**7. Inmunohistochemical markers based on tissue microarrays**

The ideal marker predictor of outcome should include the following characteristics: of low cost and easy measure, reproducibility across institutions, and measurable both before and after treatment. Most importantly, this factor would predict major differences in outcome that significantly impact treatment. A clinical example is K-ras status as a predictor of response to therapy with cetuximab, a monoclonal antibody against the EGFR. In a prospective randomized controlled trial, patients with advanced CRC were randomized to receive treatment with or without cetuximab. When stratified for K-ras status, patients with wild type K-ras tumours demonstrated a significant survival advantage compared to those with mutated K-ras tumours, who derived no benefit from the agent. Therefore, patients with mutated K-ras do not receive cetuximab therapy and are spared the toxicity associated with a treatment with no proven benefit. To date, there is no specific clinical risk score or biomarker that specifically prognosticates or guides therapy for patients with resectable CRC liver metastases to this degree. This marker may combine the immunohistochemical markers of cell proliferation and cell cycle control, p53 and Ki67.

Surface antigen CD133 is a cell membrane glycoprotein that is considered as a cell surface marker expressed in stem cells of hematopoietic immature cells, but not in mature blood cells. CD133 has also been shown to be a marker of immature neuronal stem cells (Karoui et al. 2006). Two antibodies, CD133/1 or AC133 and CD133/2 (AC141), recognize it. CD133+ cells in colon cancer are helpful markers for detection of tumour initiating cells (Karoui et al. 2006) (Figure 9).

CD44 is considered as a cell membrane marker or epithelial cell adhesion molecule (*EpCAM)*. Its phenotype *EpCAMhigh-*CD44+ is becoming established as a good marker for immature stem cells of human colon mucosa in certain series (Ieta et al. 2008) (Figure 10).

Recurrence after repeat hepatectomy has been reported in 60–80% of patients (Smith & McCall 2009). A few have resectable disease limited to the liver and may be candidates for a third or even fourth hepatic resection. In our group there are two patients with five hepatectomies. Reports of large repeated hepatectomy series show that 9-30% of patients who underwent a second hepatectomy for colorectal liver metastases had a third resection (Fong et al 1999; Söreide et al. 2008; Yamamoto et al. 1999; Petrowsky et al. 2002) and 4% of them had a fourth resection (Adam et al. 2003; Yamamoto et al. 1999). The safety of multiple repeated hepatic resections has been demonstrated in recent reports, and long-term survivors have been documented (Adam et al. 2003; Nordlinger et al, 1994; Yamamoto et al. 1999; Petroswsky et al. 2002). LiverMet Survey published the largest series (n = 251) of third hepatectomies for recurrent CRC liver metastases with a survival benefit of 29% at 5 years. Adam et al. published a large series of patients who underwent a third liver resection with zero mortality and a morbidity rate of 5%, not significantly different from those who have had only one or two liver resections. In addition, patients with a third liver resection had a survival benefit of 32–38% at 5 years (Adam et al. 2003; Yamamoto et al. 1999). Major hepatectomy is possible in a minority of these patients, who represent a small and highly

selected group (Petrowsky et al. 2002).

and Ki67.

2006) (Figure 9).

**7. Inmunohistochemical markers based on tissue microarrays** 

The ideal marker predictor of outcome should include the following characteristics: of low cost and easy measure, reproducibility across institutions, and measurable both before and after treatment. Most importantly, this factor would predict major differences in outcome that significantly impact treatment. A clinical example is K-ras status as a predictor of response to therapy with cetuximab, a monoclonal antibody against the EGFR. In a prospective randomized controlled trial, patients with advanced CRC were randomized to receive treatment with or without cetuximab. When stratified for K-ras status, patients with wild type K-ras tumours demonstrated a significant survival advantage compared to those with mutated K-ras tumours, who derived no benefit from the agent. Therefore, patients with mutated K-ras do not receive cetuximab therapy and are spared the toxicity associated with a treatment with no proven benefit. To date, there is no specific clinical risk score or biomarker that specifically prognosticates or guides therapy for patients with resectable CRC liver metastases to this degree. This marker may combine the immunohistochemical markers of cell proliferation and cell cycle control, p53

Surface antigen CD133 is a cell membrane glycoprotein that is considered as a cell surface marker expressed in stem cells of hematopoietic immature cells, but not in mature blood cells. CD133 has also been shown to be a marker of immature neuronal stem cells (Karoui et al. 2006). Two antibodies, CD133/1 or AC133 and CD133/2 (AC141), recognize it. CD133+ cells in colon cancer are helpful markers for detection of tumour initiating cells (Karoui et al.

