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ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 1  |  Page : 141-146

The diagnostic value of serum squamous cell carcinoma antigen for prediction of hepatocellular carcinoma


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

Date of Submission21-May-2014
Date of Acceptance26-Aug-2014
Date of Web Publication18-Mar-2016

Correspondence Address:
Sally S Mandour
MBBCh, Department of Clinical Pathology, National Liver Institute, Menoufia University, 5 Taiseer Street, Shebein El Kom, Menoufia Governorate, 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.179005

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  Abstract 

Objective
The aim of this study was to determine the efficacy of serum squamous cell carcinoma antigen (SCCA) in comparison with α-fetoprotein (AFP) in the detection of hepatocellular carcinoma (HCC).
Background
HCC is the most common type of primary liver cancer and shows a growing incidence worldwide, related to the increased prevalence of various risk factors for chronic liver diseases, such as hepatitis infection with hepatitis C and B viruses. Each year HCC is diagnosed in more than half a million people worldwide. Therefore, prompt diagnosis of HCC is imperative.
Patients and methods
This study was carried out at the Clinical Pathology Department, Faculty of Medicine, and Clinical Pathology Department, National Liver Institute, Menoufia University, from June 2012 to November 2013. The study included 30 patients with liver cirrhosis and 30 patients with HCC, in addition to 17 unrelated healthy adults of matched age and sex who were included as controls. Serum AFP and serum SCCA level were estimated using enzyme linked immunosorbent assay.
Results
Although SCCA has more sensitivity than AFP in the detection of HCC, AFP remains a relatively good diagnostic marker for HCC, with high specificity.
Conclusion
AFP is a relatively good diagnostic test for cirrhosis with high specificity. Moreover, a combination of the two markers may improve sensitivity, making their combination a perfect screening test for the prediction of HCC.

Keywords: Hepatocellular carcinoma, liver cirrhosis, squamous cell carcinoma antigen


How to cite this article:
El-Bassuonia MA, El-Edel RH, Gawesh EA, Mandour SS. The diagnostic value of serum squamous cell carcinoma antigen for prediction of hepatocellular carcinoma. Menoufia Med J 2016;29:141-6

How to cite this URL:
El-Bassuonia MA, El-Edel RH, Gawesh EA, Mandour SS. The diagnostic value of serum squamous cell carcinoma antigen for prediction of hepatocellular carcinoma. Menoufia Med J [serial online] 2016 [cited 2019 Sep 20];29:141-6. Available from: http://www.mmj.eg.net/text.asp?2016/29/1/141/179005


  Introduction Top


Hepatocellular carcinoma (HCC) represents the fifth most common cancer in the world, and the third most frequent oncological cause of death. Its incidence is on the increase, and it is becoming more and more significant both clinically and epidemiologically [1]. In Egypt, HCC is reported to account for about 4.7% of chronic liver disease patients [2].

HCC detected after the onset of symptoms has a dismal prognosis, with only 0-10% surviving up to 5 years. In contrast, small HCC detected by surveillance can be cured. As potentially curative treatment modalities are available, identification of HCC at an early stage with a good screening test is, therefore, imperative for a favorable outcome [3].

α-Fetoprotein (AFP) is the major biomarker used for HCC diagnosis; however, its use in the early detection of HCC is limited, especially because about one-third of patients with HCC have normal levels of serum AFP [4].

Squamous cell carcinoma antigens (SCCAs) are members of the serpin family of endogenous serine proteinase inhibitors. The first variant of SCCA, SCCA1, was originally identified in squamous cell carcinoma (SCC) of the uterine cervix. Both isoforms, SCCA1 and SCCA2, are expressed in stratified squamous epithelia and have been found to be produced in SCCs [5]. SCCA levels were significantly elevated in HCC patients compared with patients with cirrhosis only or normal individuals [6].

The current study aimed to evaluate the level of SCCA in HCC and liver cirrhosis patients in relation to AFP as a conventional marker for HCC, which lacks disease sensitivity.


  Patients and methods Top


The study was carried out at the Clinical Pathology Department, Faculty of Medicine, and Clinical Pathology Department, National Liver Institute, Menoufia University, from June 2012 to November 2013. It was a case-control study that included 30 patients with HCC and 30 patients with liver cirrhosis. In addition, 17 healthy adults, of matched age and sex, with no previous history of liver diseases, and negative for hepatitis viral markers, were included as controls and selected from healthy blood donors in the National Liver Institute blood bank unit.

