**Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements**

Natalia Olaya1 and Franck Chiappini2

*1Laboratorio clínico, de patología y banco de sangre/ Fundación Valle del Lili Cali 2Beth Israel Deaconess Medical Center, Center of Life Science, Division of Endocrinology, Diabetes and Metabolism, Boston, 1Colombia 2USA* 

**1. Introduction** 

Hepatocellular carcinoma (HCC) is responsible for significant morbidity and mortality in cirrhosis and also accounts for between 85% and 90% of primary liver cancer (Caldwell & Park 2009; Hussain & El-Serag 2009; Tandon & Garcia-Tsao 2009). Most of HCC in the world occur in the setting of cirrhosis and over half-million of people develop liver cancer every year and an almost equal number die of it (Caldwell & Park 2009; Hussain & El-Serag 2009). Liver cancer prognosis is determined by factors related to the tumor (etiologies) and factors related to the cirrhosis i.e. parameters of liver dysfunction (CLIP 1998; Llovet et al., 1999; Okuda et al., 1985; Tandon & Garcia-Tsao 2009). During the last 30 years the HCC-incidence rate increased dramatically, despite the development of the HBV-vaccine and the program for newborn vaccination against HBV, developed in European and Asian countries (El-Serag et al., 2003; Hussain & El-Serag 2009).

Since 1997, and after implementing a program for vaccination of newborns against HBV, Chinese and Japanese populations began to show a decrease in HCC incidence, especially among males (Plymoth et al., 2009; Yu S. Z. 1995). In the other hand, HCV-infection is rising around the world (Davila et al., 2004; Kong S. Y. et al., 2009; Min et al., 2009; Yoon et al., 2009; Yu M. L. & Chuang 2009) counter balancing the benefit effects of HBV-vaccination. This increase in incidence rate is observed through the world and it does not belong to a specific region. For example, the USA and Europe have the same positive incidence slope (Davila et al., 2004; Donato et al., 2006; El-Serag 2002; El-Serag 2004; El-Serag et al., 2003; Hassan et al., 2002; Hussain & El-Serag 2009; Scatton et al., 2009; Wu et al., 2000). The etiologies of HCC remain the same; the most important causes are the HBV and HCV infections, heavy alcohol consumption, aflatoxin B1, age and gender (males are more susceptible than females), race (Asian and African over 20 years old) , tobacco consumption and obesity associated with non-alcoholic fatty liver disease and the increase of the Diabetes II mellitus that rise the risk factor between 2 and 3, genetic hemochromatosis, primary biliary cirrhosis, alpha1-antitrypsin deficiency and autoimmune hepatitis (Banks et al., 2006; Borgen et al., 1998; Bostick et al., 1998; Caldwell & Park 2009; CLIP 1998; Collier & Sherman

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 329

the lymphatic system are considered to be tumoral microemboli (CTM) and represent a collective migration. The terms disseminated tumor cells (DTC) and isolated tumor cell (ITC) can be also found in the literature, but are usually used to define the cells that can be detected in both the organs and the bloodstream. The word micrometastasis is usually used to indicate tumor cells found in distant organs. (Luzzi et al., 1998; Schuler & Dolken 2006; Zieglschmid et al., 2005). The presence of circulating tumor cells reflects the aggressiveness nature of a solid tumor. Many attempts have been made to develop assays that reliably detect and enumerate these cells. The clinical results obtained with such assays suggest that in some tumor types, CTC detection and identification can be used to estimate prognosis and may serve as an early marker to assess anti-tumor activity of treatment. In addition, CTC can be used to predict progression-free survival and overall survival. CTC are an interesting source of biological information in order to understand dissemination, drug resistance and treatment-induced cell death. (Alix-Panabieres et al., 2008; Braun et al., 2005; Curry et al., 2004; Mocellin & Hoon & et al., 2006; Mocellin & Keilholz & et al., 2006; Muller et al., 2005; Nakagawa et al., 2007; Pantel et al., 2008; Pantel & Woelfle 2005; Paterlini-Brechot & Benali 2007; Riethdorf et al., 2008; Sleijfer et al., 2007; Strijbos et al., 2008;

