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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 35  |  Issue : 2  |  Page : 235-240

Chromogranin a as a serum marker for hepatocellular carcinoma


1 Professor of Hepatology, Gastroenterology and Infectious Diseases Faculty of Medicine, Benha University, Benha, Egypt
2 Gastroenterology and Infectious Diseases Faculty of Medicine, Benha University, Benha, Egypt
3 Lecturer of Clinical Pathology Faculty of Medicine, Benha University, Benha, Egypt
4 Gastro-Enterology and Hepatolgy Resident Domiat General Hospital, Domiat, Egypt

Date of Submission24-Feb-2018
Date of Acceptance20-May-2018
Date of Web Publication17-Aug-2018

Correspondence Address:
Dr. Samir H Mosallam
Domiat/Kafr El Bateekh, New Station Square
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bmfj.bmfj_29_18

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  Abstract 


Background Hepatocellular carcinoma (HCC) has an increasing incidence worldwide. The observation of neuroendocrine activity during the clinical course of HCC suggested the use of neuroendocrine serum markers to detect it and elevated serum chromogranin A (CgA) has been reported in patients with HCC.
Aim To investigate the clinical utility of serum CgA, as a diagnostic marker of HCC.
Patients and methods This study was conducted on 80 patients who were classified into two groups. Group 1: the liver cirrhosis group which included 40 patients diagnosed by clinical settings, laboratory criteria, and radiological methods. Group II: the HCC group which included 40 patients who were diagnosed by two appropriate imaging studies and serum α-fetoprotein (AFP). They were recruited from Hepatology, Gastroenterology, and Infectious Diseases Department, Benha University Hospital. Liver function tests (aspartate aminotransferase, alanine aminotransferase, total bilirubin, and serum albumin) by colorimetric assay, CgA (by enzyme-linked immunosorbent assay), and AFP (by immunometric assay) were estimated.
Results CgA levels were significantly higher in group II (HCC patients) when compared with group I (cirrhotic patients) (P<0.0001). A statistically significant positive correlation was detected in the HCC group between CgA versus aspartate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, AFP, Child score, Barcelona-Clinic Liver Cancer Classification, number and size of focal lesions and negative significant correlation versus platelets count and serum albumin (P<0.0001). CgA level shows a best cutoff of 99.95 nmol/l and it showed 90% sensitivity, 90% specificity, 90% positive predictive value, 90% negative predictive value with 95.8% accuracy for the diagnosis of HCC, while when combined with AFP, it had 90% sensitivity, 67.5 specificity, 73.5% positive predictive value, 87.1% negative predictive value with 78.8% accuracy for the diagnosis of HCC.
Conclusion CgA represents a useful diagnostic biomarker for HCC.

Keywords: chromogranin A, α-fetoprotein, hepatocellular carcinoma


How to cite this article:
Kabil SM, El-Feky HM, El-Behisy MM, Mosallam SH. Chromogranin a as a serum marker for hepatocellular carcinoma. Benha Med J 2018;35:235-40

How to cite this URL:
Kabil SM, El-Feky HM, El-Behisy MM, Mosallam SH. Chromogranin a as a serum marker for hepatocellular carcinoma. Benha Med J [serial online] 2018 [cited 2018 Nov 19];35:235-40. Available from: http://www.bmfj.eg.net/text.asp?2018/35/2/235/239194




  Introduction Top


Worldwide, hepatocellular carcinoma (HCC) has an increasing incidence and it is the second cause of cancer-related deaths [1]. In Egypt, HCC is the second most common cancer in men and the sixth most common cancer in women [2]. According to the National Institute of Cancer in Egypt, HCC is considered one of the most common malignancies in Egypt as a result of increase in the prevalence of hepatitis B and C infections, since these represent ∼45.3% of all new cases of this type of cancer [3]. Unfortunately, HCC is diagnosed in most patients at a time when curative surgical resection or organ transplantation cannot be performed because of advanced disease or substantial impairment of liver function [4]. About 30% of HCC cases with normal serum α-fetoprotein (AFP) levels are not diagnosed before the appearance of clinical manifestations, so this highlights the need for reliable biomarkers for early HCC detection [5]. Chromogranin A (CgA), the index member of the chromogranin/secretogranin protein family, is co-stored and coreleased with catecholamines from dense core vesicles in the adrenal medulla and the postganglionic sympathetic axons [6]. CgA has been identified in numerous variety of tumors, including bronchial [7], prostate [8], pancreatic [9], and gastrointestinal cancer [5]. The neuroendocrine character has been observed in some tumor cells within some HCC nodules and elevated serum CgA has also been reported in patients with HCC [10]. Different studies show high serum CgA concentrations in patients with HCC, suggesting that CgA might represent a useful marker in monitoring cirrhotic patients for the early detection of HCC [11].

