of the diagnosis of HCC. In conclusion, we have identified a high level of anti-SMP30 antibody in serum specimens from individuals infected with HCC. The anti-SMP30 antibody in serum has been detected by the indirect ELISA method, and the positive rate is significantly higher than those in patients with CH and those with LC. Anti-SMP30 antibody is expected to be a new serological marker for diagnosis of HCC; it can effectively improve the sensitivity of the diagnosis of HCC, especially in combination with other tumor markers. Abbreviations: PLC primary liver cancer HCC hepatocellular carcinoma AFP alpha-fetoprotein SMP30 senescence marker protein 30 RGN regucalcin SEREX serologic identification by recombinant expression cloning cDNA complementary DNA WB Western blotting LC liver cirrhosis CH chronic hepatitis CT computed tomography MRI magnetic resonance imaging NHS normal human serum HBsAg hepatitis B surface antigen IPTG isopropyl β-D-1-thiogalactopyranoside Ni-NTA nitrilotriacetic acid IgG-HRP horseradish peroxidase–conjugated immunoglobulin G ELISA enzyme-linked immunosorbent assay PBST phosphate-buffered saline with Tween 20 BSA bovine serum albumin HRP horseradish peroxidase TMB tetramethyl benzidine OD optical density PBS phosphate-buffered saline ROC receiver operating characteristic AUC area under the curve CIs confidence intervals SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis ALT alanine transaminase AST aspartate transaminase cDNA complementary DNA mRNA messenger RNA NA nonapplicable BCLC Barcelona Clinic Liver Cancer Acknowledgments This work is supported by the National Natural Science Foundation of China (81572994, 81460432), Guangxi Nature Science Foundation (2015GXNSFDA139017), Guangxi Science and Technology Research and Technology Development Project (15104001-7), Programs for Department of Education of Guangxi Zhuang Autonomous Region (2013ZD011), and the Guangxi Nanning Qingxiu District Science and Technology Research and Technology Development Project (2014S03). References 1. Perz JF , Armstrong GL , Farrington LA , Hutin YJ , Bell BP . The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide . J Hepatol . 2006 ; 45 ( 4 ): 529 – 538 . 2. Bei CH , Bai H , Yu HP , et al. Combined effects of six cytokine gene polymorphisms and SNP-SNP interactions on hepatocellular carcinoma risk in Southern Guangxi, China . Asian Pac J Cancer Prev . 2014 ; 15 ( 16 ): 6961 – 6967 . 3. Ferlay J , Shin HR , Bray F , Forman D , Mathers C , Parkin DM . Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008 . Int J Cancer . 2010 ; 127 ( 12 ): 2893 – 2917 . 4. El-Serag HB , Marrero JA , Rudolph L , Reddy KR . Diagnosis and treatment of hepatocellular carcinoma . Gastroenterology . 2008 ; 134 ( 6 ): 1752 – 1763 . 5. Fujita T , Shirasawa T , Uchida K , Maruyama N . Isolation of cDNA clone encoding rat senescence marker protein-30 (SMP30) and its tissue distribution . Biochim Biophys Acta . 1992 ; 1132 ( 3 ): 297 – 305 . 6. Fujita T , Uchida K , Maruyama N . Purification of senescence marker protein-30 (SMP30) and its androgen-independent decrease with age in the rat liver . Biochim Biophys Acta . 1992 ; 1116 ( 2 ): 122 – 128 . 7. Stenner-Liewen F , Luo G , Sahin U , et al. Definition of tumor-associated antigens in hepatocellular carcinoma . Cancer Epidemiol Biomarkers Prev . 2000 ; 9 ( 3 ): 285 – 290 . 8. Zhou SF , Mo FR , Bin YH , Hou GQ , Xie XX , Luo GR . Serum immunoreactivity of SMP30 and its tissues expression in hepatocellular carcinoma . Clin Biochem . 2011 ; 44 ( 4 ): 331 – 336 . 9. Mo Z , Zheng S , Lv Z , et al. Senescence marker protein 30 (SMP30) serves as a potential prognostic indicator in hepatocellular carcinoma . Sci Rep . 2016 ; 6 : 39376 . 10. Arif S , Qudsia S , Urooj S , Chaudry N , Arshad A , Andleeb S . Blueprint of quartz crystal microbalance biosensor for early detection of breast cancer through salivary autoantibodies against ATP6AP1 . Biosens Bioelectron . 2015 ; 65 : 62 – 70 . 11. Hirano N , Butler MO , Von Bergwelt-Baildon MS , et al. Autoantibodies frequently detected in patients with aplastic anemia . Blood . 2003 ; 102 ( 13 ): 4567 – 4575 . 12. Zhang H , Xia J , Wang K , Zhang J . Serum autoantibodies in the early detection of esophageal cancer: a systematic review . Tumour Biol . 2015 ; 36 ( 1 ): 95 – 109 . 13. Zhou SF , Xie XX , Bin YH , Lan L , Chen F , Luo GR . Identification of HCC-22-5 tumor-associated antigen and antibody response in patients . Clin Chim Acta . 2006 ; 366 ( 1-2 ): 274 – 280 . 14. Zhang SC , Huang P , Zhao YX , et al. Soluble expression of recombinant human SMP30 for detecting serum SMP30 antibody levels in hepatocellular carcinoma patients . Asian Pac J Cancer Prev . 2013 ; 14 ( 4 ): 2383 – 2386 . 15. Piemonte M . TNM -- classification of malignant tumors (VI edition -- 2002). Innovations in the classification of head and neck neoplasms [in Italian] . Acta Otorhinolaryngol Ital . 2003 ; 23 ( 2 ): 132 – 135 . 16. Misawa H , Inagaki S , Yamaguchi M . Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin . J Cell Biochem . 2001 ; 84 ( 1 ): 143 – 149 . 17. Tsurusaki Y , Yamaguchi M . Role of endogenous regucalcin in nuclear regulation of regenerating rat liver: suppression of the enhanced ribonucleic acid synthesis activity . J Cell Biochem . 2002 ; 87 ( 4 ): 450 – 457 . © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Laboratory Medicine Oxford University Press

Diagnostic Value of Serum SMP30 and Anti-SMP30 Antibody in Hepatocellular Carcinoma

Loading next page...
