**3.1.2 The case of the serum AFP**

The serum -fetoprotein (AFP) levels show high levels in newborns and then declines progressively below 10 ng/ml in 300 days of life. An increase of serum AFP levels can be observed during pregnancy, and in patients with mucovicidosis, acute hepatitis (30%-50%), chronic hepatitis (15%-50%), cirrhosis (11%-47%) and other cancers (gastrointestinal, pancreatic, biliary, non-seminomatous germ-cell testicular, and germ cell ovarian). Serum level AFP sensitivity is between 39% to 64% with approximately 60% for a cutoff of 20 ng/mL, and decrease to 22% if higher cutoff of 200 ng/mL is used (Bruix et al., 2001; Collier & Sherman 1998; Fujiyama et al., 2002; Okuda 1986; Saffroy et al., 2007; Trevisani et al., 2001). AFP specificity is around 76%-91% with a low predictive value between 9%-32% (Bruix et al., 2001; Collier & Sherman 1998; Okuda 1986; Saffroy et al., 2007).

In addition, the serum AFP level is correlated with the tumor size. 80% of small HCCs (<2cm) do not express AFP. In the other hand, AFP level can be elevated in patients with chronic liver disease with high degree of hepatocytes regeneration such as HCV-infection 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 their serum levels and the circulating tumor cell during the HCC development.

## **3.2 The mRNA markers**

330 Hepatocellular Carcinoma – Basic Research

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

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

The hepatocellular carcinomas can synthesize various tumor-related proteins, polypeptides and isoenzymes more or less specific of the hepatoma tissues. It is important to detect CTC in the bloodstream or lymphatic system to have tumor specific markers of HCC. Finding

Hepatocellular carcinoma tumor has shown to secret a lot of cytokines related to the development of the tumor, like vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-β1), Interleukin 8 (IL-8) or tumor-specific growth factor (TSGF). These serum markers are useful to follow-up the development and the prognosis of the

The serum -fetoprotein (AFP) levels show high levels in newborns and then declines progressively below 10 ng/ml in 300 days of life. An increase of serum AFP levels can be observed during pregnancy, and in patients with mucovicidosis, acute hepatitis (30%-50%), chronic hepatitis (15%-50%), cirrhosis (11%-47%) and other cancers (gastrointestinal, pancreatic, biliary, non-seminomatous germ-cell testicular, and germ cell ovarian). Serum level AFP sensitivity is between 39% to 64% with approximately 60% for a cutoff of 20 ng/mL, and decrease to 22% if higher cutoff of 200 ng/mL is used (Bruix et al., 2001; Collier & Sherman 1998; Fujiyama et al., 2002; Okuda 1986; Saffroy et al., 2007; Trevisani et al., 2001). AFP specificity is around 76%-91% with a low predictive value between 9%-32%

In addition, the serum AFP level is correlated with the tumor size. 80% of small HCCs (<2cm) do not express AFP. In the other hand, AFP level can be elevated in patients with chronic liver disease with high degree of hepatocytes regeneration such as HCV-infection

one of these circulating proteins does not mean that there circulating tumor cells.

HCC but useless to follow-up circulating tumor cells in blood (Zhou et al., 2006).

(Bruix et al., 2001; Collier & Sherman 1998; Okuda 1986; Saffroy et al., 2007).

diagnosis, monitoring metastasis or post treatment recurrences of HCC.

**3. HCC markers and their application for CTC** 

**3.1.1 Relevance of cytokines in HCC circulating tumor cells** 

(Mejean et al., 2000).

**3.1 Secreted proteins** 

**3.1.2 The case of the serum AFP** 

In the next sections, we focus on and describe the most specific markers of messenger RNA used to detect CTC in hepatocellular carcinoma.

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

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 obtained using AFP mRNA.

