Duration of median follow-up survey (year)
Relapse prompted by CEA (%)
Hepatic metastasis prompted by CEA (%)
The rise of CEA is earlier than relapse/metastasis (month)
In 1989, Yeatman et al.  pioneered to report the value of measuring the level of bile CEA inside the gallbladder for the diagnoses of hepatic metastasis of colorectal cancer. Among 17 patients with single and multiple hepatic metastasis, the level of bile CEA was 4.7–259 times higher than that of serum CEA (P = 0.0009). The hepatic metastasis less than 1 cm3 may generate 9–41 ng/ml bile CEA. Li Destri et al.  examined intraoperative bile CEA, preoperative serum CEA, and lymph node involvement among 89 cases after the radical operation for colorectal cancer and follow-up survey last for 3 years. Hepatic metastasis occurred in 11 cases, 9 of which had higher level of bile CEA (5 ng/ml). Among the rest of 78 cases, 73 cases had the normal level of bile CEA, so the ratio of sensitivity, specificity, and accuracy for the anticipation of hepatic metastasis can, respectively, reach 81.8 %, 93.6 %, and 92.1 %. Owing to a few caseloads, this study failed to contribute to serum CEA and lymph node involvement. Tuech et al.  examined the level of bile CEA in three groups, the hepatic metastasis group (n = 35), the non-hepatic metastasis group (n = 154), and the contractive group (n = 23), and the level of CEA was shown as follows: the hepatic metastasis group > the non-hepatic metastasis group > the contrastive group. The follow-up survey was made for the non-hepatic metastasis group for 3 years. Hepatic metastasis occurred in 22 cases, and the patients with the bile CEA >5 ng/ml were more subject to hepatic metastasis than those with the normal level of CEA (18/95 vs. 4/59). Kanellos et al.  made a 5-year follow-up survey for 73 cases with colorectal cancer. Thirteen cases suffered from hepatic metastasis and the level of bile CEA was abnormal. However, among the 60 cases with no tumor, the abnormity of bile CEA occurred in 32 cases, so the rate of false positive was quite high.
Based on the current research on bile CEA, the time to collect bile is in the course of the operation for primary colorectal cancer as a rule, but right now the bigger tumor exists in the body, and bile CEA secreted from the liver, the upmost organ to remove CEA in the body, is bound to be impacted by the primary tumor. So, the examination of bile CEA along with the primary tumor may influence the accurate diagnoses of hepatic metastasis and invasion could hardly be avoided during postoperative examination of bile CEA. Therefore, it is hard to realize clinical application to a great extent.
Besides peripheral blood serum and bile, scholars have also examined the level of CEA in the portal vein blood , vena mesenterica, duodenal juice and exudate from abdominal cavity, etc. However, further development is hindered due to the difficulty in sampling and early diagnosis as well as lower positive rate.
6.1.2 CEA-mRNA and Cytokeratin (CK)-mRNA
Tumor cells in blood circulation are considered as one of the markers to anticipate hematogenous dissemination and distant metastasis of tumor, so it is feasible to examine tumor cells in blood circulation for the risk evaluation of hepatic metastasis of colorectal cancer or early diagnosis. However, owing to fewer tumor cells in blood circulation, relevant test methods request higher sensitivity and specificity. Currently, the reverse transcription PCR (RT-PCR) technology, originated from the classic PCR, is widely applied to detect a certain gene at the mRNA level. A tumor cell can be detected from 106 to 107 normal cells, so such technology is obviously superior to traditional cytomorphology, immunohistochemistry, cytogenetics, and flow cytometer. If the RT-PCR technology is applied to examine the circulating tumor cells, the objective gene should be featured with the specificity in tumor tissue. Most common solid tumor lacks the genes with tumor specificity, so currently tissue specificity or tumor-related target gene is selected for examination. CEA and CK are mainly applied as the target gene for the colorectal cancer. CEA can be almost detected on any epithelia including cancer cell, while CK is the component protein of the interzonal fiber of epithelia as well as reliable marker for the differentiation of epithelium; for example, CK19 and CK20 exist in the malignant tumor related to glandular epithelium. Given the unstable condition of mRNA in the cell’s external environment, it is assumed that tumor cells exist if CEA-mRNA or CK-mRNA is detected in the blood of colorectal cancer patients.
