Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 2  |  Page : 341-348

Detection of circulating lamin B1 and glypican-3 for early diagnosis of hepatocellular carcinoma patients


Department of Clinical Pathology, Faculty of Medicine, National Liver Institute, Menoufia University, Menoufia Governorate, Egypt

Date of Submission17-Feb-2015
Date of Acceptance17-Mar-2015
Date of Web Publication18-Oct-2016

Correspondence Address:
Heba S Mohammed Ghanem
Department of Clinical Pathology, National Liver Institute, Menoufia University, 5 Taiseer street, Shebein El kom, Menoufia Governorate, 32511
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.192442

Rights and Permissions
  Abstract 

Objective:
To assess circulating lamin B1 (LMNB1) mRNA and glypican-3 cytotoxic T lymphocytes (CTLs) as markers for early detection of hepatocellular carcinoma (HCC).
Background:
HCC is the fifth most common form of cancer worldwide and the third most common cause of cancer-related deaths. HCC often occurs in the background of a cirrhotic liver. In recent years, surveillance strategies in patients at a higher risk of HCC have led to the diagnosis of the disease at much earlier stages. Patients in early stages have a much higher chance of curative response with different treatment options.
Materials and methods:
The study included 50 participates, of which 13 were patients with posthepatitis C liver cirrhosis (group I), 13 were patients with early stages of HCC (group IIa), 14 were patients with late stages of HCC (group IIb), and 10 were healthy volunteers with matched age and sex as a control group (group III). The patients were selected from the outpatient clinic and inpatient department of the National Liver Institute, Menoufiya University. LMNB1 mRNA was assessed by reverse transcription-PCR and glypican3 CTLs by flow cytometry and then we compared that with α±-feto protein.
Results:
Glypican3 CTLs can be used for early detection of HCC patients with 100% specificity and sensitivity. In addition, LMNB1 mRNA can be used in early detection of HCC with specificity and sensitivity 92 and 100%, respectively.
Conclusion:
Circulating LMNB1 mRNA and glypican3 CTLs can be used as markers for early detection of HCC patients.

Keywords: glypican-3 CTL, hepatocellular carcinoma, lamin B1, reverse transcription-PCR


How to cite this article:
El-Saeid GK, Al-Hendy AA, Hendy OM, Fathy WM, Mohammed Ghanem HS. Detection of circulating lamin B1 and glypican-3 for early diagnosis of hepatocellular carcinoma patients. Menoufia Med J 2016;29:341-8

How to cite this URL:
El-Saeid GK, Al-Hendy AA, Hendy OM, Fathy WM, Mohammed Ghanem HS. Detection of circulating lamin B1 and glypican-3 for early diagnosis of hepatocellular carcinoma patients. Menoufia Med J [serial online] 2016 [cited 2020 Apr 6];29:341-8. Available from: http://www.mmj.eg.net/text.asp?2016/29/2/341/192442


  Introduction Top


Hepatocellular carcinoma (HCC) is the fifth most common form of cancer worldwide and the third most common cause of cancer-related deaths. HCC often occurs in the background of a cirrhotic liver [1].

In recent years, surveillance strategies in patients at a higher risk of HCC have led to the diagnosis of the disease at much earlier stages. Patients in early stages have a much higher chance of curative response with different treatment options [1].

The only clinically proven HCC biomarker, α-feto protein (AFP), has been widely applied as part of the surveillance for screening individuals at risk of developing HCC, despite the fact that it has very limited sensitivity (39−65%) and specificity (76−94%), particularly for small and early HCC [2].

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly, and nuclear positioning [3]. Recent studies provide new insights into the role of lamins in the development, differentiation, and tissue response to mechanical, reactive oxygen species, and thermal stresses [3].

More importantly, proteolytic cleavage and subsequent degradation of lamin B1 (LMNB1) have been previously reported during radiation-induced apoptosis [4]. Some of the apoptotic cells would release LMNB1 mRNA into the circulation and that such LMNB1 mRNA might be detectable in the patients' plasma. This may explore the possibility of using reverse transcription-PCR (RT-PCR) in measuring plasma LMNB1 mRNA in the HCC patients [5].