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 333

β1 level (>1.2 µg/L) were 90% and 94% for HCC diagnosis, but no significant correlation was found between TGF-β1 expression an AFP levels or tumor size. The combined detection of TGF-β1 and serum AFP could raise the detection rate of HCC up to 97%. Both of circulating TGF-β1 and TGF-β1 mRNA could be used as sensitive biomarkers for diagnosis and prognosis of HBV-induced HCC (Dong et al., 2008; Wang Y. L. et al., 2007). Unfortunately, TGF-β1 mRNA was poor studied and further investigations have to be done

Studies using amplified fragments of IGF-II mRNA by RT-PCR showed that the lowest sensitivity with 2 ng/L of total RNA. Dong et al., showed that the positive frequencies of IGF-II mRNA were 100% in HCC, around 50% in paracancerous and 0% in noncancerous tissues respectively. But, the positive frequency of circulating IGF-II mRNA was 34% in HCC, and no amplification was found in other liver diseases, extrahepatic tumors, and normal control, meaning that IGF-II is specific of the HCC but nor really sensitive. Associated to other circulating markers IGF-II can be helpful to detect CTC. The circulating IGF-II mRNA was correlated with the stage of HCC (incidence=100%) with extrahepatic metastasis, and 35% with AFP-negative. No difference was found between tumor size and circulating IGF-II mRNA (Dong et al., 2005; Wang Y. L. et al., 2007) but these results are

For more than a decade, we know that mRNAs of hepatocytes-specific albumin genes are detected in peripheral blood by RT-PCR. It was shown that there is evidence that detection of albumin mRNA associated with the detection of AFP mRNA is strongly associated with the presence of metastases (Hillaire et al., 1994; Kar & Carr 1995; Komeda et al., 1995; Matsumura et al., 1995). Wong et al., showed that circulating hepatocellular carcinoma cells can be detected and be semi-quantified by albumin RT-PCR (Wong et al., 1997). On the other hand, Wu et al., showed that, the down regulation of alpha-albumin (ALF) specifically in HCC circulating cells can be used has a specific marker to discriminate the normal hepatocellular circulating cells that express abundantly ALF. RT-PCR ALF in association with RT-PCR AFP have been proposed to distinguish normal or malignant hepatocytes in peripheral blood, but the interpretation of the results is still debated (Resto et al., 2000).

The Prostate-Specific Antigen (PSA) had shown to be well-established reliability marker and remained a valid prostate marker in patients with acute hepatitis and HCC (Malavaud et al., 1999). But these results are controversial, PSA and mRNA PSA seem to don't be specific to the tissue and frequently detected in peripheral blood cells from healthy patients (Ishikawa et al., 1998). In addition, like the cytokines, serum PSA cannot be used as hepatocellular

Heat shock proteins (HSP) are stimulated under perturbation or stressors by the tissue. HSP are ubiquitous molecules and can be also expressed during carcinogenesis. Different HSP

to use circulating TGF-β1 mRNA as a marker of circulating tumor cells in HCC.

**3.2.3 Insulin-like growth factor (IGF)-II mRNA** 

controversial (Qian et al., 2010).

**3.2.4 Alpha-albumin (ALF) mRNA** 

**3.2.5 Prostate-specific antigen (PSA) and mRNA PSA** 

carcinoma marker for circulating tumor cells.

**3.2.6 Heat shock protein (HSP)** 


Table 1. Evaluation of serum alpha-fetoprotein as a marker of circulation tumor cell in different hepatocellular carcinoma studies. nRT-PCR, nested RT-PCR; qRT-PCR, quantitative RT-PCR.

