CHROMOGRANIN-A AS A NEW SERUM MARKER FOR DIAGNOSIS OF HEPATOCELLULAR CARCINOMA

Background: Hepatocellular carcinoma (HCC) is the world's sixth most common cancer, and it usually complicates liver cirrhosis. Its prevalence is growing globally, with annual increases ranging from 3% to 9%. Despite the fact that the alpha fetoprotein (AFP) is the gold standard for HCC serodiagnosis, it has a high rate of false positive outcomes, which are associated with a bad diagnosis. Objective: To investigate the potential role of chromogranin-A (CgA) as a diagnostic non-invasive marker for HCC and to assess its sensitivity and specificity in diagnosis of HCC compared to AFP. Material and Methods: This study was conducted from October 2017 till March 2020 at the Department of Tropical Medicine, Ain Shams University and Theodor Bilharz research institute. The study included 35 patients diagnosed with hepatocellular carcinoma HCC and 35 patients with chronic liver diseases (CLD). The study also included 20 apparently healthy individual served as a healthy control group taken from the blood donors at the blood bank of Ain Shams University Hospital. AFP and CgA serum levels were detected for all groups using enzyme linked immunosorbent assay (ELISA). Results: The results of the study showed that the median of the of CgA in the HCC group was 53.40 and in CLD group was 22.55 while in the control group was 15.30; which indicates a significant elevation of CgA in HCC patients. Conclusion: CgA is one of the promising tumor markers in hepatic focal lesions for detection of HCC.


INTRODUCTION
HCC is the sixth most common in the world, complicating liver cirrhosis (Tae- Hyung et al., 2019). Its prevalence is growing globally, with annual increases ranging from 3% to 9%. HCC has been on the rise in Egypt, with the incidence rate doubling in the last decade, and this has been due to a number of causes including (e.g. viral hepatitis B, D and C virus infections), environmental influences (e.g. aflatoxin, AF of aspergillus) and other reasons such as cigarette smoking, occupational exposure to chemicals such 710 as pesticides, and endemic infections in the community, such as bilharziasis, may have further roles in the etiology or progress of this problem (Mashaly et al.,2018).
HCC is one of the major causes of death because of its high frequency and poor prognosis; it is responsible for about one million deaths annually with only few patients surviving beyond one year. It is one of the few human cancers for which an etiological factor can be identified in most cases.
HCC commonly develops in cirrhotic livers whatever the etiology, so that liver cirrhosis by itself represents the strongest risk factor (Toyoda el al., 2015).
Early detection of patients with HCC is very important because it gives better prognosis as HCC tends to grow slowly and stay confined to the liver. So early diagnosis of HCC is not difficult if tumor markers and medical imaging are combined (Piñero et al., 2020 andToyoda et al., 2015). HCC is a potential target for cancer surveillance as it occurs in welldefined, at-risk populations and curative therapy is possible only for small tumors. Hepatic ultrasonography with or without AFP performed every 6 months is the preferred program and has been well shown to detect small tumors for curative treatment, which may be translated to improved patient survival (Biselli et al., 2015). The only diagnostic serologic test currently used in clinical practice is AFP, but it has a high rate of false positives and its concentration may be elevated in some benign liver diseases, so new, more precise and sensitive markers are needed (Piñero et al., 2020 andBiselli et al., 2015). Additionally, 20% to 30% of HCC

PATIENTS AND METHODS
This study was conducted at Ain Shams University Hospital at the clinic of Hepatoma Group and Theodor Bilharz Research Institute for pick up patients with HCC and chronic liver disease CLD.
The studied groups were classified as follows: 1. The first group included thirty five patients already diagnosed with HCC by AFP and triphasic C.T.
2. The second group included thirty five patients already diagnosed with chronic liver diseases by liver function tests and abdominal ultrasound.
3. The third group was a control group containing twenty healthy individuals.
3. Patients with any malignancy elsewhere in the body, other than liver especially those with neuroendocrine origin.
Clinical assessment included full history, clinical examinations and Child's classification for HCC and CLD were done.
Laboratory investigations included liver function tests, renal function tests, AFP and CgA ELISA assays were done for all groups.
Radiological investigations included abdominal ultrasonography and triphasic abdominal CT were done for HCC and CLD groups.

Statistical analysis:
Statistical analysis was performed using statistical package for social science (SPSS) version 25. Chi-Square test Χ² and Fisher's Exact Test were used to test the difference in qualitative data. Receiver Operating Characteristic (ROC) curve was used to compare the significant results among the studied groups.
Qualitative data were presented as mean ± standard deviation (SD) for age and as median and inter-quantitative range for non-parametric values, which were compared by Kruskal-Wallis test and Bonferroni test as a post ROC test. All Pvalues represented were two-sided, and statistical significance was declared at P< 0.05.

