Serum Serotonin as a Novel Marker for Hepatocellular Carcinoma

Aim of the Work: To investigate the role of serum serotonin as a novel marker for diagnosis of Hepatocellular carcinoma In comparison with serum alpha fetoprotein. Introduction: Hepatocellular carcinoma is the most common liver cancer and is a disease with poor prognosis. In addition to its function as a neurotransmitter and vascular active molecule, Serotonin is also a mitogen for hepatocytes and promotes liver regeneration and may be involved in tumorigenesis of HCC. Methods: The study conducted on 136 patients and 20 healthy subjects as control group. Patients classified into two groups, 68 Cirrhotic with HCC as group I and 68 only cirrhotic without HCC as group II. Patients and controls underwent through history taking, full clinical examination with Child pugh score laboratory. Investigations including Complete blood count, Liver function tests, Creatinine, serum AFP and Serotonin levels in addition to abdominal ultrasound and Triphasic CT abdomen. Results: Cirrhotic Patients presented with HCC showed significantly higher levels of serotonin, alpha fetoprotein, albumin and white blood cell count compared to pure cirrhotic group without HCC, however, HCC patients showed lower levels of AST, ALT, bilirubin and platelet count compared to cirrhotic patients. HCC patients were more in elder, associated with male gender and showed shorter prothrombin. A significant positive correlation between serum AFP and serotonin. r=0.594, P<0.001 AFP at 0.5 ng /ml is a cutoff with sensitivity 100%, specificity 70%, PPV 91.9% and Accuracy 97.6% respectively for diagnosis of HCC while Sr. Serotonin >510ng/ml is a cutoff with 89.71% sensitivity, 85% specificity, 95.3 % PPV, 70.8 % NPV and 96.6 % accuracy respectively for diagnosis of HCC. Conclusion: Serotonin could be a novel maker for diagnosis of HCC and may be used together with serum AFP for screening of HCC in cirrhotic patients.