### **3.2.2 Transforming growth factor beta-1 (TGF-β1) mRNA**

The levels of circulating TGF-β1 and TGF-β1 mRNA were significantly higher in the HCC patients than any other group of patients. The sensitivity and specificity of circulating TGF-

β1 level (>1.2 µg/L) were 90% and 94% for HCC diagnosis, but no significant correlation was found between TGF-β1 expression an AFP levels or tumor size. The combined detection of TGF-β1 and serum AFP could raise the detection rate of HCC up to 97%. Both of circulating TGF-β1 and TGF-β1 mRNA could be used as sensitive biomarkers for diagnosis and prognosis of HBV-induced HCC (Dong et al., 2008; Wang Y. L. et al., 2007). Unfortunately, TGF-β1 mRNA was poor studied and further investigations have to be done to use circulating TGF-β1 mRNA as a marker of circulating tumor cells in HCC.

### **3.2.3 Insulin-like growth factor (IGF)-II mRNA**

332 Hepatocellular Carcinoma – Basic Research

**Author PCR Sensitivity Cases Samples Positivity Predictability** 

136

25

11

RT-PCR NA 18 BM 93% No

RT-PCR 1 CHC/106 52 Blood 25% No

Table 1. Evaluation of serum alpha-fetoprotein as a marker of circulation tumor cell in different hepatocellular carcinoma studies. nRT-PCR, nested RT-PCR; qRT-PCR,

The levels of circulating TGF-β1 and TGF-β1 mRNA were significantly higher in the HCC patients than any other group of patients. The sensitivity and specificity of circulating TGF-

nRT-PCR 33 Blood 52% Extrahepatic

nRT-PCR 15 cells/mL 64 Blood 36% Extrahepatic

Blood BM

Blood BM

BM

Blood BM

18% 28%

10 % 48%

29% 43%

26% 45%

20 Blood 25% No

33 Blood 54% Yes

87 Blood 36% Yes

85 Blood 26-45% No

qRT-PCR 1 CHC/ l07 37

qRT-PCR 1 CHC/106 38

Competitive

nRT-PCR 1CHC/105

nRT-PCR 10-6μg/μLof RNA

nRT-PCR 1CHC/105

nRT-PCR 1 CHC/107

**3.2.2 Transforming growth factor beta-1 (TGF-β1) mRNA** 

mono

mono

mono

RT

nRT-PCR 5 cells/ 1mL 24 Blood

10 cells/ 9 mL 22

(Kamiyama et al., 1996)

(Morimoto et al., 2005)

(Kienle et al.,

(Aselmann et al., 2001)

(Sutcliffe et al.,

(Matsumura et al., 1995; Matsumura et al., 1994)

(Komeda et al., 1995)

(Lemoine et al., 1997)

(Miyazono et al., 2001)

(Ijichi et al., 2002)

(Witzigmann et al., 2002)

(Gross-Goupil et al., 2003)

quantitative RT-PCR.

2000)

2005)

**of recurrence** 

No Yes

Yes No

NA

Suspect

metastases

metastases

Studies using amplified fragments of IGF-II mRNA by RT-PCR showed that the lowest sensitivity with 2 ng/L of total RNA. Dong et al., showed that the positive frequencies of IGF-II mRNA were 100% in HCC, around 50% in paracancerous and 0% in noncancerous tissues respectively. But, the positive frequency of circulating IGF-II mRNA was 34% in HCC, and no amplification was found in other liver diseases, extrahepatic tumors, and normal control, meaning that IGF-II is specific of the HCC but nor really sensitive. Associated to other circulating markers IGF-II can be helpful to detect CTC. The circulating IGF-II mRNA was correlated with the stage of HCC (incidence=100%) with extrahepatic metastasis, and 35% with AFP-negative. No difference was found between tumor size and circulating IGF-II mRNA (Dong et al., 2005; Wang Y. L. et al., 2007) but these results are controversial (Qian et al., 2010).

