**2. miRNAs in the diagnostics of hepatocellular carcinoma**

**1. Introduction**

48 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

controversial cases.

straightforward even in biopsy.

Hepatocellular carcinoma is one of the most aggressive human cancers. The total oncological mortality is decreasing in many developed countries, e.g., it has been reduced by 23% since 1991 in the United States of America (USA). In contrast, death rate of HCC is increasing, along with the incidence of this tumour [1, 2]. Positive changes are expected due to risk factor eradication by vaccination against hepatitis B and improved treatment of chronic hepatitis C. The treatment of HCC has also developed significantly, including radiofrequency ablation, transarterial chemoembolisation, liver resection and transplantation as well as molecular targeted treatment by sorafenib. However, SEER-based analysis has revealed that survival has improved in early

Most of the hepatocellular carcinoma cases develop on the background of liver cirrhosis or chronic inflammatory liver disease in precirrhotic stage, e.g. chronic viral hepatitis B or C, alcohol-induced or autoimmune liver disease or non-alcoholic steatohepatitis (NASH). This might facilitate the diagnostics by screening of the risk population. Nowadays, screening by ultrasonography and non-invasive radiological diagnosis by the means of computed tomography or magnetic resonance imaging is the mainstay of HCC diagnostics. However, the radiological findings in early cases can be difficult to interpret. Biopsy is indicated in such

However, the biological course of HCC can result in diagnostic difficulties even in biopsy. HCC frequently develops in a dysplastic cirrhotic nodule. Such early HCC is typically well differentiated. Over time, it progresses to advanced dedifferentiated HCC. The resulting heterogeneity can lead to diagnostic problems and failures in biopsy due to sampling errors. For instance, if a small nodule seemed suspicious but not overtly malignant by radiological imaging, leading to biopsy, the differential diagnosis between dysplastic nodule and HCC will frequently imply the necessity to distinguish between premalignant process and welldifferentiated tumour, usually lacking marked cell atypia or clear-cut invasion. In addition, both processes can be adjacent in the tissues. Consequently, early diagnostics of HCC is not

In addition, biopsy can cause complications, including arterial hypotension, bleeding, pneumothorax, haemothorax, haemobilia, acute pancreatitis, visceral perforation, biliary fistulas, sepsis and needle breakage. Arterial hypotension is seen frequently (1.1–4.0%), mostly due to vasovagal reaction. In few cases, falling arterial blood pressure might indicate bleeding, if it is unusually severe. Bleeding can develop in the liver tissues or in the peritoneal cavity. It is seen in 4.5% of patients and is more frequent if INR is elevated: frequency of bleeding was 3.3% in patients having INR 1.3–1.5 and 7.1% among those who had INR > 1.5. Pneumothorax and haemothorax have been reported in 0.35 and 0.18% of patients, if the needle has accidentally passed through lung or diaphragmatic and intercostal blood vessels. Haemobilia has been described in 0.1% of patients and can induce acute pancreatitis via biliary obstruction. The frequency of visceral perforation, biliary fistulas, sepsis and needle breakage is 0.01% each. In addition, biopsy can result in pain, experienced in up to 84% of patients. In 40% of cases, pain lasts for 24 hours. It is attributable to skin and liver capsule damage, viscerosomatic irradiation

but not in advanced cases [2]. Thus, timely diagnostics remain an important goal.

MicroRNAs (miRNAs) are small, double-stranded, non-coding RNA molecules consisting of approximately 22 nucleotides. MiRNAs regulate gene expression at the post-transcriptional level [4, 5] acting as large-scale molecular switches. MiRNAs are found not only in cells but also in body fluids. Due to the stable and relatively simple structure, these molecules are good biomarkers for diagnostic and prognostic evaluation complying with the idea of so-called liquid biopsy—a patient-friendly blood test bringing the same information as a biopsy. In order to increase the diagnostic value of such tests, panels of miRNAs have been advocated. However, the biological course of HCC cause a possible pitfall in the elaboration of such diagnostic tests: as HCC mainly arise on the background of liver cirrhosis, inflammatory or metabolic liver diseases, these preceding pathologies can also alter the levels of miRNAs.

MiRNA-122 is attractive for its wide expression in liver tissues suggesting significant role in liver functions. MiRNA-122 is upregulated in serum of HCC patients and downregulated in HCC tissues suggesting specific excretion of miRNA-122 in blood by HCC. Although miRNA-122 shows high specificity and sensitivity for HCC in comparison with healthy controls (83.3 and 81.6%, respectively), levels of miRNA-122 change in other liver pathologies as well, limiting the diagnostic application [6].

Comparing HCC patients with those having hepatitis B or liver cirrhosis, serum levels of exosomal miRNA-18a, miRNA-221, miRNA-222 and miRNA-224 were increased, while miRNA-101, miRNA-106b, miRNA-122 and miRNA-195 were decreased. MiRNA-16 was decreased in HCC, and the levels significantly differed from those found in hepatitis C virus (HCV) infection because chronic viral hepatitis C and non-alcoholic fatty liver disease are characterised by the contrary changes—increase in miRNA-16. MiRNA-21 is characterised by positive characteristics in meta-analysis showing specificity and sensitivity for HCC diagnosis of 84.8 and 81.2%, respectively. The changes of miRNA-21 serum levels in HCC patients significantly differ from cases of chronic hepatitis; however, other malignant tumours can also yield higher serum concentration of miRNA-21 [7].