Xu et al.  reported the positive rate of CEA, CK20, and CK19-mRNA by 35.8, 28.3, and 41.9 % among 168 colorectal cancer patients. Iinuma et al.  examined CEA-mRNA and CK20-mRNA in the peripheral blood and the blood of tumor drainage area among 167 colorectal cancer patients: the positive rate of CEA-mRNA and/or CK20-mRNA in the peripheral blood was 10.2 % (17/167), while that in the blood of tumor drainage area was 34.1 % (57/167). CEA-mRNA and CK20-mRNA expressions are obviously correlated with the depth of tumor infiltration, vein encroachment, lymphatic metastasis, hepatic metastasis, and other clinical pathogenesis by stages. Also, both the survival time free from diseases and the total lifetime of positive patients are lower than those of negative patients. Moreover, CEA-mRNA and CK20-mRNA expressions in the blood of tumor drainage area belong to independent prognosis factors. Other scholars  also concluded similar findings. Also, some scholars  conducted CEA and CEA-mRNA monitoring on the patients after the resection of hepatic metastasis per month. As compared with CEA, CEA-mRNA can more sensitively prompt the postoperative relapse and metastasis at the earlier stage.
However, this method has higher false negative and false positive rate. The reasons for false negative may consist in the following: (1) Tumor cells are featured with heterogeneity and the expression of the same target gene may differ on different patients or different tumor cells of the same patient, thus resulting in low or no expression in some samples. Therefore, the combined examination with several target genes will be helpful to increase the detectable rate; (2) exfoliation or dissemination of tumor cells is intermittent, so single blood sampling is hard to find it out. If the condition permits, more blood sampling can be taken. The reasons for false positive may consist in (1) the increase of pseudo genes in hemocyte, (2) DNA contamination in the process of RNA extraction or the increase of DNA pieces due to chemotherapy, (3) CEA and CK expression of some regenerated infant cells or preexisting variant cells and low expression of non-epithelia, (4) cross contamination among samples and the contamination of skin epithelia when drawing blood, etc., and (5) other existing subclinical malignant tumors . In addition, similar to CEA in the peripheral blood serum, CEA-mRNA and CK-mRNA examinations are not featured with the specificity of hepatic metastasis, so it is more suitable for risk evaluation and prompt of hepatic metastasis rather than diagnoses.
6.1.3 C-met Gene and Hepatic Growth Factor
C-met is the gene to encode hepatic growth factor receptor (HGFR). As a kind of cross membrane receptor protein tyrosine kinase, c-met mainly exists in the epithelial tissue. The ligand of HGFR is the hepatic growth factor/scatter factor (HGF/SF), but the combination of HGFR and HGF/SF can lead to the phosphorylation of HGFR, thus activating tyrosine kinase and tyrosine phosphorylation of many substrate proteins, and then the biological effect comes into being. In addition, excessive expression of c-met genes in many malignant tumors plays a vital role in the occurrence, growth, angiopoiesis, and metastasis of tumors. As a kind of multifunctional cell factor, HGF can induce epithelia and many tumor cells to split, move, and invade as well as develop angiogenesis. High expression of c-met and HGF in tumor tissue can form positive feedback, thus leading to indeterminate growth and invasion of tumors [12, 13].