#### **3.2.4 Alpha-albumin (ALF) mRNA**

For more than a decade, we know that mRNAs of hepatocytes-specific albumin genes are detected in peripheral blood by RT-PCR. It was shown that there is evidence that detection of albumin mRNA associated with the detection of AFP mRNA is strongly associated with the presence of metastases (Hillaire et al., 1994; Kar & Carr 1995; Komeda et al., 1995; Matsumura et al., 1995). Wong et al., showed that circulating hepatocellular carcinoma cells can be detected and be semi-quantified by albumin RT-PCR (Wong et al., 1997). On the other hand, Wu et al., showed that, the down regulation of alpha-albumin (ALF) specifically in HCC circulating cells can be used has a specific marker to discriminate the normal hepatocellular circulating cells that express abundantly ALF. RT-PCR ALF in association with RT-PCR AFP have been proposed to distinguish normal or malignant hepatocytes in peripheral blood, but the interpretation of the results is still debated (Resto et al., 2000).

#### **3.2.5 Prostate-specific antigen (PSA) and mRNA PSA**

The Prostate-Specific Antigen (PSA) had shown to be well-established reliability marker and remained a valid prostate marker in patients with acute hepatitis and HCC (Malavaud et al., 1999). But these results are controversial, PSA and mRNA PSA seem to don't be specific to the tissue and frequently detected in peripheral blood cells from healthy patients (Ishikawa et al., 1998). In addition, like the cytokines, serum PSA cannot be used as hepatocellular carcinoma marker for circulating tumor cells.

#### **3.2.6 Heat shock protein (HSP)**

Heat shock proteins (HSP) are stimulated under perturbation or stressors by the tissue. HSP are ubiquitous molecules and can be also expressed during carcinogenesis. Different HSP

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 335

also can be associated to the research of AFP mRNA to increases the specificity and the frequency of the method. This group of markers seems promising and further studies have to be done first to determine the panel of CT antigens to be used as markers of HCC

To attempt the lack of CTC markers, new techniques and technologies are used such as microarray/mRNA large analyses, proteomic and "secretome" analyses and finally

DNA chips were used to measure and find new markers to diagnose HCC, but also to use these as CTC markers. The studies showed the expression of mRNAs for members of the glypican and syndecan families of heparin sulfate proteoglycans such as GPC3 can be a good CTC marker that can be used in human or in mouse models (Suzuki et al., 2010; Yao M. et al., 2011). Another interesting marker was discovered called Snail. Snail mRNA was study in blood of patients with HCC and metastasis (Min et al., 2009). But further investigation has to be done to figure out the specificity and the sensitivity of those markers.

In the process to find new markers for CTC, a number of teams started to work with proteomic analysis such as quadrupol IT-TOF, SELDI- TOF MALDI-TOF/TOF mass spectrometry. Their objective is tracking earlier the development and the progression of the HCC. Few markers or group of markers were identified by these methods such as the usual markers AFP, AFP-L3, TGF-1, and PIVKA-II but also vitronectin, alpha-1-fucosidase (AFU) and DCP, Golgi protein-73 (GP73), hepatocyte growth factor (HGF), and nervous growth factor (NGF) (Dai et al., 2009; Donati et al., 2010; Liu X. & Wan & et al., 2011; Paradis et al.,

Interestingly, the proteomic analyses were able to detect new markers in the serum secreted (which is called "secretome") by the carcinoma cells (Makridakis & Vlahou 2010; Malaguarnera et al., 2010; Niu et al., 2010). Over 90 proteins in some studies compiled with high powerful biocomputational analysis where identified and used to diagnose HCC early (Dai et al., 2009; Paradis et al., 2005; Poon et al., 2003; Zinkin et al., 2008). Unfortunately these studies did not analyze the real usefulness of these markers to identify CTC in the

A sub-group of HCC was identified by these techniques expressing stem cell markers (CD133, CD90, CD44, EpCAM, CD13 or neural cell adhesion molecule; NCAM) defining what is called now liver cancer stem cell but unfortunately these markers were not studied in the area of circulating tumor cells (Chiba et al., 2009; Huang & Geng 2010; Liu L. L. & Fu & et al., 2011). They are very promising markers. Another group of markers very promising to detect CTC belongs to the chemokine receptors such as CXCR4, CX3CR1 and CCR6 express during HCC progression (Huang & Geng 2010; Li et al., 2010), but none of them

circulating cells.