 
/lp/ou_press/diagnostic-value-of-serum-smp30-and-anti-smp30-antibody-in-XzkIFP2gSq
Publisher
Oxford University Press
Copyright
© American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
ISSN
0007-5027
eISSN
1943-7730
D.O.I.
10.1093/labmed/lmy004
Publisher site
See Article on Publisher Site

Abstract

Abstract Objective To evaluate the clinical value of senescence marker protein 30 (SMP30) and anti-SMP30 antibody in serum. Methods We used enzyme-linked immunosorbent assay (ELISA) to analytically validate serum levels of SMP30 and anti-SMP30 antibody in 143 patients with hepatocellular carcinoma (HCC), compared with those levels in serum from 137 patients with chronic hepatitis (CH), 51 with liver cirrhosis (LC), and 165 healthy control individuals. Results The positivity rate of SMP30 in the HCC group (8.39%) was significantly higher than that rate in the CH group (.73%) and in the healthy control group (1.21%). The positivity rate for anti-SMP30 antibody in patients with HCC was 25.87%, that in the CH group was 4.38%, and that in the LC group was 3.92%. Conclusion Anti-SMP30 antibody levels can be used as a biomarker for diagnosing HCC; marked results have been observed for patients with alpha-fetoprotein (AFP) negativity, in particular. hepatocellular carcinoma (HCC), senescence marker protein 30 (SMP30), serum, anti-SMP30 antibody, chronic hepatitis (CH), liver cirrhosis (LC) It has been reported1,2 that approximately 80% of liver cancer worldwide is primary liver cancer (PLC). Also, the hepatocellular carcinoma (HCC) histological subtype is highly common in China; this subtype ranks first among all malignant tumors in Guangxi province. Despite the improvement in diagnosing and treatment of HCC, 5-year overall survival for the patients is only 3% to 5%.3 This finding is partly attributed to the lack of a reliable method for early diagnosis (to our knowledge). So far, alpha-fetoprotein (AFP) has been used as the most common serum biomarker for diagnosis of HCC. However, its sensitivity is only 25% to 65% at the standard cutoff of 25 ng per mL, especially when diagnosing in the early stages of HCC.4 Therefore, it is important to develop serological markers for effectively diagnosing HCC at early stages and to monitor aggressiveness, responsiveness to treatments, tumor recurrence, and patient survival rate. It has been shown that senescence marker protein 30 (SMP30), otherwise known as regucalcin (RGN), a calcium-binding protein discovered by Fujita et al in 1992,5 plays a crucial role in protective effects against apoptosis and oxidative stress.6 Using the serologic identification by recombinant expression cloning (SEREX) approach, SMP30 was identified in the Guangxi HCC complementary DNA (cDNA) expression library as being an HCC-associated antigen.7 The results of immunohistochemical analysis showed that the levels of SMP30 expression in HCC paracancerous tissues were higher than those in the corresponding HCC.8 The low expression of SMP30 protein in HCC is closely related to the survival prognosis for patients.9 The aforementioned studies have shown that expressions of SMP30 are important in the progression of liver cancer. However, whether the concentration of SMP30 in serum can be used as a diagnostic indicator of liver cancer has not been reported, to our knowledge. Numerous studies10,11 have shown that the tumor itself can induce the immune response of the body to produce antibody related to the antitumor antigen. This response can be tested even when the tumor antigen expression is low. Therefore, the antibody related to the antitumor antigen is understood as the symbol of tumor diagnosis.12 Previously, Western blotting (WB) has been used to confirm the presence of SMP30 and anti-SMP30 antibody in the serum of patients with HCC, which suggests that detection of SMP30 and anti-SMP30 antibody levels may be understood as auxiliary indices for HCC diagnosis.13 However, as a serological diagnostic index of HCC, the specificity and sensitivity of those methods for HCC diagnosis still requires greater nontumor serological verification. Based on the aforementiond analyses, we have examined the levels of serum SMP30 together with anti-SMP30 antibody in 495 serum specimens from 143 patients with HCC, 51 patients with liver cirrhosis (LC), 137 patients with chronic hepatitis (CH), and 165 healthy control individuals to validate the diagnostic value. Materials and Methods Patients and Specimen Obtaining Procedures The BL21 (DE3) pLysS-pColdIII-SMP30-pTf16 recombinant genetic-engineering strain was constructed by our research team and stored at −80°C.14 This study included 331 patients, of whom 143 with HCC were recruited by the Affiliated Tumor Hospital of Guangxi Medical University, China. A total of 137 patients with CH and 51 with LC were followed up at the first Affiliated Hospital of the same university. The diagnoses of CH and LC were carried out by abdominal ultrasound scanning and laboratory testing. Patients with HCC were diagnosed by histology and laboratory testing, including computed tomography (CT) scanning, abdominal ultrasound scanning, and magnetic resonance imaging (MRI). The clinical stages