Study of the Serum Level of Annexin A5 as a Marker for Hepatocellular Carcinoma in Hepatitis C Virus Cirrhosis Patients

INTRODUCTION

Hepatocellular carcinoma (HCC) is considered one of the most common cancers all over the world moreover epidemiologic studies showed that HCC is termed the fifth in men and the seventh in women most common in malignancy and the sixth cancer causing death all over the world [1]. hepatocellular carcinoma (HCC) causes death to about eight hundred thousand people worldwide in 2018 [1].

Many studies reported that the enlargement of the number of HCC patients could be related to advancement in the programs of screening and diagnosis of the cirrhotic patients and their follow up moreover, increasing the survival of cirrhotic patients leads to more chances of having HCC, and high incidence of hepatitis C virus (HCV) complications[2].

HCV infection causes cirrhosis in (93%) Globally. For HCV virus strains, 7genotypes and 67 subtypes are present. The infection causes inflammation fibrosis leading to the development of HCC. HCV protein causes the Mutation and malignant changes of the infected cells moreover, slow progression to cirrhosis-related HCC [3].

To decrease the high mortality rate from HCC, There is an increase in the demand for sensitive blood tests to detect HCC early to facilitate early treatment [4].

Annexins are proteins which are soluble in water and are found in many tissues in animals. Those proteins form ion voltage channels by binding calcium with phospholipids. these channels are calcium-dependent [5]. They are present in the signals of the cell, apoptosis, more over control of different inflammatory  components, like the cytokines. Annexin A5 is one of the members of the annexin family with a molecular weight of 36 kDa which depends on binding to the Calcium of the cell membrane [5].

Annexin A5 appears to be efficient in inhibition of the inflammatory changes that happened in apoptosis, Annexin-A5 is thought to act as a powerful anti-inflammatory agent with high selectivity to decrease inflammatory response that happened in the apoptosis process [6].

AnxA5 arises from liver secretory cells, splenic cells, myocardial tissue when injured, endothelium of blood vessels, and cells of smooth muscles. It binds to platelets, red blood cells, and endothelial cells when it is in the plasma [7].Annexin A5 reflects the degree of damage to the blood cells as it is also released from apoptotic blood cells [7].

In patients with HCC, cirrhosis mostly  prevalent [8]. About 80% of patients recently diagnosed with HCC have preexisting cirrhosis. Recent analysis showed that, HCC occurrence without preexisting cirrhosis is not common and occurs specifically in 15% of people infected with HBV [2].

An increase in the prevalence of hepatocellular carcinoma (HCC) in Egypt in cirrhotic patients has been documented. The explanation for this rising may be related to the presence of hepatitis C virus (HCV) infection as a risk factor, improvement of the diagnostic modalities of HCC as well as increase the survival in patients with cirrhosis allowing time for some of them to develop HCC [9].

PATIENTS AND METHODS

Study design: this study is a retrospective  study .

Study settings: The present study has been done in Menoufia university hospital, Egypt, between March 2022 and April 2023.

Study patients: The present study patients were selected from outpatient and inpatients of the tropical medicine department (Menoufia university), Egypt.

Endpoints: the study was designed to evaluate the level of Annexin A5 in cirrhotic patients (due to HCV infection) with and without HCC and in healthy volunteers.

Sample size: The present study included 60 patients, 30 patients having cirrhosis due to HCV having hepatocellular carcinoma and 30 patients with cirrhosis due to HCV not having hepatocellular carcinoma and 20 normal controlled patients of the same age and sex

Inclusion criteria:

-       The study included cirrhotic patients due to HCV infection either having HCC or not having HCC.

-       Any age and both sex.

Exclusion criteria: Any patient with malignancy that can rise the serum Annexin A5 level as cancer of colorectum, cancer of the pancreas, and breast cancer.

Patient assessment: Patients were subjected to the following:

-    History and examination clinically: the age at the diagnosis, sex, abdominal examination (ascites, liver examination, spleen examination). Liver disease severity calculated by Model for end stage liver disease (MELD) score, AST to Platelet Ratio Index (APRI) score, FIB4 score, and (Child- Pugh) classification included.

-    Laboratory investigations:done during fasting and the specimens were the serum except the complete blood count (CBC), the specimens were the whole blood collected in EDTA tubes. Investigations included, the enzymes aspartate transaminase (AST) and alanine transaminase (ALT), albumin, bilirubin, CBC, creatinine, blood urea nitrogen, prothrombin time, The international normalized ratio (INR), alkaline phosphatase (ALK), gamma glutamyl transferase (GGT), sodium& alpha-fetoprotein.