**3.3 New approaches** 

microRNA testing.

patients with HCC.

were tested during a clinical trial.

**3.3.1 Microarray/mRNA large analyses** 

**3.3.2 Proteomic and secretome analyzes** 

2005; Peng X. Q. et al., 2009; Poon et al., 2003; Zinkin et al., 2008).

have been related to the development of the hepatocellular carcinoma like gp96 or GRP94, HSP70 and HSP27, but none of them were used as a specific marker of circulating tumor cell (Wang Y. L. et al., 2007).

#### **3.2.7 Human telomerase reverse transcriptase mRNA or hTERT mRNA**

Human telomerase is a ribonucleic protein composed by the association of three structures: human telomerase RNA component (hTERC); human telomerase-associated protein 1 (hTEP1); and human telomerase reverse transcriptase (hTERT). hTERT is the catalytic unit of the complex. Also, telomerase is expressed in embryonic cells, in most human cancer cells or immortal cell lines, but not in normal somatic cell lines or tissues. For these reasons, hTERT was investigated as a marker of diagnosis and prognosis of HCC, but the results are controversial and appear that false-positive results can be observed because of lymphocytes, precancerous liver parenchymal cells and micrometastasis maybe responsible (for review (Grizzi et al., 2007; Wang Y. L. et al., 2007; Zhou et al., 2006)). Recently, Kong et al., investigated hTERT in peripheral blood in HCC from 343 Korean patients. There is no association between hTERT expression and clinical features and nor relationship between AFP and hTERT mRNA. Their conclusion is that AFP and hTERT mRNA expression in peripheral blood is useless as HCC prognostic markers (Kong S. Y. et al., 2009).

#### **3.2.8 Cancer-testis antigens (CTA)**

Cancer-testis antigens (CTA) represent a category of tumor-associated antigens normally expressed in male germ cells but not in adult somatic tissues (Scanlan et al., 2002). CTA are heterogeneous group of antigens. Actually, more than 44 distinct CT "gene" or "antigen" families have been reported in literature. Certain CT gene families contain multiple members, as well as splice variants and today more than 89 distinct transcripts are known to be encoded by CT genes (Scanlan et al., 2002). A number of CT antigens have been found expressed with high percentage and specificity in HCC. The expression of the CT antigens mRNA was investigated by Wu et al., in the HCC and corresponding peripheral blood of 37 patients with HCC, 15 samples of cirrhotic tissues and 15 normal tissues with the same method. Two CT antigens SSX-2 and SSX-5 showed in this study high specific and high frequent expression only in HCC tissues. In corresponding peripheral blood of HCC tissues, the positive expressions rate of these two CT antigens mRNA was not very high (Benoy et al., 2006). The same group of researchers used another two CT antigens SSX-1 and NY-ESO1 in the same group of patients and with the same methods (RT-PCR) with the corresponding peripheral blood. They showed that SSX-1 can be potential used in peripheral blood, with short term recurrence rat at 46% (6/13) in patients whose peripheral blood expressed SSX-1 mRNA, while the recurrence rate in patients with negative SSX-1 mRNA was 28.6% (4/14) (Bergamaschi et al., 2008). In another study, Peng et al., showed that specific expression of CT antigens was observed in AFP-negative HCC, suggesting the application of their mRNA as tumor markers to detect circulating HCC cells (Peng J. R. et al., 2005). Yang et al., showed that FATE/BJ-HCC-2 (another CTA) mRNA expression was detected in the peripheral blood mononuclear cells (PBMCs) of 46.67% patients, whose HCC tissue samples were cut off and positive for FATE/BJ-HCC-2 mRNA, which implicated tumor cell dissemination in blood circulation and related to the metastasis of HCC. These studies suggest that CT antigens expressions can be used in peripheral blood to detect HCC circulating cells, but also can be associated to the research of AFP mRNA to increases the specificity and the frequency of the method. This group of markers seems promising and further studies have to be done first to determine the panel of CT antigens to be used as markers of HCC circulating cells.