were classified according to the International Union against Cancer TNM classification.15 Neither chemotherapy nor surgical treatments had been given to the patients. Physical examinations and laboratory tests were performed at the time that blood specimens were obtained. The normal human serum (NHS) specimens were obtained from 165 healthy college students, aged 17 through 23, who had tested negative for hepatitis B surface antigen (HBsAg) and whose liver function and abdominal ultrasound test results revealed no abnormalities (Table 1). The investigation procedures were conducted in compliance with the guidelines of the Ethical Committee of Guangxi Medical University. Oral informed consent was obtained from all participating patients and healthy donors. The serum specimens were divided into aliquots and stored at −80°C until they were analyzed. Table 1. Test Results From Specimens, by Donor Characteristics Variable HCC (n = 143) CH (n = 137) LC (n = 51) NHS (n = 165) Age, y Mean (SD) 50.06 (11.7) 41.28 (11.05) 45.47 (12.01) 19.13 (1.56)  Range 15–81 15–69 23–70 17–23 Sex  Male 123 120 42 95  Female 20 17 9 70 ALT  <40 U/L 70 29 18 165  ≥ 40 U/L 73 108 33 NA AFP  <5 ng/mL 60 77 27 NA  ≥ 25 ng/mL 83 60 24 NA  HBsAg 121 131 28 NA BCLC  A 46 NA NA NA  B 42 NA NA NA  C 55 NA NA NA Size of Tumor (Diameter)  <3 cm 20 NA NA NA  ≥ 3 cm 123 NA NA NA Variable HCC (n = 143) CH (n = 137) LC (n = 51) NHS (n = 165) Age, y Mean (SD) 50.06 (11.7) 41.28 (11.05) 45.47 (12.01) 19.13 (1.56)  Range 15–81 15–69 23–70 17–23 Sex  Male 123 120 42 95  Female 20 17 9 70 ALT  <40 U/L 70 29 18 165  ≥ 40 U/L 73 108 33 NA AFP  <5 ng/mL 60 77 27 NA  ≥ 25 ng/mL 83 60 24 NA  HBsAg 121 131 28 NA BCLC  A 46 NA NA NA  B 42 NA NA NA  C 55 NA NA NA Size of Tumor (Diameter)  <3 cm 20 NA NA NA  ≥ 3 cm 123 NA NA NA HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis; NHS, normal human serum; ALT, alanine transaminase; NA, nonapplicable; AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer View Large Table 1. Test Results From Specimens, by Donor Characteristics Variable HCC (n = 143) CH (n = 137) LC (n = 51) NHS (n = 165) Age, y Mean (SD) 50.06 (11.7) 41.28 (11.05) 45.47 (12.01) 19.13 (1.56)  Range 15–81 15–69 23–70 17–23 Sex  Male 123 120 42 95  Female 20 17 9 70 ALT  <40 U/L 70 29 18 165  ≥ 40 U/L 73 108 33 NA AFP  <5 ng/mL 60 77 27 NA  ≥ 25 ng/mL 83 60 24 NA  HBsAg 121 131 28 NA BCLC  A 46 NA NA NA  B 42 NA NA NA  C 55 NA NA NA Size of Tumor (Diameter)  <3 cm 20 NA NA NA  ≥ 3 cm 123 NA NA NA Variable HCC (n = 143) CH (n = 137) LC (n = 51) NHS (n = 165) Age, y Mean (SD) 50.06 (11.7) 41.28 (11.05) 45.47 (12.01) 19.13 (1.56)  Range 15–81 15–69 23–70 17–23 Sex  Male 123 120 42 95  Female 20 17 9 70 ALT  <40 U/L 70 29 18 165  ≥ 40 U/L 73 108 33 NA AFP  <5 ng/mL 60 77 27 NA  ≥ 25 ng/mL 83 60 24 NA  HBsAg 121 131 28 NA BCLC  A 46 NA NA NA  B 42 NA NA NA  C 55 NA NA NA Size of Tumor (Diameter)  <3 cm 20 NA NA NA  ≥ 3 cm 123 NA NA NA HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis; NHS, normal human serum; ALT, alanine transaminase; NA, nonapplicable; AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer View Large Preparation of Soluble Recombinant HIS-SMP30 Protein We prepared soluble recombinant HIS-SMP30 protein in our laboratory. In brief, the BL21(DE3)pLysS-pColdIII-SMP30-pTf16 recombinant genetic-engineering strain was cultured and induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) to express HIS-SMP30; the fusion protein is 165 amino acids near the C-terminal. The supernatant of bacteria with sonication was harvested and loaded into the nitrilotriacetic acid (Ni-NTA) affinity column, and then purified HIS-SMP30 genetic-engineering protein was obtained. We performed verification testing of the target protein by WB in which the primary antibody was the murine-origin anti-SMP30 monoclonal antibody and the secondary antibody was goat anti-mouse horseradish peroxidase–conjugated immunoglobulin G (IgG-HRP). The aliquots of target protein were frozen and stored at −80°C for further use. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for Serum Anti-SMP30 Antibody We used 2.5 μg per mL purified HIS-SMP30 protein to coat the ELISA plate at 4°C overnight. The wells were then emptied and washed 3 times with phosphate-buffered saline with Tween 20 (PBST) and with 0.5% bovine serum albumin (BSA) closure at 37°C for 1 hour. We then added serum diluted (1:1024) with PBST at 37°C for 1 hour; all serum specimens underwent this process twice. After washing, we added biotin goat anti–human IgG antibody (Boster Biological Engineering) to the wells and then incubated the resulting product for 1 hour at 37°C, followed by washing. The horseradish peroxidase (HRP)–avidin was added for 40 minutes at 37°C, the plates were washed, and 100 μL of tetramethyl benzidine (TMB) substrate solution was added. The TMB color development was conducted for 15 minutes and then 2 M of sulfuric acid was used to terminate the reaction. The optical densities were read at 450 nm using a microplate reader. The optical density (OD) values of all the results were deducted from the blank control values, and the result was determined by the X¯+3SD of the NHS group as