The participants were classified into three groups on the basis of clinical findings, laboratory findings, serological tests, and abdominal ultrasound as follows:

  1. Group I (patients with liver cirrhosis): This group included 30 patients with cirrhotic liver disease.
  2. Group II (patients with HCC): This group included 30 patients with HCC.
  3. Group III (controls): This group included 17 healthy individuals of matched age and sex.


Exclusion criteria

Patients with SCC other than HCC, patients with acute liver disease, and patients with autoimmune hepatitis were excluded.

Ethical consideration

Consent was taken from both patients and controls or from their relatives before the samples were taken, and they had the right to refuse at any time. The study was approved by the ethics board of the National Liver Institute.

All individuals were subjected to clinical assessment including full history and clinical examination, abdominal ultrasonography, and computerized tomography or liver biopsy when possible. Laboratory investigations including complete blood count, renal functions tests, liver function tests, hepatitis viral markers - including hepatitis B virus surface antigen (HBsAg) and hepatitis C virus antibodies (HCV Ab) - serum AFP level, and serum level of SCCA by enzyme linked immunosorbent assay technique using the CanAg SCC EIA (Fujirebio Diagnostics AB, ElofLindδlvsgata 13 SE-414 55 Göteborg, Sweden) kit were carried out.

Determination of serum squamous cell carcinoma antigen concentration

Principle of the test

The CanAg SCC EIA is a solid-phase, noncompetitive immunoassay based on the direct sandwich technique. Calibrators and patient samples are incubated together with biotinylated anti-SCC monoclonal antibody and horseradish peroxidase (HRP)-labeled anti-SCC monoclonal antibody in streptavidin-coated microstrips. After washing, buffered substrate/chromogen reagent is added to each well, and the enzyme reaction is allowed to proceed. During the enzyme reaction a blue color develops if the antigen is present. The intensity of the color is proportional to the amount of SCCA present in the samples. The color intensity is determined using a microplate spectrophotometer at 620 nm. Calibration curves are constructed for each assay by plotting absorbance value versus the concentration for each calibrator. The SCCA concentrations of patient samples are then read from the calibration curve.

Assay steps

The assay was performed in two sets for calibrators and patient samples. A calibration curve was run with each assay. All reagents and samples were brought to room temperature (20-25°C) before use:

  1. SCC calibrators, wash solution, and antibody solution were prepared.
  2. The required number of microplate strips were transferred to a strip frame. Each strip was washed once with the wash solution.
  3. A volume of 25 μl of the SCC calibrators (CAL A, B, C, D, E) and patient samples was pipetted into the strip wells.
  4. A volume of 100 μl of antibody solution was added to each well. Carry-over was avoided by holding the pipette tip slightly above the well and avoiding contact with the plastic strip or the surface of the liquid.
  5. The frame containing the strips was incubated for 1 h (±5 min) at room temperature (20-25°C) with constant shaking of the plate using a microplate shaker, and then each strip was washed six times using the wash procedure described in procedural notes item 4.
  6. A volume of 100 μl of TMB HRP substrate was added to each well using the same pipetting procedure as in item 4. The TMB HRP substrate should be added to the wells as quickly as possible, and the time between the addition to the first and last well should not exceed 5 min.
  7. The frame containing the strips was incubated for 30 min (±5 min) at room temperature with constant shaking. Direct sunlight was avoided. The absorbance was read immediately at 620 nm using a microplate spectrophotometer.


For calculation of results, manual evaluation was used. A calibration curve was constructed by plotting the absorbance (A) values obtained for each SCC calibrator against the corresponding SCC concentration (μg/l). The unknown SCC concentrations were then read from the calibration curve using the mean absorbance value of each patient specimen.

Statistical analyses

The data collected were tabulated and analyzed by statistical package for the social sciences (SPSS, version 17.0; SPSS Inc., Chicago) on an IBM compatible computer. Two types of statistics were determined:

Descriptive statistics: This comprised percentage, range, mean (x), and SD.

Analytical statistics: This comprised the following:

The ν2 -test, which was used to study the association between two qualitative variables.