#### **3.3 New approaches**

334 Hepatocellular Carcinoma – Basic Research

have been related to the development of the hepatocellular carcinoma like gp96 or GRP94, HSP70 and HSP27, but none of them were used as a specific marker of circulating tumor cell

Human telomerase is a ribonucleic protein composed by the association of three structures: human telomerase RNA component (hTERC); human telomerase-associated protein 1 (hTEP1); and human telomerase reverse transcriptase (hTERT). hTERT is the catalytic unit of the complex. Also, telomerase is expressed in embryonic cells, in most human cancer cells or immortal cell lines, but not in normal somatic cell lines or tissues. For these reasons, hTERT was investigated as a marker of diagnosis and prognosis of HCC, but the results are controversial and appear that false-positive results can be observed because of lymphocytes, precancerous liver parenchymal cells and micrometastasis maybe responsible (for review (Grizzi et al., 2007; Wang Y. L. et al., 2007; Zhou et al., 2006)). Recently, Kong et al., investigated hTERT in peripheral blood in HCC from 343 Korean patients. There is no association between hTERT expression and clinical features and nor relationship between AFP and hTERT mRNA. Their conclusion is that AFP and hTERT mRNA expression in

**3.2.7 Human telomerase reverse transcriptase mRNA or hTERT mRNA** 

peripheral blood is useless as HCC prognostic markers (Kong S. Y. et al., 2009).

Cancer-testis antigens (CTA) represent a category of tumor-associated antigens normally expressed in male germ cells but not in adult somatic tissues (Scanlan et al., 2002). CTA are heterogeneous group of antigens. Actually, more than 44 distinct CT "gene" or "antigen" families have been reported in literature. Certain CT gene families contain multiple members, as well as splice variants and today more than 89 distinct transcripts are known to be encoded by CT genes (Scanlan et al., 2002). A number of CT antigens have been found expressed with high percentage and specificity in HCC. The expression of the CT antigens mRNA was investigated by Wu et al., in the HCC and corresponding peripheral blood of 37 patients with HCC, 15 samples of cirrhotic tissues and 15 normal tissues with the same method. Two CT antigens SSX-2 and SSX-5 showed in this study high specific and high frequent expression only in HCC tissues. In corresponding peripheral blood of HCC tissues, the positive expressions rate of these two CT antigens mRNA was not very high (Benoy et al., 2006). The same group of researchers used another two CT antigens SSX-1 and NY-ESO1 in the same group of patients and with the same methods (RT-PCR) with the corresponding peripheral blood. They showed that SSX-1 can be potential used in peripheral blood, with short term recurrence rat at 46% (6/13) in patients whose peripheral blood expressed SSX-1 mRNA, while the recurrence rate in patients with negative SSX-1 mRNA was 28.6% (4/14) (Bergamaschi et al., 2008). In another study, Peng et al., showed that specific expression of CT antigens was observed in AFP-negative HCC, suggesting the application of their mRNA as tumor markers to detect circulating HCC cells (Peng J. R. et al., 2005). Yang et al., showed that FATE/BJ-HCC-2 (another CTA) mRNA expression was detected in the peripheral blood mononuclear cells (PBMCs) of 46.67% patients, whose HCC tissue samples were cut off and positive for FATE/BJ-HCC-2 mRNA, which implicated tumor cell dissemination in blood circulation and related to the metastasis of HCC. These studies suggest that CT antigens expressions can be used in peripheral blood to detect HCC circulating cells, but

(Wang Y. L. et al., 2007).

**3.2.8 Cancer-testis antigens (CTA)** 

To attempt the lack of CTC markers, new techniques and technologies are used such as microarray/mRNA large analyses, proteomic and "secretome" analyses and finally microRNA testing.