This study aimed to determine the clinical utility of CgA serum level as a diagnostic marker of HCC and correlates its level with demographic, clinical, and laboratory features of the studied cases and with HCC characters.


  Patients and methods Top


The study population consisted of 80 patients; 52 men (65%) and 28 women (35%) who attended the Hepatology, Gastroenterology and Infectious Diseases Department, Benha University Hospital, during from January 2016 to July 2016 after approval from the Research and Ethics Committee of Benha Faculty of Medicine and its University Hospitals. Their age ranged from 60 to 79 years. This study included two groups. Group 1: the liver cirrhosis group which included 40 patients who were diagnosed by clinical settings, laboratory criteria, and radiological methods. Group II is the HCC group which included 40 patients documented to have HCC on top of liver cirrhosis who were diagnosed by two appropriate imaging studies and serum AFP. Patients with renal failure, cardiac failure, hypertension, carcinoma elsewhere and those who received proton pump inhibitors were excluded from the study. All included patients were subjected to the following: (a) full medical history and thorough clinical examination; (b) laboratory investigations included: liver panel tests to determine the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum bilirubin (total and conjugated), serum albumin, prothrombin time; international normalized ratio and viral markers including hepatitis B surface antigen, and hepatitis B virus by enzyme-linked immunosorbent assay (ELISA); and detection of the serum AFP level by immunoenzymometric assay. Normal reference range (2–10.9 ng/ml) obtained by the manufacturer instruction and serum CgA was measured by ELISA. Kits were provided by Epitope Diagnostics Inc. (San Diego, California, USA) (normal reference range <36.4 ng/ml). Abdominal ultrasonographic examination was done for all cases and abdominal triphasic spiral CT examination was performed for HCC cases to confirm focal lesion characters (number, size, shape). Also HCC cases were stratified according to Barcelona-Clinic Liver Cancer Classification (BCLC) and Child–Pugh classification system was done for all cases.

Samples

A measure of 5 ml of blood was collected from antecubital vein of each patient after being diagnosed by venipuncture and then centrifuged for separation of the serum. Serum samples were divided into two aliquots: one aliquot was used for immediate routine tests and serum AFP detection, while the remaining portion of sera was stored in an aliquot at −20°C until used for measurements of serum CgA.

Serum chromogranin A detection

A sufficient number of antibody microwell strips was placed in a holder and then centrifuged. A measure of 15 µl of standard, controls, and patient samples was added into the designed microwell and then 200 µl of assay buffer is added to each well, then the plate is covered with a plate sealer and aluminum foil and is incubated on an ELISA plate with shaking with at a rate of 350–450 rpm at room temperature for 1 h.The aluminum foil was removed and the contents of each well was aspirated and then 100 µl of CgA tracer antibody was added to each well and then the plate was covered with a plate sealer and aluminum foil and was incubated. The plate is an ELISA plate and shaken at a rate of 350–450 rpm at room temperature for 1 h, following which the aluminum foil was removed and the contents of each well was aspirated. One hundred microliter of ELISA HRP substrate was added to each well and then covered with a plate sealer and aluminum foil to avoid exposure to light and then incubated at room temperature for 20 min. The aluminum foil and the plate sealer were removed and 100 ml µl of ELISA stop solution was added into each well and gently mixed. Then the absorbance at dual wave length at 450/650 nm within 10 min was read.