-    Color doppler ultrasonography on the portal vein: was done to all the patients to detect if there is portal vein thrombosis or not.

-    Triphasic CT abdomen and pelvis: was done to all patients to evaluate the hepatocellular carcinoma, and exclude other malignancies.

-    Biopsy for the focal lesions in the liver:to confirm the diagnosis of hepatocellular carcinoma.

-    Serum level of Annexin A5: The level of the serum anxA5 was measured by kits, which are immunoassay of a sandwich enzyme for quantitative measurements of the in vitro of ANXA5 in human serum. Blood samples of the present study were taken while fasting, in the morning.

Normal range: 0.6- 6.7 ng /ml

Statistical analysis

The data collected was analyzed by SPSSVERSION 19. For quantitative variables mean & SD or median& (student t-test) were used, while for qualitative variables chi- square, Pearson correlation (r) were used. (Tukey) is the post hoc test that was used to compare between the groups. (ANOVA) used for the differentiation of three classes of variants according to the mean. The receiver operating characteristic curve (ROC): is a graphic representation of the relationship between sensitivity and specificity at different cutoff  points for a diagnostic test. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the area under curve (AUC), were calculated by using the ROC curve.

RESULTS

The  present study included 60 patients and they were divided into three groups; the  first group  including cirrhotic patients (due to hepatitis C virus) with hepatocellular carcinoma, the second group  including cirrhotic patients (due to hepatitis C virus) without hepatocellular carcinoma and  the third group including  control healthy with no liver disease.

There was no statistically significant difference among  the studied groups according to age and sex as shown in (table 1) , there was a high statistically significant difference among the studied groups (P<0.001) according to  the serum level  of AST, ALT, albumin, platelets count, bilirubin, creatinine, urea, INR, ALK& sodium, there was only significant difference among the studied groups according to GGT (P 0.002) and no statistically significant difference of the previous parameter between the group of cirrhotic with HCC and cirrhotic without HCC as shown in (table 2).

There was highly significant difference between studied groups according to serum level of Annexin (P< 0.001 ) and alpha-fetoprotein (P 0.001) as the serum Annexin A5 was 12.553+ 3.962 ng/ml in cirrhotic patients with HCC and it was 6.040+ 1.227 ng/ml in cirrhotic patients without HCC while, it was 5.014+0.901 ng/ml in the  control group as shown in ( Table 3 , figure 1) moreover, alpha-fetoprotein serum level was 670.667+40.385ng/ml  in cirrhotic group  with HCC and it was 63.773+ 18.366ng/ml while it was 7.050+1.905 ng/ml in the control group as shown in( table 3).there was also a highly significant difference between cirrhotic patients with HCC and cirrhotic patients without HCC (P1<0.001) according to the serum level of Annexin A5 and alpha-fetoprotein (table 3) .

There was no statistically significant difference between the cirrhotic patients with HCC & cirrhotic patients without HCC according to Child Pugh and MELD score, there is a high statistically significant difference between the three studied groups according to APRI&FIBS score as shown in (table 4).

There was a positive correlation between the presence of portal vein thrombosis and a high level of Annexin A5 as, there was 17 patients having portal vein thrombus with high Annexin A5 serum level (14.053+ 4.023) while, there were 43 patients having no portal vein thrombus with Annexin A5 serum level (7.416+2.843) as shown in ( table 5). There was a positive correlation between serum Annexin A5 and alpha-fetoprotein level (P<0.001) as shown in (table 6).

The ROC curve between cirrhosis with HCC group & cirrhosis without HCC group shows the ability of Annexin A5 to predict the HCC with a cutoff point >8.7. Sensitivity of 83.33 %, specificity of 100%, PPV of 100 %, NPP of 85,7% and AUC 0.916, as shown in (Table 7 and Figure 2). The ROC curve between cirrhosis with HCC group& normal group shows cutoff point >6.4, sensitivity of 90 %, specificity of 95 %, PPV of 96.4 %, NPP of 86.4%, and AUC 0.962, as shown in (Table 8 & Figure3) while, the ROC curve between cirrhosis without HCC group &normal group shows a cutoff point >5.8, sensitivity of 63.33%, specificity of 85 %, PPV of 86.4 %, NPP of 60.7% and AUC 0.755, as shown in ( table 9 & figure 4).

Table 1: Age &sex distribution among studied groups