The Mann-Whitney U-test, which is a test of significance and was used for between-group comparison of nonparametric quantitative variables.

The Kruskal-Wallis test, which is a test of significance used for comparison of nonparametric quantitative variables between three or more groups.

ANOVA (f ) test, which is a test of significance used for comparison of parametric quantitative variables between three or more groups.

Pearson's correlation, which is a test used to measure the association between two quantitative variables.

The data were considered statistically significant when P-value was less than 0.05.


  Results Top


The age of patients with cirrhosis ranged from 22 to 71 years with a mean of 48.7 ± 15.4 years and that of patients with HCC was 30-70 years with a mean of 47.8 ± 14.2 years. Control ages ranged between 20 and 51 years with a mean age of 33.7 years. There was a high statistical difference between studied groups with respect to age (P = 0.004). The study included 19 (63.3%) male patients and 11 (36.7%) female patients with cirrhosis, and 22 (73.3%) male patients and eight (26.7%) female patients with HCC, showing no statistical difference between studied groups (P = 0.55) [Table 1].
Table 1: Comparison between the studied groups with respect to their sociodemographic characteristics

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Hepatitis C Ab was positive in 29 of 30 patients in groups I and II, but HBsAg was positive in 29 out of 30 in group I and negative in all patients of group II [Table 2].
Table 2: Serological hepatitis markers in the three studied groups

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Serum level of AFP showed the highest levels in the HCC group (mean value was 16079.2) compared with the other two groups. The liver cirrhosis group showed a higher level of AFP compared with the controls (mean value was 7.41 for the cirrhotic group and 3.27 for the control group) with a high significant increase (P < 0.001) [Table 3] and [Figure 1]. For SCCA, the highest levels were reported in the HCC group (mean value was 2.0) compared with the other two groups; also the liver cirrhosis group showed a high level of SCCA compared with the control group (mean value was 1.20 for the cirrhotic group and 1.01 for the control group) with no significant differences between the studied groups (P = 0.49) [Table 4] and [Figure 2].
Figure 1: The mean α-fetoprotein levels showed that the highest levels were found in the hepatocellular carcinom a group.

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Figure 2: The mean squamous cell carcinoma antigen levels showed that the highest levels were found in the hepatocellular carcinom a group.

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With respect to tumor size, AFP level was higher in patients with tumor size greater than 6 cm (mean 24337.7) than in patients with tumor size less than 6 cm (mean 5279.6), showing a significant increase (P = 0.007). SCCA level was also higher in patients with tumor size greater than 6 cm (mean 2.71) than in patients with tumor size less than 6 cm (mean 1.08) but with no significant increase (P = 0.89) [Table 5] and [Figure 3].
Figure 3: Mean level of á-fetoprotein (AFP) in the hepatocellular carcinoma group as regards tumor size showed that AFP level was higher in patients with tumor size greater than 6 cm than in patients with tumor size less th an 6 cm.

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Table 3: Comparison between levels of ¦Á-fetoproteins in the studied groups

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The receiver operating characteristic curve for AFP was closer to the upper left corner. This reflects its accuracy as a good screening test for HCC compared with SCCA. The area under the curve of SCCA was 0.56; the cutoff value was established at 0.5 μg/l with 73% sensitivity and 44% specificity. The area under the curve of AFP was 0.87; the significant cutoff value was established at 258 μg/l with 60% sensitivity and 100% specificity. On combination of the two cutoff values the sensitivity improved to 100% and the specificity was 47% [Table 6], [Table 7] and [Figure 4].
Figure 4: Receiver operating characteristic curve with sensitivity of 73% and specifi city of 44% for squamous cell carcinoma antigen at cutoff 0.5 μg/l, and sensitivity of 60% and specifi city of 100% for α-fetoprotein at cutoff 258 μg/l.