CD44 is considered as a cell membrane marker or epithelial cell adhesion molecule (*EpCAM)*. Its phenotype *EpCAMhigh-*CD44+ is becoming established as a good marker for immature stem cells of human colon mucosa in certain series (Ieta et al. 2008) (Figure 10).

Fig. 9. Immunohistochemical pathological study. Positive membrane staining for stem cell markers CD133, CS-130127, CD133 (32AT1672) in most cells of metastatic adenocarcinoma. Santa Cruz Biotechnology®, Inc (x100).

Fig. 10. Immunohistochemical pathological study. Positive membrane staining for stem cell markers CD44, *EpCAMhigh-*CD44+ in most cells of metastatic adenocarcinoma. Santa Cruz Biotechnology®, Inc (x400).

CD166 is considered as a marker for both cell membrane or epithelial cell adhesion molecule (*EpCAM)* and cytoplasm (Figure 11). It is a marker of mesenchymal stem cells whose role in carcinogenesis is not fully clear (Ieta et al. 2008). Its phenotype *EpCAMhigh-*CD166+ added to *EpCAMhigh-*CD44+ is starting to be considered as an additional marker of immature stem cells in human colon mucosa (Dalerba et al. 2007).

Fig. 11. Immunohistochemical pathological study. Positive membrane staining for stem cell markers CD166, 35264, CD166 LYO 1 mL *EpCAMhigh-*CD166+ in most cells of metastatic adenocarcinoma. Menarini Diagnostics® (x40).

Borrego et al. (2010) analyzed, as did Kokudo et al. (2002), p53 expression. The immunohistochemical markers tested in their study (p53, Ki-67) were not poor prognostic factors, in agreement with Saw et al. (2002). By contrast, authors such as Tanaka et al. 2004 reported that p53 or Ki-67 expression had a negative impact on survival. It should be noticed, however, that in Borrego-Estella et al.'s study survival was longer than 5 years in patients with high Ki-67 levels and in those with a high mitotic index (>10 mitosis/mm2), which is also another expression of the tumour proliferation index. However, no significant relationship was found between cell proliferation, as measured by Ki-67 and p53, whose changes express a loss of cell cycle control, and survival.

For O'Brien et al*.* (2007), most CD133+ stem cells had a 200-fold greater oncogenic potential than CD133- cells for development of CRC. In addition, this subpopulation is able to maintain itself and to differentiate and become established again as a tumour when transplanted in certain solid organs of experimental animals. For Borrego-Estella et al. significant trends were found in their series with regard to membrane CD133 and CD166 markers. According to O'Brien et al*.* and Ricci-Vitiani et al*.* 2007, in several CRC, CD44 was more determinant than CD133, because CD44 was expressed in tumour lines not expressing CD133.

To compare the results of Borrego et al. with other groups is difficult since many authors (Fong et al. 1999; Pawlik et al. 2005) did not performed immunohistochemical studies. However, regarding immunohistochemical markers, more significant results were not achieved probably because the only technique performed was immunohistochemistry array, but not flow cytometry or other molecular biology techniques.

Another interesting marker, microsatellite instability, is a measure of the inability of the DNA mismatch repair system to correct errors that occur during DNA replication. It is the alternative pathway to chromosomal instability with loss of heterozygosity in colorectal carcinogenesis. Microsatellite instability has been suggested to be prognostic for survival and predictive for response to therapy in patients with colorectal cancer.

In conclusion, many studies have analyzed pre-operative prognostic factors in patients undergoing liver resection for hepatic metastases from CRC in order to select patients for surgery. However, intraoperative and post-operative factors have been poorly analyzed. Future studies should establish post-operative prognostic factors through histological and immunohistochemical tests based on the tissue microarray technique.