Glypican-3 (GPC3) is a member of the glypican family of glycosylphosphatidylinositol-anchored cell-surface heparan sulfate proteoglycans. GPC3 is highly expressed in the HCC cells and tissues. It is thought that GPC3 stimulates the growth of HCC cells by upregulating autocrine/paracrine canonical Wnt signaling [6]. The specific function of a glypican in a particular cell context depends on its structural features and on the set of growth factors and growth factor receptors present in that cellular context [7]. The prediction of the overexpression of GPC3 in HCC is related to the development of HCC in a background of chronic hepatitis (CH) and/or liver cirrhosis (LC) [7]. To date, few studies were concerned about the role of noninvasive markers in early detection of HCC, and therefore this study was designed.


  Aim of the Study Top


The study aimed to assess the levels of LMNB1 mRNA and GPC3 cytotoxic T lymphocytes (CTLs) in the peripheral blood of LC and HCC patients as markers for early detection of HCC.


  Materials and Methods Top


The study included 50 persons, of which 13 were patients with posthepatitis C LC (group I), 13 were patients with early stages of HCC (group IIa), 14 were patients with late stages of HCC (group IIb, according to The Barcelona Clinic Liver Cancer staging system), and 10 were healthy volunteers with matched age and sex as a control group (group III). The patients were selected from the outpatient clinic and inpatient department of the National Liver Institute, Menoufiya University.

Exclusion criteria (excluded subjects): patients who received antiviral or immunomodulatory therapy, patients who had a history of excessive alcohol consumption, patients who were treated with hepatotoxic drugs, and patients who had other chronic liver diseases (such as Wilson's disease, hemochromatosis) were excluded.

All individuals were subjected to:

  1. History and clinical examination.
  2. Abdominal ultasonography.
  3. Spiral computed tomography for HCC cases.


Laboratory investigations

Fifteen milliliters venous blood samples were taken by sterile venipuncture, without frothing and after minimal venous stasis using a disposable syringes. The blood samples were distributed as follows: 5 ml of venous blood were delivered in a vacutainer plain test tube. Blood was left for a sufficient time to clot, serum was then separated after centrifugation at 3000 rpm/min for 10 min for liver function tests, AFP level, hepatitis viral markers. Five milliliters of venous blood were delivered in a vacutainer plastic tube containing EDTA for CBC, GPC3 analysis. Five milliliters of venous blood were delivered in a vacutainer plastic tube containing EDTA for LMNB1 mRNA by RT-PCR.

The following laboratory tests were conducted:

Complete blood picture was done on Sysmex KX-21 (Wakinohama-kaigandori, Kobe, Hyogo, Japan).

Liver function tests [alanine transaminase, aspartate transaminase, albumin, total protein, total bilirubin, direct bilirubin, alkaline phosphates, gamma-glutamyl transferase (GGT)] were conducted using Integra 400 autoanalyzer (Roche-Diagnostics, Mannheim, Germany).

HBsAg and hepatitis C virus antibody tests were conducted by the electrochemiluminescence immunoassay 'ECLIA' using Elecsys 2010 autoanalyzer (Roche-Diagnostics).

Measurement of serum AFP was taken by Elecsys 2010 autoanalyzer (Roche-Diagnostics).

Detection of LMNB1 mRNA by real time PCR:

The test is based on these major processes: sample preparation, RNA extraction, RT-PCR amplification of target DNA using LMNB1 specific complimentary primers, hybridization of the amplified products to SYBER green dye and detection of the SYBER green dye-bound amplified products by colorimetric determination.

  1. RNA extraction from whole blood: Total RNA was extracted from 0.5 ml up to 1.0 ml of whole blood using innuPREP Blood RNA Analytik Jena AG Kit. (Wakinohama-kaigandori, Chuo-ku, Kobe, Hyogo, Japan)
  2. Reverse transcription: Verso cDNA Synthesis Kit is tested functionally for use in RT-PCR. This is done by Thermo Scientific Verso cDNA Synthesis Kit.
  3. PCR amplification was done by the following steps:

    1. Denaturation: generally, a 2 min initial denaturation step at 95°C is sufficient.
    2. Annealing: optimize the annealing conditions by performing the reaction starting ∼5°C below the calculated melting temperature of the 95 primers (63°C) and increasing the temperature in increments of 1°C to the annealing temperature. The annealing step is typically 1 min at 65°C.
    3. Extension: the extension reaction is typically performed at the optimal temperature for DNA polymerase, which is 72°C. A final extension of 2 min at 72°C is recommended.Soak cycle: Hold the reactions at 4°C overnight. Long-term storage of cDNA at –20°C is recommended.