Introduction
Cirrhosis is a consequence of chronic liver disease characterized by replacement of liver tissue by fibrosis, scar tissue and regenerative nodules leading to loss of liver function. Cirrhosis is most commonly caused by hepatitis B and C, alcoholism, and fatty liver disease, but has many other possible causes [1]. In developing countries viral hepatitis is the leading cause of cirrhosis and in the developed countries alcoholic liver disease (ALD), HCV and non-alcoholic steatohepatitis (NASH) are the most significant causes of cirrhosis [2]. Some studies have demonstrated that patients who present with cirrhosis related complications (for example, hepatic encephalopathy, esophageal varices, bleeding and HCC) have a decreased survival rate compared with those without complications [3].
Liver biopsy is the only definite method for confirming a diagnosis of cirrhosis. It also helps to determine its cause, Advanced Research in Gastroenterology & Hepatology treatment possibilities, the extent of damage, and the long-term outlook. For example, hepatitis C patients who show no significant liver scarring when biopsied may have a low risk for cirrhosis. The histopathological landmarks of cirrhosis are nodularity (regenerating nodules), fibrosis (deposition of connective tissue creates pseudolobules), abnormal hepatic architecture, and hepatocellular abnormalities (pleomorphism, dysplasia, and regenerative hyperplasia) [4]. Because Liver Biopsy is an uncomfortable and sometimes risky procedure, it is unsuitable to incorporate it into the routine follow-up examinations of chronic liver disease patients. There is therefore a demand for serum markers that can routinely assess progression of liver fibrosis and reliably detect the stage of liver cirrhosis. Models such as Fibro test, which is based on a range of clinical chemistry analytes, have recently been intensively studied for these purposes [5].
Serotonin is known as 5-hydroxytryptamine (5-HT), a biogenic amine that function as a ligand for a large family of 5-HT receptors. [6] The majority of serotonin in the body (90%) is synthesized by enterochromaffin cells of the gastrointestinal (GI) tract, where it regulates intestinal motility [7]. It plays a major role in neurotransmission within the central nervous system (CNS) and the autonomic nervous system (ANS), In the CNS serotonin is known to control mood, behavior, learning, sleep and anxiety. Peripherally, serotonin is able to mediate vascular contraction and relaxation, cell proliferation, apoptosis and platelet aggregation [8]. Serotonin is actively taken up by cells expressing the Na+/Cl− dependent serotonin transporter (SERT) where it is stored in intracellular vesicles and released in response to various stimuli. Once bound to target receptors or taken up by the SERT, internalised serotonin can be metabolised by monoamine oxidase (MAO) leading to the generation of 5-hydroxyindoleacetaldehyde (5-HIAA) which is excreted in urine [8]. Liver cirrhosis is one of various pathological conditions in which serotonin homeostasis can change. In cirrhotic patients, secondary changes in the GI tract occur. In the altered intestinal wall, disturbances in serotonin synthesis and metabolism are observed. Serotonin leakage to systemic circulation cannot be excluded. It could be manifested by anxiety, sleep disorders, and other emotional disturbances. Then, the liver should play the role of a filter, where serotonin is catabolized. This mechanism fails in the case of liver diseases [9].
The chronic hepatic insufficiency gives rise to serotonin system changes, contributing to the development of hepatic encephalopathy, portal hypertension, and hyperdynamic circulation. In patients with liver cirrhosis, low whole-blood serotonin levels depend probably on reduced uptake, retention of serotonin by platelets, and low platelet number. Also, concentration of circulating serotonin in liver cirrhosis can be influenced by other factors, such as altered serotonin co-oxidase and impaired metabolism of tryptophan, as a precursor of serotonin [10]. Patients with cirrhosis are known to have platelet storage pool defects, significantly lower intraplatelet serotonin concentrations when compared to healthy individuals. It is therefore tempting to propose that the haemorrhagic tendency of cirrhotic patients [11]. Each year, hepatocellular carcinoma (HCC) (SEER 2010) is diagnosed in more than half a million people worldwide, including approximately 20,000 new cases in the United States. Liver cancer is the fifth most common cancer in men and the seventh in women. Most of the HCC cases (85%) are present in developing countries. [12] Incidence of HCC in Egypt is currently increasing, which may be the result of a shift in the relative importance of HBV and HCV as primary risk factors in addition to exposure to aflatoxin as an additional risk factor [13]. HCC is the second most frequent cause of cancer incidence and mortality among men in Egypt [14]. Egypt has the highest prevalence of HCV in the world, with estimates ranging from 6 to 28% and a reported average of ∼ 13.8%, also investigations in Egypt have also shown the increasing importance of HCV infection in the etiology of HCC, account for 40-50% of cases [15].
Hence platelets are not expected to function properly in diseased liver. Platelets harbour important growth factors for liver regeneration, e.g. Hepatocyte growth factor (HGF). Platelets contain transforming growth factor α (TGF-α), which is required for termination of liver regeneration. Thus, it is plausible that platelets may participate in orchestrating liver regeneration through stimulation and inhibition of growth-related signals. The ability of serotonin to modulate all of these factors renders it crucial in times of hepatic injury and repair [16]. Platelet derived serotonin has been shown to be beneficial in terms of stimulating hepatocyte proliferation following hepatic ischaemia in mice [17]. In addition over proliferation of hepatocytes can lead to HCC and this would raise the possibility that serotonin may play a role in HCC pathogenesis [18]. Serotonin is emerging as a mediator of different pathological conditions. It contributes to liver fibrosis, mediates oxidative stress in nonalcoholic steatotic hepatitis, and aggravates viral hepatitis promoting the progression of steatohepatitis by oxidative stress [18]. It promotes tumor growth in a mouse model of subcutaneous colon cancer allografts. 5HT deficiency led to decreased vascularity and increased necrosis reflecting cell death of the tumor.

Subjects and Methods
This study conducted on 136 patients and 20 healthy subjects as control group, Subjects selected from the outpatient of our Menoufia University Hospital and after oral and written consent from all, Patients and control subjects classified into 3 groups Patients who are taking benzodiazepines, narcotics, or other agents that can alter gastrointestinal motility such as prokinetic agents like metoclopramide, erythromycin, cisapride, opioids, adsorbents like bismuth subsalicylate and anti-emetic drugs like promethazine.

•
Patients with cardiopulmonary, renal and endocrinal diseases.
All patients were subjected to the following: I-Thorough history taking with special emphasis on the following: • Age and Sex.
• Manifestations of liver cirrhosis history of bleeding, encephalopathy and previous medication II-Thorough physical examination with special emphasis on: • Signs of liver cell failure (e.g. jaundice, pallor, ascites, hepatomegaly, splenomegaly, and lower limb edema).
Signs of hepatic encephalopathy: all patients will be examined carefully for the presence of asterixis