Statistical analysis

The collected data were tabulated and analyzed using SPSS version 16 software (SPSS Inc., Chicago, Illinois, USA). Categorical data were presented as number and percentages, while quantitative data were expressed as mean and SD, median, range, and interquartile range. χ2 That compares between two or more categorical groups, Fisher’s exact test that is used when comparing two nominal variables, Student’s ‘t’-test that compares between two means of two independent groups, Mann–Whitney U-test was used instead of the t-test to compare a quantitative variable between two independent groups when the data were nonparametric, Kruskal–Wallis test nonparametric test that was used to compare quantitative variables among more than two independent groups and Spearman’s correlation coefficient (ρ) that was used to evaluate the linear association between two quantitative variables (one was the independent variable X and the other was the dependent variable Y). The receiver operating characteristic curve was used to determine the cutoff values of serum CgA with optimum sensitivity and specificity. The accepted level of significance in this study was stated to be less than 0.05 (P<0.05 was considered significant).


  Results Top


The demographic features of the included cases were as follows: 26 (65%) men and 14 (35%) women with a mean age of 55.3±6.74 in the cirrhotic group and 25 (62.5%) men and 15 (37.5%) women with a mean age of 62.40±6.75 in the HCC group, so the patients of the HCC group were older than those of the cirrhotic group with highly significant difference (P<0.001). Except for prothrombin time and international normalized ratio that were significantly higher in the cirrhotic group, the rest of the laboratory results had nonsignificant differences between both groups. Comparative statistics between the studied groups in terms of AFP and serum CgA using the Mann–Whitney U-test showed that the serum CgA level was higher in the HCC than in the cirrhotic group with a highly significant difference (P<0.001) and AFP was significantly higher in the HCC group than the cirrhotic one (P=0.026). All studied cases were hepatitis B virus positive and hepatitis B virus negative. According to the BCLC staging system; 13 (32.5%) patients of the HCC group were BCLC stage A; two (5%) patients were of BCLC stage B, six (15%) patients were of BCLC stage C; and 19 (47.5%) patients were of BCLC stage D. According to the Child–Pugh classification system; 47% of HCC patients were of Child C, 47% were of Child B, and 5% were of Child A, while 60.0% of the cirrhotic group were of Child C, 25% were of Child B, and 15% were of Child A. CgA had a positive significant correlation with AFP, and the Child score in the cirrhotic group while in the HCC group, it had a positive significant correlation with AST, ALT, total bilirubin, direct bilirubin, AFP, Child score, BCLC, and negative significant correlation with platelets count and serum albumin. There were positive highly significant correlations between CgA on one hand and the number and size of focal lesions of the HCC group on the other hand (P<0.001). CgA at a cutoff level=99.95 ng/ml had 90% sensitivity, 90% specificity, 90% positive predictive value (PPV), 90% negative predictive value (NPV) with 95.8% accuracy for the diagnosis of HCC while when combined with AFP, it had 90% sensitivity, 67.5 specificity, 73.5% PPV, 87.1% NPV with 78.8% accuracy for the diagnosis of HCC ([Table 1],[Table 2],[Table 3] and [Figure 1],[Figure 2],[Figure 3],[Figure 4]).
Table 1 Demographic and laboratory characters of the studied groups

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Table 2 Correlation between serum chromogranin A levels and different variables among the studied groups

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Table 3 Diagnostic performance of serum chromogranin A for the diagnosis of hepatocellular carcinoma

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Figure 1 Box-plot diagram for median and interquartile range of serum chromogranin A (CgA) levels in the studied groups. HCC, hepatocellular carcinoma.

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Figure 2 Barcelona-Clinic Liver Cancer Classification (BCLC) among the hepatocellular carcinoma (HCC) patients.

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Figure 3 Correlation between serum chromogranin A (CgA) levels and number of focal lesions.

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Figure 4 Correlation between serum chromogranin A (CgA) levels and size of focal lesions.