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Table 4: Comparison between levels of squamous cell carcinoma antigen in the studied groups

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Table 5: Comparison between α-fetoprotein and squamous cell carcinoma antigen in the hepatocellular carcinoma group as regards tumor size

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Table 6: Sensitivity, specificity, positive predictive value, and negative predictive value of α-fetoprotein and squamous cell carcinoma antigen at various cutoff values in cirrhotic and hepatocellular carcinoma groups

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Table 7: Sensitivity, specifi city, positive predictive value, and negative predictive value for combination of á-fetoprotein
and squamous cell carcinoma antigen at cutoff values 258 and 0.5 ng/ml, respectively


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  Discussion Top


Detection of HCC at an early stage may reduce mortality significantly. Mass screening may be justified as the population at-risk can be identified easily and tumor ablation or resection at an early stage can increase survival. However, mass screening should be justified only when sensitive and specific diagnostic procedures are available [7].

AFP is the major biomarker used for HCC diagnosis; however, its use in the early detection of HCC is limited, especially because many patients with HCC have normal levels of serum AFP [4]. Other biomarkers failed to discriminate between liver cirrhosis and HCC in a satisfactory manner in terms of diagnostic accuracy, reproducibility of the results, or technical issues related to the biomarker detection method [8]. For this reason, the simultaneous use of different tests seems to offer a promising approach that warrants further investigation [9].

SCCA, the serine protease inhibitor, is physiologically expressed in the skin and other squamous epithelial cells. SCCA levels were significantly elevated in HCC patients compared with patients with cirrhosis only or normal individuals [6].

This study aimed to evaluate the significance of SCCA serum level in HCC and liver cirrhosis patients in relation to AFP.

In the present study, the mean serum level of each AFP and SCCA showed great variation between the three studied groups, with the highest increase in the HCC group. Despite the presence of this variation, there was no significant statistical difference with respect to the mean serum level of SCCA between any of the studied groups (P = 0.49). In contrast, AFP showed significant statistical increase between the three groups (P < 0.001).

On dividing HCC patients into two groups with respect to tumor size, AFP showed significant association with tumor size, being higher in patients with tumor size greater than 6 cm (P = 0.007), whereas SCCA level showed no significant difference between the two studied groups(P = 0.89).

The best cutoff value for SCCA to differentiate HCC patients from cirrhotic patients was 0.5 μg/l showing 73% sensitivity and 44% specificity, and the cutoff value for AFP was 258 μg/l with 60% sensitivity and 100% specificity. On combining the two cutoff values, sensitivity improved to 100% and specificity was 47%.

In accordance with this study, Soyemi et al. [7] obtained a mean serum level for SCCA of 0.64 ± 0.56 and 0.71 ± 0.65 for cases and controls, respectively, with no significant statistical difference between the two groups (P = 0.631), and a mean serum level for AFP of 393.21 ± 386.97 and 5.60 ± 13.03 for cases and controls, respectively, with high significant statistical difference (P = 0.001). Contradictory to this finding was that of Hussein et al. [4], El-Ezawy et al. [10], and Giannelli et al. [6]. They found that SCCA serum levels were significantly more elevated in HCC than in cirrhotic patients (P < 0.001).

Beale et al. [11] in his study highlighted the role of AFP in the early prediction of HCC and its correlation with tumor size especially when it is combined with other markers like glypican-3 (GPC3), protein induced by vitamin K absence/antagonist-II (PIVKAII), and SCCA. Absence of correlation between SCCA level and size of the tumor in the current study was in accordance with the results of Hussein et al. [4].

The higher SCCA sensitivity compared with AFP sensitivity was comparable to the study by Giannelli et al. [6] in which SCCA sensitivity was 84.2%. However, the specificity of SCCA in our study was poor (44%), compared with AFP specificity of 100%. The specificity was also poor in the study by Giannelli et al. [6] (48.9%).

Presently, SCCA is still a research tool and has not been approved for widespread clinical use in the screening of patients with HCC. It is also very expensive. Unfortunately, as shown in this study, it also lacks good discriminatory power between HCC patients, liver cirrhosis patients, and apparently healthy controls, and may not be useful in these patients. This antigen is noted to be common in the skin, saliva, and sweat of apparently normal people, which may account for the levels found in the controls and for the poor specificity of this marker. The advantage of SCCA may be its higher sensitivity compared with AFP. The earlier appearance of serum SCCA may be attributed to the observation made by Uemura et al. [12] that SCCA is distributed mainly in the cytosol and is not associated with membrane-bound vesicles and therefore is not properly secreted but rather released in the serum as a consequence of cell lysis.

