**2. Definition of CTC and HCC**

In the field of biology of tumors, some expressions have been coined for the different types of circulating cellular elements. The term *circulating tumor cells* (CTC) defines specifically the tumor cells detected in blood or lymphatic vessels. Circulating cells in the bloodstream or in

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.,

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;

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;

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

In the field of biology of tumors, some expressions have been coined for the different types of circulating cellular elements. The term *circulating tumor cells* (CTC) defines specifically the tumor cells detected in blood or lymphatic vessels. Circulating cells in the bloodstream or in

2007; Yu M. C. et al., 2000; Yu S. Z. 1995).

Hussain & El-Serag 2009; Mendizabal & Reddy 2009).

Racila et al., 1998; Sanchez Antolin et al., 2009; Yao D. F. et al., 2007).

tumor cells in the bloodstream.

**2. Definition of CTC and HCC** 

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; Willipinski-Stapelfeldt et al., 2005).

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

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

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

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 331

that show a high level of AFP in absence of malignancy (Toyoda et al., 2004; Vejchapipat et al., 2004). For these reasons, some additional serological markers used in combination with AFP seem to improve the performance of this biomarker, especially in terms of sensitivity. Studies were done by using one or two more markers like des- carboxyprothrombin (DCP) also called prothrombin induced by vitamin K absence II (PIVKAII) and glycosylated AFP-L3 (*Lens culinaris* Agglutinin-Reactive AFP) fraction serum levels to diagnose earlier HCC and increasing the sensitivity especially when HCC is associate with cirrhosis, HCV or HBV infection (Dohmen et al., 2003; Fujiyama et al., 2002; Gonzalez & Keeffe 2011; Lok et al., 2009; Min et al., 2009; Saffroy et al., 2007; Suehiro et al., 1994). But there is no study that correlates

In the next sections, we focus on and describe the most specific markers of messenger RNA

Initially, the albumin gene has been proposed as a biological marker to track CTC during HCC development but has been rapidly abandoned since many groups have reported illegitimate transcription of albumin gene in peripheral-blood leukocytes. The relevance of AFP mRNA as a marker of circulating tumor cells is better but is also controversial because these cells have not been further characterized and it has been shown that they may correspond to normal circulating hepatocytes (Lemoine et al., 1997; Louha et al., 1999; Minata et al., 2001). Furthermore, these tumor cells have mostly been sought and detected shortly after liver resection (Kienle et al., 2000; Lemoine et al., 1997; Louha et al., 1999; Matsumura et al., 1994; Minata et al., 2001). This finding suggests that CTC could spread following liver mobilization or manipulation. Although the mechanisms leading to intra and extrahepatic recurrences are still unknown, some observations suggest that bone marrow (BM) could also be a specific reservoir of CTC. Indeed, several reports have suggested that tumor cells are of BM origin (Agarwal et al., 2004; Houghton et al., 2004; Sell 2002). Hepatic tumor stem cells may take advantage of the potential for stem cell support of the BM microenvironment. The amplification of AFP mRNA by means of reverse transcription (RT) and a nested polymerase chain reaction (PCR) is the highly sensitive method for the detection of residual HCC cells in peripheral blood. The qualitative (positive versus negative) detection of HCC circulating tumor cells in blood samples from individual patients is of limited value in predicting the risk of disease progression. Because the level of AFP mRNA is increased in HCC tissue compared with in normal hepatocytes, the quantification of AFP transcripts seems to be a more reliable indicator of disease progression. A more highly sensitive assay based on TaqMan® technology to quantify AFP mRNA in "real time" should be preferred (Cheung et al., 2006; Gross-Goupil et al., 2003; Lu Y. et al., 2007; Matsumura 2001). Even using this methodology, reported results are not homogeneous and contradictory (Bruix et al., 2001). The main studies which have evaluated AFP mRNA are summarized in Table 1. The false-positive results can be

their serum levels and the circulating tumor cell during the HCC development.

**3.2.1 Relevance of -fetoprotein (AFP) messenger RNA (mRNA)** 

**3.2 The mRNA markers** 

obtained using AFP mRNA.

used to detect CTC in hepatocellular carcinoma.

useful, the method used to identify circulating tumor cells must also detect all tumor cells and discriminate them from non-tumor cells. (Alix-Panabieres et al., 2008; Becker et al., 2005; Braun et al., 2005; Curry et al., 2004; Mocellin & Hoon & et al., 2006; Nakagawa et al., 2007; Paterlini-Brechot & Benali 2007; Ross et al., 1993; Schuler & Dolken 2006; Smerage & Hayes 2006). Before considering the technical problems, it is important to have circulating hepatoma-specific biomarkers to be able to detect the CTC and further to be useful to early diagnosis, monitoring metastasis or post treatment recurrences of HCC.

Currently, CTC detection is mainly based on alpha-fetoprotein (AFP) messenger RNA (mRNA) assessment or quantification, and in few reported cases using cytomorphometric technology, especially the ISET devise. A high sensibility could be obtained using flow cytometry assays but high blood volumes (200 ml) and long analysis time (40 h for one sample) are required. Consequently, its use has been discouraged as a routine technique (Mejean et al., 2000).