In general, intra- or extrahepatic metastases appear at a late tumor stage or after treatment suggesting that earlier during the development of the HCC, the tumor spread circulating of liver-derived cells or circulating tumoral cells also named in literature "micrometastasis". In HCC animal models showed that 10 to 10 000 CTC are capable to initiate new metastasis (Groom et al., 1999; Hermanek et al., 1999; Liotta et al., 1974; Luzzi et al., 1998). Even after curative resection, the tumor recurrence rate remains high. Although CTC detection has been applied and well documented in different types of cancer, especially breast cancer, CTC detection is not routinely performed in HCC follow-up and remains in the experimental field. However, CTC detection might bring new interesting information of metastatic process, might be used as diagnostic tool of early recurrence and may allow a better patient selection for liver transplantation. Mechanisms of tumor recurrence are still poorly understood. Several arguments point out that HCC tumor cells can infiltrate the blood system as shown by the presence of alpha-fetoprotein mRNA (Aselmann et al., 2001; Kamiyama et al., 1996; Kienle et al., 2000; Komeda et al., 1995; Lemoine et al., 1997; Matsumura 2001; Matsumura et al., 2001; Matsumura et al., 1995; Matsumura et al., 1994; Matsumura et al., 1999; Morimoto et al., 2005). CTC seem to be correlated with poor survival in many types of tumors (Aselmann et al., 2001; Kamiyama et al., 1996; Komeda et al., 1995;

However, HCC circulating cells are still difficult to detect, and their presence and amount are poorly correlated with either long-term survival or recurrence in the setting of HCC. Given the lack of specific biological markers, few studies focused on CTC detection. The challenge of CTC detection is related to the requirement of both high sensitivity and specificity. A wrong labeling of ''non-tumor cells'' (epithelial non tumor cells or normal

Methods of CTC detection have to be highly sensitive and specific. The first technical challenge in this field consists of finding exceptional cells. Just a few CTC are mixed with the approximately 10 million leukocytes and 5 billion erythrocytes in 1 ml of blood. To be

Lemoine et al., 1997; Matsumura et al., 1994; Morimoto et al., 2005).

hepatocytes, for instance) as ''tumor cells' could generate poor clinical interest.

Willipinski-Stapelfeldt et al., 2005).

1998; Davila et al., 2004; Donato et al., 2006; El-Serag 2002; El-Serag 2004; El-Serag et al., 2003; Hassan et al., 2002; Hussain & El-Serag 2009; Idilman et al., 1998; Ishikawa et al., 1998; Jung et al., 1998; Llovet et al., 1999; Luzzi et al., 1998; Mendizabal & Reddy 2009; Mocellin & Hoon & et al., 2006; Mocellin & Keilholz & et al., 2006; Naume 1998; Naume et al., 1998; Okuda et al., 1985; Racila et al., 1998; Schutte et al., 2009; Wu et al., 2000; Yao D. F. et al., 2007; Yu M. C. et al., 2000; Yu S. Z. 1995).

Usually, HCC develops during a long process of inflammation and fibrosis, eventually leading to cirrhosis. (Britto et al., 2000; Hussain & El-Serag 2009; Idilman et al., 1998; McMahon 2009). The majority of liver masses are detected incidentally in asymptomatic patients. These lesions are identified using imaging tools such as ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI). A diagnostic approach to HCC has been developed based on the literature and expert consensus, and incorporates serology, cytohistology and radiology/imaging characteristics. (Assy et al., 2009; Bruix & Sherman 2005; Bruix et al., 2001; Byrnes et al., 2007; Durand et al., 2001; Gomaa et al., 2009; Hussain & El-Serag 2009; Mendizabal & Reddy 2009).

HCC is one of the most aggressive cancers. Patients who show progress over the terminal stage have a 1-year survival of less than 10%. In the other hand, patients presenting as earlystage HCC with preserved liver function, a solitary HCC or up to three nodules, each less than 3cm have 5-years survival figures up to 75%. The choice of the therapy and the prognosis are dictated by the severity of the liver function, portal hypertension and medical co-morbidities. National and internationals consensus were established to choose the best treatment adapted for each case and obtain the best prognostic. Milan, Barcelona Clinic Liver Cancer, Cancer of the Liver Italian Program, and San Francisco criteria had determined the prognosis of patients based on the number and size of nodules, the metastasis and the state of liver function leading to the best choice for the patient (Banks et al., 2006; Borgen et al., 1998; Bostick et al., 1998; Caldwell & Park 2009; CLIP 1998; Collier & Sherman 1998; Davila et al., 2004; Donato et al., 2006; El-Serag 2002; El-Serag 2004; El-Serag et al., 2003; Freeman R. B. et al., 2006; Hassan et al., 2002; Idilman et al., 1998; Ishikawa et al., 1998; Jung et al., 1998; Llovet et al., 1999; Luzzi et al., 1998; Naume 1998; Naume et al., 1998; Racila et al., 1998; Sanchez Antolin et al., 2009; Yao D. F. et al., 2007).

Based on these data, the physician can choose resection, orthotopic liver transplantation (OLT), percutaneous ethanol injection or radiofrequency ablation, chemoembolisation, systemic chemotherapy and symptomatic therapy. The first 3 treatments are potentially curative, the second following treatments are palliative and the last one usually is applied at the terminal stage of the disease (Mendizabal & Reddy 2009). At the time the HCC will be treated an important question that the physician has to face is the risk of recurrence and metastasis. To answer this question the best approach would be able to detect the circulating tumor cells in the bloodstream.