We recommend more studies on a large scale and with larger sample sizes to assess the diagnostic as well as the prognostic value of SCCA in patients with HCC with different etiologies of liver disease, not only post viral cirrhosis, with inclusion of more hepatitis B patients in the study. Moreover, further studies focusing on the quantitative assessment of SCCA in tumor and peritumoral tissues should be correlated with serum studies to improve the discriminatory power of SCCA between HCC patients and controls. Inclusion of a new group of patients with different cancer etiologies other than HCC is recommended for better assessment of SCCA specificity as a tumor marker for HCC.


  Conclusion Top


We conclude that, despite the slightly high sensitivity of SCCA, AFP remains a relatively good diagnostic marker for HCC with high specificity because SCCA lacks accuracy as a diagnostic test for HCC in Egyptians. Moreover, a combination of the two markers may improve the sensitivity, making their combination a better screening test for liver cancer.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Stefaniuk P, Cianciara J, Wiercinska-Drapalo A. Present and future possibilities for early diagnosis of hepatocellular carcinoma. World J Gastroenterol 2010; 16 :418-424.  Back to cited text no. 1
    
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Hassan ZK, Hafez MM, Mansor TM, Zekri ARN. Occult HBV infection among Egyptian hepatocellular carcinoma patients. Virol J 2011; 8 :90.  Back to cited text no. 2
    
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Sarwar S, Khan A, Tarique S. Validity of alpha fetoprotein for diagnosis of hepatocellular carcinoma in cirrhosis. J Coll Physicians Surg Pak 2014; 24 :18-22.  Back to cited text no. 3
    
4.
Hussein MM, Ibrahim AA, Abdella HM, Montasser IF, Hassan MI. Evaluation of serum squamous cell carcinoma antigen as a novel biomarker for diagnosis of hepatocellular carcinoma in Egyptian patients. Indian J Cancer 2008; 45 :167-172.  Back to cited text no. 4
    
5.
Catanzaro JM, Guerriero JL, Lui J, Ullman E, Sheshadri N, Chen JJZong WX. Elevated expression of squamous cell carcinoma antigen (SCCA) is associated with human breast carcinoma. PLoS One 2011; 6 :e19096.  Back to cited text no. 5
    
6.
Giannelli G, Marinosci F, Sgarra C, Lupo L, Dentico P, Antonaci S. Clinical role of tissue and serum levels of SCCA antigen in hepatocellular carcinoma. Int J Cancer 2005; 116 :579-583.  Back to cited text no. 6
    
7.
Soyemi OM, Otegbayo JA, Ola SO, Akere A, Soyemi T. Comparative diagnostic efficacy of serum squamous cell carcinoma antigen in hepatocellular carcinoma. BMC Res Notes 2012; 5 :403.  Back to cited text no. 7
    
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Marrero JA, Lok AS. Newer markers for hepatocellular carcinoma. Gastroenterology 2004; 127 :S113-S119.  Back to cited text no. 8
    
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Giannelli G, Fransvea E, Trerotoli P, Beaugrand M, Marinosci F, Lupo L, et al. Clinical validation of combined serological biomarkers for improved hepatocellular carcinoma diagnosis in 961 patients. Clin Chim Acta 2007; 147-152.  Back to cited text no. 9
    
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El-Ezawy HM, Shebil N, El-Hasanin S, Obada MA, El-Waraay M. Assessment of squamous cellular carcinoma antigen (SCCA) and KL-6 as a tumor markers and their correlation to tumor size in HCC. J Am Sci 2012; 8 :3.  Back to cited text no. 10
    
11.
Beale G, Chattopadhyay D, Gray J, Stewart S, Hudson M, Day C, et al. AFP, PIVKAII, GP3, SCCA-1 and follisatin as surveillance biomarkers for hepatocellular cancer in non-alcoholic and alcoholic fatty liver disease. BMC Cancer 2008; 8 :200.  Back to cited text no. 11
    
12.
Uemura Y, Pak SC, Luke C, Cataltepe S, Tsu C, Schick C, et al. Circulating serpin tumor markers SCCA1 and SCCA2 are not actively secreted but reside in the cytosol of squamous carcinoma cells. Int J Cancer 2000; 8:368-377.  Back to cited text no. 12
    


    Figures

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

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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Introduction
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