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  Discussion Top


HCC is considered as the sixth most common neoplasm globally, with more than 600 000 deaths per year worldwide [12]. Despite the major advances achieved in the diagnostic workup of HCC, only one-third of the newly diagnosed patients are presently eligible for curative treatments [13]. Even in the curative setting, 5-year survival rates after resection for early-stage HCC ranges between 17 and 53%, and the recurrence rates can be as high as 70% [14]. Two diagnostic tests are routinely used to detect HCC in clinical practice: serum AFP and ultrasonography. The sensitivity of AFP as a diagnostic tool is restricted by the existence of non-AFP-secreting tumors [15]. The imaging-based diagnosis of small tumors is relatively inaccurate, as cirrhotic dysplastic nodules can resemble HCC, so a given imaging modality cannot always differentiate between benign hepatic lesions and HCC; as a result, early and small lesions might be overlooked [16]. So, the availability of a more sensitive serological marker that distinguishes between HCC and benign hepatic lesions would therefore be very useful and essential for early and specific diagnosis [17]. CgA is a 50-kµ acid glycoprotein originally described in the catecholamine storage vesicles of the adrenal medulla [18]. Clusters of cells containing CgA have been demonstrated within the HCC tissue [19]. One study, reported high serum CgA values in approximately half of the patients with HCC [20]. Massironi et al. [21] confirm the presence of elevated plasma levels of CgA in both patients with chronic liver disease (chronic hepatitis and liver cirrhosis) and those with HCC, but HCC showed the highest levels of CgA, up to 30 times the upper limit of normal. This finding suggests that the determination of CgA serum values is useful in monitoring patients with cirrhosis of the liver for early detection of HCC. The present study revealed a highly significant elevation in the median of serum CgA level in the HCC group (median=294.3 ng/ml) when compared with the cirrhotic group (median=47.25 ng/ml) (P<0.01). These results were consistent with another study which reported that HCC patients showed a significant increase in CgA (P<0.001) compared with controls [22]. On the same hand, Spadaro et al. [18] and Massironi et al. [21] reported a statistically significant elevation of CgA serum levels in HCC when compared with those in cirrhotic patients. The fact that plasma CgA levels are higher than normal in the early status of chronic liver disease suggests that this protein could be secreted through an activation of endocrine cells during hepatitis and not necessary reflect reduced CgA catabolism due to progressive hepatocellular failure [21]. Moreover, Leone et al. [20] reported elevated CgA serum values in 43% of patients with cirrhosis and superimposed HCC suggesting rising CgA levels, likely due to a the neuroendocrine component of the tumor. Circulating CgA levels in HCC patients could reflect CgA expression in HCC tissues [23]. In the current study, AFP was significantly higher in the HCC group (median=174.5 ng/ml) than the cirrhotic group (median=88.9 ng/ml) .This was in agreement with Abbasi et al. [24] who reported that HCC patients had an AFP level more higher than the cirrhotic group. Page et al. [25] declared that one of the limitations in the use of AFP for the diagnosis of HCC is its increase in CLD patients who do not have HCC. On the same hand, El-Serag [26] stated that hepatic injury and regeneration alone (such as during active hepatitis C virus infection) can increase the serum levels of AFP in patients who do not have HCC. In 2011, AASLD guidelines [27] omitted AFP from the algorithm for surveillance and diagnosis of HCC. In the current study, highly significant positive correlations were found in the HCC group between serum CgA level on one hand and AST, ALT, total bilirubin, direct bilirubin, AFP, Child score, BCLC, number, and size of focal lesions on the other hand, while negative significant correlations were found between CgA and platelets count and serum albumin. On the other side, significant positive correlations were detected between CgA level and AFP and Child score in the cirrhotic group. In agreement with the current results, Biondi et al. [28] reported that serum CgA level was correlated with AFP and BCLC in patients with HCC. The clinical utility of serum CgA level for HCC diagnosis in this study showed that at a cutoff=99.95 ng/ml, it had a sensitivity of 90%, specificity of 90%, NPV of 90%, PPV of 90% with an accuracy of 95.8%, and an area under the curve (AUC) of 0.96.This result was in agreement with Wafaa et al. [22], who reported a sensitivity and specificity of 100% for serum CgA in HCC diagnosis. On the other hand, serum AFP level at a cutoff=95.3 ng/ml had a sensitivity of 62.5%, specificity of 52.5%, NPV of 56.8%, PPV of 58.3% with an AUC of 0.64 for HCC diagnosis in the present study. This result came in agreement with Song et al. [29], who reported that AFP had a sensitivity of 47.5–94.0% and a specificity of 53.3–98.5% in detecting HCC, when CgA combined with AFP showed a sensitivity of 90% and specificity of 67.5% and AUC of 0.79 compared with AFP alone.


  Conclusion Top


The present data showed that CgA was significantly higher in patients with HCC than in cirrhotic patients. CgA had higher sensitivity, specificity, PPV, and NPV than AFP for HCC diagnosis. So, CgA might be a good biomarker for HCC detection. However, future trial that includes a larger number of patients is emphasized.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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