RESULTS
There were no significant differences in demographic and clinical characteristics between the studied groups except for the lower limb edema which was increased among CLD group (<0.05). As regarding the ultra-sonographic findings, no statistically significant data were detected among the studied groups except for the presence of portal vein thrombosis among HCC group (<0.05).
A positive triphasic CT criteria was noticed among 29 patients of HCC group (82.9%) only.
There was a statistical significant difference between the three groups as regards the level of CgA, AFP, AST, bilirubin and prothrombin time (P<0.05). There was no statistical significant difference between the three groups as regards the ALT, albumin levels ( Table  1). There was a statistical significant difference between the three groups as regards the distribution of +ve CgA, AFP (Table 2). AFP was normal in 12 cases of HCC and showed positive results in 23 cases, but CgA showed a positivity in 10 cases only out of 23 diagnosed by AFP and in 6 cases out of 12 noted as normal by AFP, when this 6 was added to 23 cases by AFP the results were 29+ve cases This combined use of CgA with AFP showed a sensitivity and specificity of 82% and 90%, respectively PPV 95% and NPV 75% with a diagnostic accuracy of 88% so, when AFP was normal, CgA serum values represented a complementary diagnostic tool (Table 3). On comparing HCC with CLD group: AFP: showed a sensitivity and specificity of 66% and 97%, respectively, PPV 95% and NPV 74% with a diagnostic accuracy of 81%. CgA: showed a sensitivity and specificity of 46% and 74%, respectively, PPV 64% and NPV 58% with a diagnostic accuracy of 60%.
The combined use of CgA with AFP did not improve the diagnostic indices of individual each marker it showed a sensitivity and specificity of 83% and 74%, respectively, PPV 76% and NPV 81% with a diagnostic accuracy of 79% figures (1, 2).

Figure (1): ROC curve for differentiation between normal and HCC is weak as AUC is 74%
*(AUC) Area under the curve.

Figure (2): ROC curve for differentiation between CLD and HCC is weak as AUC is 66%
1 -Specificity Patients with neuroendocrine tumours such as carcinoids and endocrine pancreatic tumours have chromogranin A (CgA) throughout their serum. CgA levels in the blood have also been shown to be high in patients with other cancers such as colon, lung, breast, and prostate cancer, likely due to a neuroendocrine distinction Aluri and Dillon. (2017).
Although it is not clear why a nonneuroendocrine tumor such as HCC express CgA, it was noted that neuroendocrine differentiation can occur in carcinomas that lack neuroendocrine cells in their normal epithelial counterparts, such as hepatocellular carcinoma (Aluri &Dillon. 2017, andMikkelsen et al., 2017). The present study aimed at assessing the potential usefulness of CgA and its use as a marker in diagnosis of hepatocellular carcinoma.
In this study, there was a statistically significant elevation in the median serum AFP in HCC group (40ng/ml) when compared with control group (3.1 ng/ml) and significant elevation when compared with CLD group (4.37ng/ml). These findings were similar to many other studies but with different figures as Awadallah et al. (2011) who reported a significant elevation in the median serum level of AFP (26.5ng/ml) in HCC group when compared with the control group (2.75ng/ml) and significant elevation when compared with the CLD group (5.35ng/ml). The same findings reported by Biondi et al. (2012) in the current study, the sensitivity of AFP was (66%) and the specificity was (97%). When using the receiver operator curve (ROC) to improve the specificity and sensitivity of AFP at a cutoff value of 20.4 ng/dl the detected sensitivity and specificity was 68.7% and 98.2%, respectively (best cutoff). These results were near to that detected by Wu et al. (2019), who reported a sensitivity of 63% and specificity of 91% for AFP in diagnosis of HCC with a cut off value of 13.5 ng/dl.
In terms of using CgA as a marker for HCC diagnosis, our findings showed a statistically significant (P<0.05) increase in the median serum level of CgA in the HCC group (53.4ng/ml) when compared to the control group (15.3ng/ml) and a highly significant (22.55ng/ml) increase when compared to the cirrhotic group. Chromogranin-A's sensitivity and specificity were found to be 46% and 74%, respectively, in this study. At a cutoff value of 4.45 ng/dl, the yielded sensitivity and specificity were 49.7% and 80%, respectively, when using the ROC curve to increase the specificity and sensitivity of CgA (best cutoff). These findings were close to those of The combined use of CgA with AFP did not improve the net diagnostic results for detection of HCC cases. These findings were in line with those of Biondi et al. (2012) who concluded that CgA alone is a useful diagnostic marker for HCC and should be evaluated with or without AFP especially in chronic liver disease patients. This showed that simultaneous measurements of serum AFP and CgA are of value in detecting HCC.
In the current study, there was no statistically significant difference between Barcelona HCC staging and CgA levels in the three groups. However, In a study performed by Biondi et al. (2012), CgA levels in the Barcelona staging of HCC were higher in stage D relative to stage C (p<0.01), stage B , and stage A.
According to Lv et al. (2018), the association between CgA and the degree of neuroendocrine inflammation indicates that CgA excretion could be involved during chronic inflammatory diseases as that in hepatitis and pancreatitis. These findings concluded that CgA levels are higher than usual in the early stages of chronic liver disease, which is why it should be tested. As a consequence, it should be viewed as a diagnostic marker for people with chronic liver diseases in order to diagnose early HCC and enhance their management outcomes.

CONCLUSION
CgA is one of the most promising tumour markers for detecting HCC in hepatic focal lesions. Chromogranin A serum levels can be used as a supplementary diagnostic instrument in the evaluation of chronic liver disease patients for the identification of HCC. It could also be used to diagnose HCC patients with focal lesions larger than 2 cm in diameter, especially when AFP levels were ambiguous despite the presence of a hepatic focal lesion on abdominal ultrasound and triphasic CT.