  4. The step of reverse transcription is repeated using house keeping gene GAPDH.
  5. Hybridization of the amplified products to SYBR green dye: This step is done using 5´ Hot Firepol Eva Green qPCR Mix Plus catalog number: (08-25-0001). Pack size: 1 ml (250 reactions). Purchased from (Solis BioDyne, Tartu, Estonia).
  6. Calculation of relative expression level: The mean expression levels of LMNB1 mRNA relative to GAPDH mRNA level were calculated using the quantitation-comparative Ct(ΔΔCt) method on the ABI 7500 fluorescence quantitative PCR analyzer. After correction with the corrector sample, the relative quantitation values of the expression levels were used for the statistical analysis.


  1. GPC3 CTLs: flow cytometry was carried out by two monoclonal antibodies are used, one directed against human leukocyte surface marker CD8-fluorescein isothiocyanate. The other is monoclonal anti-human GPC3-phycoerythrin (PE).


Principle of the test

Washed cells are incubated with the PE-labeled monoclonal antibody, which binds to cells expressing GPC3. Unbound PE-conjugated antibody is then washed from the cells. Cells expressing GPC3 are fluorescently stained, with the intensity of staining directly proportional to the density of expression of GPC3. Cell surface expression of GPC3 is determined by flow cytometric analysis using 488 nm wavelength laser excitation and monitoring emitted fluorescence with a detector optimized to collect peak emissions at 565–605 nm.

Statistical analysis

The data were collected, tabulated, and analyzed by statistical package for social science, version 17.0 (SPSS Inc., Chicago, Illinois, USA) on an IBM-compatible computer. MannWhitney U-test: a nonparametric test of significance used for comparison between two groups non-normally distributed having quantitative variables. Kruskal–Wallis test was used for comparison of more than two groups of non-normally distributed variables Pearson correlation (r) was used to detect the relationships among variables.


  Results Top


The levels of ALP and GGT were significantly higher in the late HCC patient group than in the cirrhotic group ([Table 1]). In comparison between the early HCC group and late HCC group, a higher significant level of total bilirubin and direct bilirubin, was reported in the late HCC group than in the early HCC group. In comparison between the early HCC group, late HCC group, cirrhotic group, and control group, there was a high significant increase in the levels of aspartate transaminase, alanine transaminase, total bilirubin and direct bilirubin, and GGT were reported in these groups than in the control group. Lower level of albumin were reported in the late HCC patient group than in the control group.
Table 1: Comparison between the studied groups with regard to liver functions

Click here to view


The level of Hb and platelets were significantly lower in the early HCC group and late HCC groups than in the control group ([Table 2]).
Table 2: Comparison between the studied groups with regard to CBC

Click here to view


In comparison between the early HCC group and cirrhotic group, a higher significant increase in the level of AFP, LMNB1, and GPC3 were reported in the early HCC patient group and late HCC group than in the cirrhotic group ([Table 3]). In addition, their levels were significantly increased in the early HCC patient group than in the control group ([Figure 1]).
Table 3: Comparison between the studied groups as regards a-feto protein, lamin B1, glypican-3

Click here to view
Figure 1: Comparison between the studied groups with regard to lamin B1 and glypican-3

Click here to view


[Table 4] demonstrates the receiver operator characteristics (ROC) of AFP, LMNB1 mRNA, and GPC3 for differentiation between HCC and cirrhosis showed that: the best cutoff value of AFP was 13.2 ng/ml with a sensitivity of 85.2% and specificity of 76.9%, and the area under the curve (AUC), which represents the overall accuracy of the test, was 0.90. The best cutoff value of LMNB1 was 2.86 with a sensitivity of 100% and specificity of 92.3%, and the AUC was 0.99. The best cutoff value of GPC3 was 1.285 ng/ml with a sensitivity of 100% and specificity of 100%, and the AUC was 1.0 ([Figure 2]).
Table 4: The value of AFP, lamin B and glypican for differentiation between HCC and cirrhosis

Click here to view
{Figure 1}
Figure 2: ROC curve of α-feto protein, lamin B and glypican for differentiation between early and late HCC cases. HCC, hepatocellular carcinoma; ROC, receiver operator characteristic