the cutoff value. Double Antibody Sandwich ELISA for Serum SMP30 ELISA plates were prepared by coating them with 100 μL per well of rabbit anti–human SMP30 polyclonal antibody at 4°C overnight. The wells were emptied, followed by washing 3 times with PBST, and with 0.5% BSA closure at 37°C for 1 hour. All serum specimens were diluted to 1:5 with PBST and loaded with 100 μL of diluted serum per well. The plates were incubated for 1 hour at 37°C and washed 3 times. The plates were incubated with 100 μL per well of mouse anti–human SMP30 monoclonal antibody for 1 hour at 37°C. The plates were once again washed 3 times and then incubated with 100 μL per well of goat anti-mouse IG-HRP solution in phosphate-buffered saline (PBS). After all the aformentioned treatments, the plates were further incubated for 1 more hour at 37°C plus washing. We then added 100 μL of TMB substrate solution. The TMB color development was conducted for 15 minutes, and then 2 M of sulfuric acid was used to terminate the reaction. We used a microplate reader to read the OD values at 450 nm. The OD values of all the results were deducted from the blank control results, and the result was determined by the X¯+3SD of the NHS group as the cutoff value. Statistical Analysis We adapted SPSS statistical software, version 17.0 (IBM) and GraphPad Prism software (GraphPad Software, Inc) for analyses. We conducted receiver operating characteristic (ROC) analyses to assess sensitivity, specificity, and area under the curve (AUC) with 95% confidence intervals (CIs). The sensitivity and specificity of each biomarker were calculated separately from the concentrations in 496 clinical serum specimens. We used the Pearson χ2 bilateral detection method to test the relationship between the serum test results from the HCC group, various clinical indices, and the positive rate for each group. Test results were considered significant when the P value was less than .05. Results Expression and Identification of the Interest Protein We used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis on the identified genetic-engineering protein HIS-SMP30. The results (Figure 1A) showed that the target product could be well separated and purified under NTA-100 conditions. A protein fragment of approximately 21 kDa can appear in the purified product; this finding is consistent with the previous results.14 To verify whether the expressed protein is the protein of interest, anti-SMP30 antibody has been used simultaneously in carrying out WB experiments. The results of WB have shown that the purified protein can be combined with anti-SMP30 antibody (Figure 1B). The HIS-SMP30 antigenic protein fragment that can be combined with the anti-SMP30 antibody has been expressed efficiently. Figure 1 View largeDownload slide Results of testing of genetic-engineering protein HIS-SMP30 and anti-SMP30 antibody. A, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) testing on genetic engineering protein HIS-SMP30. B, Western blotting. SMP30 indicates senescence marker protein 30. Figure 1 View largeDownload slide Results of testing of genetic-engineering protein HIS-SMP30 and anti-SMP30 antibody. A, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) testing on genetic engineering protein HIS-SMP30. B, Western blotting. SMP30 indicates senescence marker protein 30. The results of WB confirmed that the extracted protein and the protein were effectively bound with anti-SMP30 antibody. SMP30 protein meets the experimental requirements of ELISA and can be used to detect the presence of antibody in serum. Positive Rates of SMP30 and Anti-SMP30 Antibody in Serum Specimens ELISA has been used to detect the absorbance of SMP30 and anti-SMP30 antibody in the serum of patients with HCC, CH, and LC, as well as the healthy control group. According to X¯+3SD of absorbance in the healthy control group, the cutoff values of SMP30 and anti-SMP30 antibody in serum are defined as 0.340 and 0.049, respectively, and serum specimens that tested positive have been divided. The positive rate of SMP30 in HCC group was 8.39%, and the positive rates in CH group and healthy control group were 0.55% and 1.21%, respectively. The positive rate of SMP30 in LC group was negative (Figure 2A). The rate of positivity for anti-SMP30 antibody in patients with HCC was 25.87% and that of the CH group was 4.38%. In the LC group, the rate of positivity for anti-SMP30 antibody was 3.92%; no positive results were detected in the healthy group (Figure 2B). Figure 2 View largeDownload slide Senecence marker protein 30 (SMP30) and anti-SMP30 results in patients with various conditions and in healthy control individuals. A, Results for SMP30 in hepatocellular carcinoma (HCC), chronic hepatitis (CH), liver cirrhosis (LC), and healthy control groups. B, Results for anti-SMP30 in HCC, CH, LC, and healthy control groups. OD indicates optical density; NHS, normal human serum. Figure 2 View largeDownload slide Senecence marker protein 30 (SMP30) and anti-SMP30 results in patients with various conditions and in healthy control individuals. A, Results for SMP30 in hepatocellular carcinoma (HCC), chronic hepatitis (CH), liver cirrhosis (LC), and healthy control groups. B, Results for anti-SMP30 in HCC, CH, LC, and healthy control groups. OD indicates optical density; NHS, normal human serum. Diagnostic Value of SMP30 and Anti-SMP30 Antibody in Patients With HCC We measured SMP30 and anti-SMP30 antibodies in serologic specimens from the cohort with HCC and the control cohort and report that the levels of SMP30 and anti-SMP30 antibody are noticeably higher in the HCC group than in other groups. We used ROC curves to analyze the positive test results from the HCC group (Figure 3). The AUC of SMP30 is 0.461, indicating that there is a low degree of diagnostic value, with 8.39% sensitivity and 99.15% specificity. Although SMP30 antigen yields poor diagnostic performance in patients with HCC, its corresponding anti-SMP30 antibody has a high diagnostic value, yielding AUC of 0.814, with 25.87% sensitivity and 97.73% specificity (Table 2). Table 2. AUC, Sensitivity, and Specificity of Different Variables and Variable Combinations Variable AUC (95% CI) Sensitivity Specificity P Value SMP30 0.461 (0.400-0.522) 8.39% 99.15% .17 Anti-SMP30 antibody 0.814 (0.773-0.856) 25.87% 97.73% <.001 AFP 0.731 (0.679-0.783) 58.04% 70.25% <.001 SMP30 + anti-SMP30 antibody 0.816 (0.775-0.858) 34.27% 96.88% <.001 SMP30 + AFP 0.737 (0.682-0.791) 61.54% 69.41% <.001 Anti-SMP30 antibody + AFP 0.856 (0.819-0.893) 69.23% 67.99% <.001 Variable AUC (95% CI) Sensitivity Specificity P Value SMP30 0.461 (0.400-0.522) 8.39% 99.15% .17 Anti-SMP30 antibody 0.814 (0.773-0.856) 25.87% 97.73% <.001 AFP 0.731 (0.679-0.783) 58.04% 70.25% <.001 SMP30 + anti-SMP30 antibody 0.816 (0.775-0.858) 34.27% 96.88% <.001 SMP30 + AFP 0.737 (0.682-0.791) 61.54% 69.41% <.001 Anti-SMP30 antibody + AFP 0.856 (0.819-0.893) 69.23% 67.99% <.001 AUC, area under the curve; SMP30, senescence marker protein 30; AFP, alpha-fetoprotein. View Large Table 2. AUC, Sensitivity, and Specificity of Different Variables and Variable Combinations Variable AUC (95% CI) Sensitivity Specificity P Value SMP30 0.461 (0.400-0.522) 8.39% 99.15% .17 Anti-SMP30 antibody 0.814 (0.773-0.856) 25.87% 97.73% <.001 AFP 0.731 (0.679-0.783) 58.04% 70.25% <.001 SMP30 + anti-SMP30 antibody 0.816 (0.775-0.858) 34.27% 96.88% <.001 SMP30 + AFP 0.737 (0.682-0.791) 61.54% 69.41% <.001 Anti-SMP30 antibody + AFP 0.856 (0.819-0.893) 69.23% 67.99% <.001 Variable AUC (95% CI) Sensitivity Specificity P Value SMP30 0.461 (0.400-0.522) 8.39% 99.15% .17 Anti-SMP30 antibody 0.814 (0.773-0.856) 25.87% 97.73% <.001 AFP 0.731 (0.679-0.783) 58.04% 70.25% <.001 SMP30 + anti-SMP30 antibody 0.816 (0.775-0.858) 34.27% 96.88% <.001 SMP30 + AFP 0.737 (0.682-0.791) 61.54% 69.41% <.001 Anti-SMP30 antibody + AFP 0.856 (0.819-0.893) 69.23% 67.99% <.001 AUC, area under the curve; SMP30, senescence marker protein 30; AFP, alpha-fetoprotein. View Large Figure 3 View largeDownload slide Receiver operating characteristic (ROC) curve measurement of positive results for senecence marker protein 30 (SMP30) in patients with hepatocellular carcimona (HCC). OD indicates optical density; HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis; NHS, normal human serum; AUC, area under the curve; AFP, alpha-fetoprotein. Figure 3 View largeDownload slide Receiver operating characteristic (ROC) curve measurement of positive results for senecence marker protein 30 (SMP30) in patients with hepatocellular carcimona (HCC). OD indicates optical density; HCC, hepatocellular carcinoma; CH, chronic hepatitis; LC, liver cirrhosis; NHS, normal human serum; AUC, area under the curve; AFP, alpha-fetoprotein. The result of Pearson χ2 bilateral detection of various indices of case individuals with HCC is shown in Table 3. The P values of the groups are all greater than .05, which means that there is no statistical significance between SMP30 and anti-SMP30 antibody and the clinic parameters of the patients, including age, sex, pathologic grading, AFP value, HbsAg, alanine transaminase (ALT) and aspartate transaminase (AST) values, and tumor size. Table 3. Results of Pearson χ2 Bilateral Detection of Various Indices of Case Individuals With HCC Variable Value SMP30 Anti-SMP30 Antibody Positive Result/ Total No. in Cohort Positivity Rate (%) x2 P Value Positive Positivity Rate (%) x2 P Value Age, y ≥ 50 5/74 6.75 0.533 .46 21/74 28.37 0.501 .48 <50 7/69 10.14 16/69 23.19 Sex Male 11/123 8.94 0.024 .88 32/123 26.02 0.009 .92 Female 1/20 5.00 5/20 25.00 AFP <25 6/60 10.00 0.348 .56 16/60 26.67 0.034 .85 ≥ 25 6/83 7.22 21/83 25.30 <400 7/86 8.14 0.018 .89 20/86 23.26 0.771 .38 ≥ 400 5/57 8.77 17/57 29.82 ALT <40 6/70 8.52 0.006 .94 18/70 25.71 0.002 .97 ≥ 40 6/73 8.22 19/73 26.03 AST <40 5/60 8.33 0 .98 11/60 18.33 3.065 .08 ≥ 40 7/83 8.43 26/83 31.33 BCLC A 4/46 8.70 1.179 .55 11/46 23.91 0.482 .79 B 2/42 4.76 10/42 23.81 C 6/55 10.91 16/55 29.09 HBsAg + 2/22 9.09 0.017 .90 7/22 31.82 0.479 .49 − 10/121 8.26 30/121 24.79 Tumor size <3 cm 2/20 10.00 0.078 .78 6/20 30.00 0.206 .65 ≥ 3 cm 10/123 8.13 31/123 25.20 Variable Value SMP30 Anti-SMP30 Antibody Positive Result/ Total No. in Cohort Positivity Rate (%) x2 P Value Positive Positivity Rate (%) x2 P Value Age, y ≥ 50 5/74 6.75 0.533 .46 21/74 28.37 0.501 .48 <50 7/69 10.14 16/69 23.19 Sex Male 11/123 8.94 0.024 .88 32/123 26.02 0.009 .92 Female 1/20 5.00 5/20 25.00 AFP <25 6/60 10.00 0.348 .56 16/60 26.67 0.034 .85 ≥ 25 6/83 7.22 21/83 25.30 <400 7/86 8.14 0.018 .89 20/86 23.26 0.771 .38 ≥ 400 5/57 8.77 17/57 29.82 ALT <40 6/70 8.52 0.006 .94 18/70 25.71 0.002 .97 ≥ 40 6/73 8.22 19/73 26.03 AST <40 5/60 8.33 0 .98 11/60 18.33 3.065 .08 ≥ 40 7/83 8.43 26/83 31.33 BCLC A 4/46 8.70 1.179 .55 11/46 23.91 0.482 .79 B 2/42 4.76 10/42 23.81 C 6/55 10.91 16/55 29.09 HBsAg + 2/22 9.09 0.017 .90 7/22 31.82 0.479 .49 − 10/121 8.26 30/121 24.79 Tumor size <3 cm 2/20 10.00 0.078 .78 6/20 30.00 0.206 .65 ≥ 3 cm 10/123 8.13 31/123 25.20 SMP30, senescence marker protein 30; AFP, alpha-fetoprotein; ALT, alanine transaminase; AST, aspartate transaminase; BCLC, Barcelona Clinic Liver Cancer; HBsAg, hepatitis B surface antigen. View Large Table 3. Results of Pearson χ2 Bilateral Detection of Various Indices of Case Individuals With HCC Variable Value SMP30 Anti-SMP30 Antibody Positive Result/ Total No. in Cohort Positivity Rate (%) x2 P Value Positive Positivity Rate (%) x2 P Value Age, y ≥ 50 5/74 6.75 0.533 .46 21/74 28.37 0.501 .48 <50 7/69 10.14 16/69 23.19 Sex Male 11/123 8.94 0.024 .88 32/123 26.02 0.009 .92 Female 1/20 5.00 5/20 25.00 AFP <25 6/60 10.00 0.348 .56 16/60 26.67 0.034 .85 ≥ 25 6/83 7.22 21/83 25.30 <400 7/86 8.14 0.018 .89 20/86 23.26 0.771 .38 ≥ 400 5/57 8.77 17/57 29.82 ALT <40 6/70 8.52 0.006 .94 18/70 25.71 0.002 .97 ≥ 40 6/73 8.22 19/73 26.03 AST <40 5/60 8.33 0 .98 11/60 18.33 3.065 .08 ≥ 40 7/83 8.43 26/83 31.33 BCLC A 4/46 8.70 1.179 .55 11/46 23.91 0.482 .79 B 2/42 4.76 10/42 23.81 C 6/55 10.91 16/55 29.09 HBsAg + 2/22 9.09 0.017 .90 7/22 31.82 0.479 .49 − 10/121 8.26 30/121 24.79 Tumor size <3 cm 2/20 10.00 0.078 .78 6/20 30.00 0.206 .65 ≥ 3 cm 10/123 8.13 31/123 25.20 Variable Value SMP30 Anti-SMP30 Antibody Positive Result/ Total No. in Cohort Positivity Rate (%) x2 P Value Positive Positivity Rate (%) x2 P Value Age, y ≥ 50 5/74 6.75 0.533 .46 21/74 28.37 0.501 .48 <50 7/69 10.14 16/69 23.19 Sex Male 11/123 8.94 0.024 .88 32/123 26.02 0.009 .92 Female 1/20 5.00 5/20 25.00 AFP <25 6/60 10.00 0.348 .56 16/60 26.67 0.034 .85 ≥ 25 6/83 7.22 21/83 25.30 <400 7/86 8.14 0.018 .89 20/86 23.26 0.771 .38 ≥ 400 5/57 8.77 17/57 29.82 ALT <40 6/70 8.52 0.006 .94 18/70 25.71 0.002 .97 ≥ 40 6/73 8.22 19/73 26.03 AST <40 5/60 8.33 0 .98 11/60 18.33 3.065 .08 ≥ 40 7/83 8.43 26/83 31.33 BCLC A 4/46 8.70 1.179 .55 11/46 23.91 0.482 .79 B 2/42 4.76 10/42 23.81 C 6/55 10.91 16/55 29.09 HBsAg + 2/22 9.09 0.017 .90 7/22 31.82 0.479 .49 − 10/121 8.26 30/121 24.79 Tumor size <3 cm 2/20 10.00 0.078 .78 6/20 30.00 0.206 .65 ≥ 3 cm 10/123 8.13 31/123 25.20 SMP30, senescence marker protein 30; AFP, alpha-fetoprotein; ALT, alanine transaminase; AST, aspartate transaminase; BCLC, Barcelona Clinic Liver Cancer; HBsAg, hepatitis B surface antigen. View Large Combined Detection of AFP and Anti-SMP30 Antibody in Patients With HCC Through the data analyses, we report 16 cases of AFP negativity in patients who tested positive for anti-SMP30 antibody in the HCC group. With AFP of 25ng per mL or greater as a positive value, the positive rate of AFP in the diagnosis of HCC is 58.0%. We have assessed whether combined detection of anti-SMP30 antibody and AFP can improve the sensitivity of HCC detection. As we expected, testing of anti-SMP30 antibody and AFP have effectively increased the diagnostic accuracy for HCC with AUC of 0.856, compared with either test by itself (Figure 3). The positive rate in HCC is 69.23% for combined detection of AFP and Anti-SMP30 antibody (Table 2). Our results have shown that the combined detection has clinical significance and that it can significantly improve the detection rate of liver cancer (P <.01). Discussion Stenner-Liewen et al7 have used the SEREX technique for screening of HCC antigens from the serum of patients with HCC by using the complementary DNA (cDNA) library of HCC tissues, located in Guangxi, China. One of the antigens screened is homologous with human messenger RNA (mRNA) for 165 amino acids near the C-terminal of SMP30. Serologic tests have demonstrated that HCC ranging from 3 through 5 cm has an autoantibody-positive reaction to SMP30. All 20 serum specimens from different kinds of malignant neoplasms and 4 from hepatocirrhosis yielded negative reactions. The results of our serum analyses have suggested that SMP30 is a new HCC-associated antigen and thus can be of great value in the diagnosis of HCC. In a previous study,13 the research group had obtained BMP-SMP30 recombinant fusion protein by using genetic-engineering technology. The BMP-SMP30 fusion protein has shown strong water solubility and antigenicity and can be used to detect serum antibody. Our results have shown a 39% positive rate for autoantibody to SMP30 in serum specimens from patients with HCC. However, the molecular weight of BMP-labeled protein is too large for its 45-kD size. As a result, large-tag protein may be affecting the combining capacity of BMP-SMP30 fusion protein and anti-SMP30 antibody. HIS-SMP30 recombinant protein, when expressed, is water-insoluble. To