Results
The present study was conducted 136 patient and 20 healthy subjects as control group classified as follows: G1: 68 Cirrhotic patients , 38 males and 30 females with age 53.51±7.21, classified according to child pugh classification into child A 11patients , child B 10 patients and child C 47 patients. Patients presented with HCC showed significantly higher levels of serotonin, alpha fetoprotein, albumin and white blood cell count compared to cirrhotic groups. However, HCC patients showed lower levels of AST, ALT, bilirubin and platelet count compared to cirrhotic patients. However, HCC patients were of older age associated with male gender and showed shorter prothrombin time compared to cirrhotic patients (Table 1). Serum serotonin was significantly higher in HCC patients with metastases than patients without metastases (Table 2) with Mean± SD 1134±140.641 compared to that of isolated HCC without metastases 852.524± 283.965. Serotonin level in patients with isolated HCC and patients with metastasis are shown in Table 2. Serotonin level in different etiology of liver cirrhosis is shown in Table 3. Correlation between serotonin and different parameters in HCC patients are shown in Table 4. Comparison between Cirrhotic patients groups (A,B,C)according to Child classification as regard blood serotonin shown in Table  5. Sensitivity, specificity and accuracy for AFP and serotonin HCC group are shown in Table 6. ROC curve for α fetoprotein level in HCC patients is shown in Figure 1. AFP at cutoff 5 ng/ml in the area under ROC curve showing high sensitivity and specificity. ROC curve for serotonin level in HCC group is shown in Figure  2. Sr .Serotonin at cutoff > 510 under ROC curve showed high sensitivity and specificity in HCC group

Discussion
Serotonin (5HT) a well known neurotransmitter within the central nervous system, also regulate a wide range of physiological actions in the gastrointestinal tract [19]. 5HT a potent mitogen for many different cell types including hepatocytes [20]. Within the liver 5HT has the ability to regulate hepatic blood flow at both portal vein and sinusoidal levels and it may play a role in hepatic regeneration [8]. Serotonin has been shown to mediate the pathology of many liver diseases, such as steatohepatitis, chronic cholestasis, viral hepatitis and liver cirrhosis [8]. All these conditions are involved in the tumorigenesis of HCC [21]. Although Serotonin promoting liver regeneration, it is involved in many liver diseases and tumors such as steatohepatitis, chronic cholestasis, development of portal hypertension, aggrevation of viral hepatitis and progression of hepatic fibrosis and facilitate tumor growth as cholangiocarcinoma and HCC [22].
HCV infection was the main etiology of liver cirrhosis in the majority of our cases, about 60 patients (88.2%) and HBV in 8 patients (11.76 %) of cirrhotic group while 78% and 4% respectively in HCC group. In our study Serotonin level was correlated with degree of portal hypertension, splenic size and portal vein diameter as with statistically significant difference between serotonin level in cirrhotic patients and healthy control subjects and between cirrhotic and HCC groups. Abdu Elmoety et al. [23] found that ROC curve for serotonin cut off 75ng /ml, 93.33 % sensitivity with 100% PPV and 100 % specificity with 83.3% NPV and 95% accuracy, in the present study sensitivity of serotonin 95.27 % and specificity 88.24%, PPV 97.1% and accuracy 97.6% for liver cirrhosis. Farintai et al. [24] concluded that AFP was not a sensitive marker to detect the presence of HCC. Also, the prognostic value of AFP is limited, but correlated with the overall survival in untreated patients. AFP is less specific and can be elevated in liver cirrhosis and other malignancies, it is recommended that it is no longer be used for diagnosis of HCC [25].
In our study, Sr. serotonin was significantly higher in Child A cirrhotic patients than child C (p< 0.005) and also with statistical significant difference between HCC group and cirrhotic patients. We found serum serotonin is also correlated with liver synthetic capacity, child score, sr. bilirubin and prothrombin time. Abdo -Elmoety et al. [23] found in the cirrhotic group that ROC for AFP at cut off 10 ng /ml the area under ROC curve was 0.733, p=0.074 showing 51.11% sensitivity, 100% PPV and 31.25% NPV and 60 % accuracy .Found also serotonin AUROC with cut off 75ng /ml (0939) p=0.031 showed 86.67% sensitivity and 100% specificity with 62.50 % NPV and 89.09% accuracy. And serotonin in the HCC group at cut off 75ng/ml by ROC the diagnostic performance arrived 100%. In the present study sensitivity of serotonin 89.71%and specificity 85%and sensitivity of AFP is 100% and specificity 70%.

Conclusion
Combined use of serotonin and AFP is better in diagnosis of HCC. A significant positive correlation was found between Sr. serotonin and AFP in HCC patients (r =0.954), p<0.001.So higher AFP is associated with higher serotonin and this signify the association between AFP and serotonin and so the importance of sr. Serotonin as marker of HCC and together with the result of ROC curve can be considered as a good marker for diagnosis of HCC.