Click here to view


ROC of AFP, LMNB1 mRNA, and GPC3 for differentiation between early and late HCC showed that: the best cutoff value of AFP was 64 ng/ml with sensitivity of 64.3% and specificity of 53.8%, and the AUC was 0.47 ([Table 5]). The best cutoff value of LMNB1 was 5.785 with sensitivity of 78.6% and specificity of 61.5%, and the AUC was 0.7. The best cutoff value of GPC3 was 5.15 ng/ml with sensitivity of 85.7% and specificity of 69.2%, and the AUC was 0.77 ([Figure 3]).
Table 5: The value of AFP, lamin B and glypican for differentiation between early and late HCC cases

Click here to view
Figure 3: ROC curve of α-feto protein, lamin B and glypican for differentiation between HCC and cirrhosis. HCC, hepatocellular carcinoma; ROC, receiver operator characteristic

Click here to view


ROC of AFP, LMNB1 mRNA, and GPC3 for differentiation between LC and early HCC showed that: the best cutoff value of AFP was 13.2 ng/ml with sensitivity of 92.3% and specificity of 76.9%, and the AUC was 0.91 ([Table 6]). The best cutoff value of LMNB1 was 2.86 with sensitivity of 100% and specificity of 100%, and the AUC was 0.99. The best cutoff value of GPC3 was 1.29 ng/ml with sensitivity was 100% and specificity was 92.9%, and the AUC was 1.0 ([Figure 4]).
Table 6: The value of AFP, lamin B and glypican for differentiation between cirrhosis and early HCC cases

Click here to view
Figure 4: Cutoff value of GPC3 was 1.29 ng/ml with sensitivity was 100% and specificity was 92.9%, and the AUC was 1.0

Click here to view



  Discussion Top


Liver cancer on men is the fifth most frequently diagnosed cancer worldwide, and is the second leading cause of cancer-related death in the world [8]. In recent years, surveillance strategies in patients at a higher risk of HCC have led to the diagnosis of the disease at much earlier stages. Patients in early stages have a much higher chance of curative response with different treatment options [1] Therefore, the current study was designed to assess circulating LMNB1 mRNA and GPC3 CTLs as noninvasive markers for early detection of HCC ([Figure 5] and [Figure 6]).
Figure 5: Housekeeping (GAPDH) gene amplification plot

Click here to view
Figure 6: Target gene (lamin B1)amplification plot

Click here to view


In this study, higher levels of GGT and ALP were reported in the HCC patient group than in the cirrhotic group. This is in agreement with Mini [9], who reported that ALP and GGT of HCC patients were significantly increased compared with the LC patients. Serum GGT in healthy adults is mainly secreted by the hepatic Kupffer cells and endothelial cells of the bile duct, and its activity increases obviously in the tissues of HCC and fetal liver. Abnormal expression of total GGT activities can be found in patients with different liver diseases and extrahepatic tumors [0]. Higher levels of total bilirubin and direct bilirubin were detected in the HCC patient group than in the cirrhotic group, and this result was agreed with Baghdadya et al. [1] who reported that bilirubin showed a highly significant increased in the HCC than in the non-HCC groups.

The obtained results of the present study showed that a high significant increase in the level of LMNB1 was reported in the HCC patient group than in the cirrhotic and control groups. In addition, there was a highly significant level of LMNB1 was reported in the early HCC patient group than in the cirrhotic group. These results are agreed with Sun et al. [2], who reported that LMNB1 is a potential HCC marker that acts intracellularly or extracellularly in the form of protein and mRNA, respectively. The expression of this marker could be detected in the earliest stage of HCC.

Salway et al. [3] added that the overexpression of LMNB1 is positively correlated with the tumor development. Some of the apoptotic cells would release LMNB1 mRNA into the circulation and that such LMNB1 mRNA might be detectable in the patients' plasma [3].

The current study showed that the GPC3 was significantly increased in the HCC patient group as compared with the cirrhotic and control groups. Its level was significantly higher in the early HCC patient group than in the cirrhotic group. This result agreed with Hayashi et al. [5] who reported that GPC3-specific CTLs were found to be present not only in patients with HCC, but also in patients with CH and LC. This suggests the possibility of GPC3-specific CTLs serving as a marker for the early diagnosis of imaging-invisible HCC. The increase in the frequency of GPC3-specific CTLs and titers of anti-GPC3 IgG in the peripheral blood might be related to the expression of GPC3 in CH with high-grade inflammation and LC [4].

Although serum AFP level is a useful tumor marker for the detection and monitoring of HCC, the false-negative rate with AFP level alone may be as high as 40% for patients with early-stage HCC. Even in patients with advanced HCC, the AFP levels may remain normal in 15–30% of the patients [6].