resolve this problem, a molecular chaperone coexpression system (BL21[DE3] pLysS- pColdIII-SMP30 -pTf16) has been applied to express HIS-tag SMP30-soluble protein successfully.14 The label of such a fusion protein is small and does not have a great impact on the exposure of the SMP30 functional epitopes. Despite this finding, anti-SMP30 antibody levels from 143 patients with HCC have been detected by indirect ELISA, with soluble HiS-SMP30 fusion protein as antigen. Our results have shown that the positive rate is 25.87%, lower than the positive rate of 39% detected in the previous study. There are 3 possible reasons. First, the serum specimens from patients with HCC were tested in different batches, which may have influenced the rate of positivity. Second, the time spent, degree of freshness, and time saved with HCC serum specimens are different, leading to the differences in cutoff values. Third, although the molecular chaperone can promote the soluble expression of SMP30, it is worth considering the effect of molecular chaperone on the antigenic site of SMP30. CH and LC are important risk factors for the occurrence of HCC. In our study, we further detected the level of serum anti-SMP30 antibody in 137 patients with CH and 51 patients with LC. However, 8 serum specimens tested positive in patients with CH and 2 cases of a positive result in patients with LC. The positivity rate for the presence of anti-SMP30 antibody in the CH and LC groups was significantly lower than that in the HCC group (P <.05). Tumor material itself can induce the immune response of the human body to produce antibody related to the antitumor antigen; however, the specific mechanism is unclear. Part of the HCC serum anti-SMP30 antibody is negative for 2 possible reasons. First, the results of previous studies have shown that anti-SMP30 antibody appears in the early stages of HCC. The positivity rate for tumors less than 3 cm in diameter was higher than that of patients with tumors of 5 cm in diameter, which suggests that the presence of anti-SMP30 antibody may be an early indicator of HCC. In our current work, most of our serum specimens were donated by patients with advanced HCC. That type of patient has a lower immune response to tumor antigens, so some of the serum can not detect anti-SMP30 antibody. Second, anti-SMP30 antibody content is low and cannot be detected by ELISA assay. In this work, we detected anti-SMP30 antibody in some AFP-negative serum from patients with HCC. Therefore, anti-SMP30 antibody has great value in the diagnosis of HCC. It suggests that anti-SMP30 antibody production is closely related to the process of liver-cell malignant transformation and further shows that SMP30 autoantibody can be used as a biomarker for aiding with diagnosis of HCC. We report that the expression of SMP30 decreases in HCC-infected tissues but increases in adjacent cancerous tissues.8 Further, cell proliferation assay and migration assay results have shown that SMP30 can effectively inhibit the proliferation and migration of HCC cells, suggesting that SMP30 may function as a tumor suppressor.16,17 Because of low expression of SMP30 in liver cancer tissues and high expression in adjacent cancer tissues, it is valuable to study whether the appearance of serum anti-SMP30 antibody in patients with HCC is related to the protective reaction for HCC in the early stages. To study the relationship between serum anti-SMP30 antibody production and SMP30 expression levels, we have developed a method of double-antibody sandwich ELISA to detect SMP30 levels in serum. The results have shown that only 12 cases test positive for SMP30 in 143 cases of HCC, the positive rate is 8.39%, and the positive rate of SMP30 is lower than the antibody levels. Autoantibody to SMP30 has been detected in some HCC serum; however, the corresponding SMP30 has not been detected. Therefore, the mechanism of anti-SMP30 antibody appearance needs to be further investigated. Possible reasons for the low concentration of SMP30 in serum are that SMP30 mainly exists in cells, and normal tissue is rarely secreted into the serum; SMP30 expression in HCC tissues is too low to release in serum; and SMP30 from early stage liver cancer is released into the blood but as a foreign protein, making it easy to be removed by the body. At present, serum AFP testing is most commonly used for the diagnosis of HCC. However, the sensitivity of AFP is only 25% to 65%, which suggests that approximately 40% of patients with HCC may test false negative.4 We report that the positive rate of combined detection of anti-SMP30 antibody and AFP has increased to 69.23%, higher than the positive rate of 58.04% yielded by AFP single testing; the difference is statistically significant (P <.01). The combination of 2 biomarkers can improve the sensitivity of the diagnosis of HCC. In conclusion, we have identified a high level of anti-SMP30 antibody in serum specimens from individuals infected with HCC. The anti-SMP30 antibody in serum has been detected by the indirect ELISA method, and the positive rate is significantly higher than those in patients with CH and those with LC. Anti-SMP30 antibody is expected to be a