Several panels of miRNAs have been recommended. Exploring nine serum miRNAs (miRNA-21, miRNA-30c, miRNA-93, miRNA-122, miRNA-125b, miRNA-126, miRNA-130a, miRNA-193b and miRNA-222) in HCC and chronic viral hepatitis C patients, nine markers were decreased in chronic hepatitis C *versus* healthy controls, while seven markers (miRNA-21,

miRNA-30c, miRNA-93, miRNA-122, miRNA-125b, miRNA-130a and miRNA-222) were significantly (p < 0.05) decreased in HCC *versus* chronic viral hepatitis C patients and four (miRNA-93, miRNA-122, miRNA-125b and miRNA-130a) in HCC *versus* non-HCC patients [8]. Panel of three miRNAs (upregulated miRNA-92a-3p, downregulated miRNA-3126-5p and upregulated miRNA-107) could discriminate HCC from healthy controls [9] Evaluating serum levels of 13 miRNAs in HCV-associated chronic hepatitis, liver cirrhosis and HCC *versus* healthy controls, panel of three miRNAs (miRNA-122, miRNA-885-5p and miRNA-29b) in association with serum alpha-fetoprotein (AFP) level could identify HCC *versus* healthy persons, while four miRNAs (miRNA-122, miRNA-885-5p, miRNA-221 and miRNA-22) and AFP were recommended for HCC diagnostics in liver cirrhosis and two (miRNA-22, miRNA-199a-3p) along with AFP—in chronic hepatitis [10]. In another study of HCV-infected patients including cases of HCV-related chronic hepatitis, liver cirrhosis and HCC, serum levels of miRNA-126, miRNA-129, miRNA-155, miRNA-203 and miRNA-223 were significantly decreased in HCC *versus* non-HCC patients [11]. Panel of eight miRNAs was assessed in hepatitis B virus-infected patients diagnosed with HCC or liver cirrhosis as well as in healthy controls. The levels of hsa-miRNA-206, hsa-miRNA-141-3p, hsa-miRNA-433-3p and hsamiRNA-1228-5p were significantly increased in HCC *versus* control group comprising both healthy and cirrhosis patients, while hsa-miRNA-199a-5p, hsa-miRNA-122-5p, hsa-miRNA-192-5p and hsa-miRNA-26a-5p were downregulated [4].

The systemic effects of cancer include alterations in bone marrow function, especially myelopoiesis. Besides the increased production and release of leukocytes, immature myeloid cells, including the precursors of granulocytes and monocytes, are retained in early stages of differentiation. Immature myeloid cells can act as immune suppressors and generate pre-metastatic niches, among other pathogenetic processes [13]. Thus, it has even been stated that cancer is an inflammatory disease [14]. Further, neutrophils can form neutrophil extracellular traps developed from externalised DNA network. These nets are bidirectionally associated with platelet activation and can contribute to cancer progression via several mechanisms; there-

Innovative Blood Tests for Hepatocellular Carcinoma: Liquid Biopsy and Evaluation of Systemic…

http://dx.doi.org/10.5772/intechopen.76599

51

fore, neutrophil extracellular traps also represent an attractive treatment target [15].

Cancer-related SIR involves cells of innate and adaptive immunity as well as soluble factors. Macrophages are recruited in tumour by hypoxia and tumour-released molecular agents including growth factors and cytokines [16]. Macrophage phenotype switch from tumoursuppressing classical M1 to tumour-promoting M2 subtype promotes angiogenesis and immunosuppression. Platelet activation contributes to cancer progression and patient mortality [15]. Neutrophils are locally recruited in the cancer via chemokine signalling. Neutrophil activation can contribute to angiogenesis and increased blood vessel permeability locally and metastatic spread systemically. In addition, immature myeloid cells and neutrophil extracellular traps might have tumour-promoting activity. These molecular events also highlight the association between infection or surgery-induced inflammation [17] and cancer relapse or metastatic spread. Thus, innate immunity is generally thought to act as tumour enhancers. In contrast, lymphocytes representing the adaptive immunity are considered to have tumour-suppressing effects [16], although contrary effects have been ascribed to certain subpopulations [18].

HCC can be considered a classical inflammation-induced cancer, as its most common risk factors are hepatitis B and hepatitis C virus infections. Inflammation is also present in liver tissues in patients affected by alcohol-induced hepatitis or NASH. Thus, SIR is not expected to have diagnostic value. Indeed, SIR parameters change before the tumour develops, e.g., increased NLR has been observed in chronic viral hepatitis C [19]. NLR is an independent

Wide variety of inflammation-based markers could be used as indicators of HCC prognosis, tumour recurrence and response to specific treatment. In particular, neutrophil, lymphocyte and platelet counts as well as C-reactive protein and albumin and their combinations, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and Glasgow prognostic

Neutrophil-to-lymphocyte ratio is the most extensively evaluated parameter of SIR in HCC. NLR, calculated as the ratio between count of neutrophils and lymphocytes in blood, seems to reflect the intensity of systemic pro- and anti-tumour reaction. NLR has shown prognostic importance in surgically treated HCC cases, including early stage tumours; in liver

Several meta-analyses have been devoted to NLR in HCC patients. Thus, baseline NLR was associated with overall survival and recurrence-free survival, while post-treatment NLR was significantly associated with overall survival [21]. In meta-analysis of NLR in HCC patients

transplantation; sorafenib treatment and different ablation techniques.

prognostic factor in liver cirrhosis [20].

score (GPS) have prognostic value.

**3.1. Neutrophil-to-lymphocyte ratio in HCC**

In addition to the diagnostic role, miRNAs have been evaluated in the prognostic aspect. The influence of miRNAs upon HCC stem cells has been exploited. It has also been suggested that miRNAs could become treatment targets [12].