In this study, ROC of AFP for differentiation between HCC and cirrhosis showed that the best cutoff value was 13.2 ng/ml with a sensitivity of 85.2% and specificity of 76.9%, and the AUC was 0.90. In addition, ROC of LMNB1 mRNA and GPC3 for differentiation between HCC and cirrhosis showed that the best cutoff value of LMNB1 was 2.86 with a sensitivity of 100% and specificity of 92.3%. The best cutoff value of GPC3 was 1.285 ng/ml with a sensitivity of 100% and specificity of 100%. These results agreed with Sun et al., 2010 who reported elevation of circulating LMNB1 marker in plasma could detect early stages of HCC patients, with 76% sensitivity and 82% specificity.


  Conclusion Top


The increasing levels of circulating LMNB1 mRNA and GPC3 CTLs in early HCC patients more than cirrhosis patients with a high specificity and sensitivity could imply these markers in the early detection of HCC patients. Further large-scale study must be conducted to clarify the role of these markers in tumor pathogenesis and that their antagonist may be implicated as target therapy of HCC.

Conflicts of interest

There are no conflicts of interest.[14]

 
  References Top

1.
Raza A, Sood GK. Hepatocellular carcinoma review: current treatment, and evidence-based medicine. World J Gastroenterol 2014; 95:4115–4127.  Back to cited text no. 1
    
2.
Chan AC, Poon RT, Ng KK, Lo CM, Fan ST, Wong J. Changing paradigm in the management of hepatocellular carcinoma improves the survival benefit of early detection by screening. Ann Surg 2008; 95:666–673.  Back to cited text no. 2
    
3.
Zuela N, Bar DZ, Gruenbaum Y. Lamins in development, tissue maintenance and stress. EMBO Rep 2012; 95:1070–1078.  Back to cited text no. 3
    
4.
Capurro M, Shi W, Sandal S, Filmus J. Processing by convertases is not required for glypican-3-induced stimulation of hepatocellular carcinoma. J Biol Chem 2005; 95:41201–41206.  Back to cited text no. 4
    
5.
Hayashi E, Motomura Y, Shirakawa H, Yoshikawa T, Oba N, Nishinakagawa S, et al. Detection of glypican-3-specific CTLs in chronic hepatitis and liver cirrhosis. Oncol Rep 2009; 95:149–154.  Back to cited text no. 5
    
6.
Yao D, Dong Z. Hepatoma-related gamma-glutamyl transferase in laboratory or clinical diagnosis of hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int 2007; 95:9–11.  Back to cited text no. 6
    
7.
Filmus J, Capurro M. Glypican-3: a marker and a therapeutic target in hepatocellular carcinoma. FEBS J 2013; 95:2471–2476.  Back to cited text no. 7
    
8.
Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 95:69–90.  Back to cited text no. 8
    
9.
Mini K. Investigation on enzymes and acute phase proteins in liver diseases [thesis]. Calicut, India: Department of Life Sciences, University of Calicut; 2008.  Back to cited text no. 9
    
10.
Dechat T, Pfleghaar K, Sengupta K, Shimi T, Shumaker DK, Solimando L, Goldman RD. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev 2008; 95:832–853.  Back to cited text no. 10
    
11.
Baghdadya I, Fouada F, Sayedb M, Shoaiba A, Salaha Y, Elshayeba E, Hasana AF. Serum markers for the early detection of hepatocellular carcinoma in patients with chronic viral hepatitis C infection. Menoufia Med J 2014; 95:544–550.  Back to cited text no. 11
    
12.
Sun S, Xu MZ, Poon RT, Day PJ, Luk JM. Circulating Lamin B1 (LMNB1) biomarker detects early stages of liver cancer in patients. J Proteome Res 2010; 9:70–78.  Back to cited text no. 12
    
13.
Salway F, Day J, Ollier E, Peakman C. Levels of 5 ′ RNA tags in plasma and buffy coat from EDTA blood increase with time. Int J Epidemiol 2008; 95:111–115.  Back to cited text no. 13
    
14.
Baumhoer D, Tornillo L, Stadlmann S, Roncalli M, Diamantis EK, Terracciano LM. Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples. Am J Clin Pathol 2008; 95:899–906.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Aim of the Study
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed712    
    Printed2    
    Emailed0    
    PDF Downloaded83    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]