new serological marker for diagnosis of HCC; it can effectively improve the sensitivity of the diagnosis of HCC, especially in combination with other tumor markers. Abbreviations: PLC primary liver cancer HCC hepatocellular carcinoma AFP alpha-fetoprotein SMP30 senescence marker protein 30 RGN regucalcin SEREX serologic identification by recombinant expression cloning cDNA complementary DNA WB Western blotting LC liver cirrhosis CH chronic hepatitis CT computed tomography MRI magnetic resonance imaging NHS normal human serum HBsAg hepatitis B surface antigen IPTG isopropyl β-D-1-thiogalactopyranoside Ni-NTA nitrilotriacetic acid IgG-HRP horseradish peroxidase–conjugated immunoglobulin G ELISA enzyme-linked immunosorbent assay PBST phosphate-buffered saline with Tween 20 BSA bovine serum albumin HRP horseradish peroxidase TMB tetramethyl benzidine OD optical density PBS phosphate-buffered saline ROC receiver operating characteristic AUC area under the curve CIs confidence intervals SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis ALT alanine transaminase AST aspartate transaminase cDNA complementary DNA mRNA messenger RNA NA nonapplicable BCLC Barcelona Clinic Liver Cancer Acknowledgments This work is supported by the National Natural Science Foundation of China (81572994, 81460432), Guangxi Nature Science Foundation (2015GXNSFDA139017), Guangxi Science and Technology Research and Technology Development Project (15104001-7), Programs for Department of Education of Guangxi Zhuang Autonomous Region (2013ZD011), and the Guangxi Nanning Qingxiu District Science and Technology Research and Technology Development Project (2014S03). References 1. Perz JF , Armstrong GL , Farrington LA , Hutin YJ , Bell BP . The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide . J Hepatol . 2006 ; 45 ( 4 ): 529 – 538 . 2. Bei CH , Bai H , Yu HP , et al. Combined effects of six cytokine gene polymorphisms and SNP-SNP interactions on hepatocellular carcinoma risk in Southern Guangxi, China . Asian Pac J Cancer Prev . 2014 ; 15 ( 16 ): 6961 – 6967 . 3. Ferlay J , Shin HR , Bray F , Forman D , Mathers C , Parkin DM . Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008 . Int J Cancer . 2010 ; 127 ( 12 ): 2893 – 2917 . 4. El-Serag HB , Marrero JA , Rudolph L , Reddy KR . Diagnosis and treatment of hepatocellular carcinoma . Gastroenterology . 2008 ; 134 ( 6 ): 1752 – 1763 . 5. Fujita T , Shirasawa T , Uchida K , Maruyama N . Isolation of cDNA clone encoding rat senescence marker protein-30 (SMP30) and its tissue distribution . Biochim Biophys Acta . 1992 ; 1132 ( 3 ): 297 – 305 . 6. Fujita T , Uchida K , Maruyama N . Purification of senescence marker protein-30 (SMP30) and its androgen-independent decrease with age in the rat liver . Biochim Biophys Acta . 1992 ; 1116 ( 2 ): 122 – 128 . 7. Stenner-Liewen F , Luo G , Sahin U , et al. Definition of tumor-associated antigens in hepatocellular carcinoma . Cancer Epidemiol Biomarkers Prev . 2000 ; 9 ( 3 ): 285 – 290 . 8. Zhou SF , Mo FR , Bin YH , Hou GQ , Xie XX , Luo GR . Serum immunoreactivity of SMP30 and its tissues expression in hepatocellular carcinoma . Clin Biochem . 2011 ; 44 ( 4 ): 331 – 336 . 9. Mo Z , Zheng S , Lv Z , et al. Senescence marker protein 30 (SMP30) serves as a potential prognostic indicator in hepatocellular carcinoma . Sci Rep . 2016 ; 6 : 39376 . 10. Arif S , Qudsia S , Urooj S , Chaudry N , Arshad A , Andleeb S . Blueprint of quartz crystal microbalance biosensor for early detection of breast cancer through salivary autoantibodies against ATP6AP1 . Biosens Bioelectron . 2015 ; 65 : 62 – 70 . 11. Hirano N , Butler MO , Von Bergwelt-Baildon MS , et al. Autoantibodies frequently detected in patients with aplastic anemia . Blood . 2003 ; 102 ( 13 ): 4567 – 4575 . 12. Zhang H , Xia J , Wang K , Zhang J . Serum autoantibodies in the early detection of esophageal cancer: a systematic review . Tumour Biol . 2015 ; 36 ( 1 ): 95 – 109 . 13. Zhou SF , Xie XX , Bin YH , Lan L , Chen F , Luo GR . Identification of HCC-22-5 tumor-associated antigen and antibody response in patients . Clin Chim Acta . 2006 ; 366 ( 1-2 ): 274 – 280 . 14. Zhang SC , Huang P , Zhao YX , et al. Soluble expression of recombinant human SMP30 for detecting serum SMP30 antibody levels in hepatocellular carcinoma patients . Asian Pac J Cancer Prev . 2013 ; 14 ( 4 ): 2383 – 2386 . 15. Piemonte M . TNM -- classification of malignant tumors (VI edition -- 2002). Innovations in the classification of head and neck neoplasms [in Italian] . Acta Otorhinolaryngol Ital . 2003 ; 23 ( 2 ): 132 – 135 . 16. Misawa H , Inagaki S , Yamaguchi M . Suppression of cell proliferation and deoxyribonucleic acid synthesis in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin . J Cell Biochem . 2001 ; 84 ( 1 ): 143 – 149 . 17. Tsurusaki Y , Yamaguchi M . Role of endogenous regucalcin in nuclear regulation of regenerating rat liver: suppression of the enhanced ribonucleic acid synthesis activity . J Cell Biochem . 2002 ; 87 ( 4 ): 450 – 457 . © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Laboratory MedicineOxford University Press

Published: Mar 17, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off