**3.3.1 Microarray/mRNA large analyses**

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.

#### **3.3.2 Proteomic and secretome analyzes**

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

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 patients with HCC.

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 were tested during a clinical trial.

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements 337

**Non-Specific** 

1. Isolation of whole and living

2. Can use another method of enrichment more specific (immunomagnetic beads), 3. Cytopathology, Cytological staining, ICH, FISH...etc can be

4. RNA, DNA extractions followed by RT-PCR or PCR respectively can be done.

2. Precise counting of the cells per ml of blood, independently of the volume of the blood

3. Allows Cytopathology, Cytological staining, ICH,

4. Allows Microdissection

2. Allows Cytopathology, Cytological staining, ICH,

3. Allows Microdissection follow

4. RNA, DNA extraction follow by RT-PCR or PCR respectively.

2. The cells harvested by this method can be re-enriched or analysed by the methods already described, 3. Low cost.

5. RNA, DNA extraction follow by RT-PCR or PCR respectively 6. Avoids multiple steps, 7. Increases sensitivity (1 single CTC can be detect from 1ml of

CTC,

performed,

1. Easy

treated,

FISH...etc,

followed by

blood).

FISH...etc,

by

1. Easy,

**Cytospin** 1. Easy,

**Lysis Buffer**  (Qiagen)

**Advantages Disadvantages References** 

1. Non-specific,

2. Rare CTC can be lost in the plasma fraction or trapped among erythrocyte and neutrophils,

(Balic et al., 2005; Becker et al., 2005; Mankin et al.,

(Li et al., 2010; Mejean et al.,

(Becker et al., 2005; Farina et al., 2004; Kallergi et al.,

Kollermann et al., 1999; Saraiva-Romanholo et al., 2003)

(Aryal et al., 2004; Khan et al., 2000; Wharton et al., 1999)

2008;

2002; Paterlini-Brechot & Benali 2007)

2000; Paterlini-Brechot & Benali 2007; Pinzani et al., 2006; Vona et al., 2004)

3. Low and variable

4. Depends of the type of CTC, temperature,

2. CTC can go through

3. Cells can be damaged, 4. Expensive, but less

immunomagnetic beads.

1. Increase the mortality

1. Increase the mortality

of the CTC.

of the CTC, 2. Low sensitivity.

sensitivity,

centrifuge, 5. Expensive.

the filter,

than the

1. Non-specific,

**Methods of Enrichment** 

**Density**  (OncoQuick®, Ficoll, UNI-Set®)

**Size** 

(MEMS, ISET)

#### **3.3.3 MicroRNA markers: A new hope**

Around 28 years ago, microRNA (miRNA) was discovered and showed the regulation of genes (Lee et al., 1993; Reinhart et al., 2000) such as oncogenes or tumor suppressor genes at the level of the mRNA. More than 35 studies focused on the identification of miRNA or a group of miRNAs to be used as marker of early diagnosis or metastasis. miR-122/-122a, miR-221/222, miR-145, miR-146a, miR-26 (NFB pathway), miR-199a-3p (mTOR pathway) and miR-26 (MYC pathway) were strongly linked to the development and metastasis of the HCC. Also a group of miRNAs were used to identify and classify HCC (Hoshida et al., 2010; Ji & Wang 2009; Kerr et al., 2011; Kojima et al., 2011; Kong G. et al., 2011; Sato et al., 2011), but non of them were used as a CTC marker and tested during a clinical trial.

#### **3.4 Conclusion**

In Conclusion, we can observe that not too many specific HCC markers are available and useful for the detection of the CTC. This is certainly due to the heterogeneity of the hepatocellular carcinoma. The most important marker used in clinical routine is the detection of serum AFP mRNA expression (Table 1). But this marker is not expressed in all HCC and by consequence in all CTC leading false negative results. Some propose to combine the research of more than one marker to increase the specificity and the sensitivity of CTC detection method. One of promising marker is Cancer-Testis Antigens but more studies need to be done to select one or more CTA combined (or not) with the detection of the AFP mRNA expression. As we notice previously, CTC are very rare in peripheral blood. We saw also that real-time polymerase chain reaction is a method that in addition to be specific by the nature of the primers used, it can amplify the signal by increasing the number of copies of mRNA originally presents in the sample. But before using RT-PCR, it's necessary to concentrate the number of CTC from the peripheral blood in a smaller volume. The next chapter will describe these methods.