Di Renzo et al.  discovered that excessive expression of c-met protein, 5–50 times the normal level, exists in 50 % primary colorectal cancer and 70 % hepatic metastasis. This phenomenon occurs at each stage of colorectal cancer, so it may stimulate the metastasis thereof. Xu Jianmin et al. in China  also reported that the expression of c-met protein in primary tumor and synchronous hepatic metastasis is more than that in primary tumor with no hepatic metastasis (P < 0.05); but the expression of c-met protein in metachronous hepatic metastasis is not at odds with that in primary tumor with no hepatic metastasis or synchronous hepatic metastasis. Lately, Zeng et al.  made use of the quantitative PCR/joining enzyme reaction technology to directly examine c-met gene copy number in the tissue sample. This study collected 247 pairs of primary colorectal cancer/normal intestinal mucosa and 147 pairs of hepatic metastasis/normal hepatic tissue. The c-met gene copy number in normal intestinal mucosa and hepatic tissue is similar, while that in primary tumor is apparently higher than that in normal tunica mucosa and the c-met gene copy number in hepatic metastasis is apparently higher than that in normal hepatic tissue (P < 0.001). Moreover, the c-met gene copy number in hepatic metastasis is apparently higher than that in primary tumor (P < 0.0001). The rate of c-met gene amplification in the patients with local tumor is only 2 % (3/177), while that in the patients with other distant metastases and the patients with hepatic metastasis, respectively, reaches 9 % (6/70, P < 0.02) and 18 % (25/147, P < 0.01). After the operative treatment for hepatic metastasis, the patients with c-met gene amplification may have a shorter 3-year survival rate (P = 0.07).
Shi Weijian et al. in China  reported 52 cases of colorectal cancer, whose serum HGF was apparently higher than that of healthy persons. Higher HGF value was closely related to later clinical stadium. Seventeen cases with hepatic metastasis had higher HGF than those with no hepatic metastasis. Yoon et al.  kept monitoring plasma HGF of 26 cases with hepatic metastasis, whose preoperative HGF was apparently higher than healthy persons. Three days after the resection, plasma HGF began to rise. Three months later, plasma HGF recovered to the preoperative level. During the 19-month median follow-up survey, ten patients suffered relapse, which might be associated with higher preoperative HGF (P = 0.021).
The positive feedback between c-met gene products and HGF doesn’t belong to the specificity of colorectal cancer or hepatic metastasis, which widely exists in a great variety of tumors.
6.1.4 Matrix Metalloproteinase (MMP) and Tissue Inhibitors of Metalloproteinases (TIMP)
Matrix metalloproteinase (MMP) is a series of hydrolytic enzyme that is featured with high autopolyploid and can degrade extracellular matrix, but such metal ions as calcium and zinc are needed as the accessory factor and that is what its name stems from; MMP can degrade various proteins in extracellular matrix and destroy tumor cells, so it is deemed as the protective screen against tumor cell invasion. MMP, as the main proteolytic enzyme in the process, plays a critical role against tumor cell invasion and metastasis. Extracellular matrix is mainly composed of basilar membrane and interstitial substance; the former is made of membrana basilaris collagen, i.e., type IV collagen, which is the protective screen against tumor cell invasion and metastasis. In view of type IV collagenase (MMP-2 and MMP-9) that can degrade this protective screen, there are quite a lot of research resources available. Tissue inhibitors of metalloproteinases (TIMP) are the main repressors of MMP, so the degree of degradation as well as tumor cell invasion and metastasis depends on the dynamic equilibrium of MMP and TIMP .
Currently, most of studies aim at primary tumor or metastasis, for example, German scholars , reported that the expression of MMP-1, MMP-2, MMP-3, and MMP-12 in hepatic metastasis was apparently lower than those in primary colorectal cancer; also, the tumors with high expression of MMP-7, TIMP-1, and TIMP-2 were less sensitive to 5-FU chemotherapy, while the tumors with high expression of MMP-2, MMP-9, MMP-11, and MMP-14 were quite sensitive to chemotherapy.
However, the studies on MMP and TIMP in blood are relatively less. Waas et al.  reported the examination results on MMP-2, MMP-9, and TIMP-1 in the plasma of 57 patients with hepatic metastasis, 94 patients with no hepatic metastasis, and 51 healthy persons. The level of MMP-2 in the patients with hepatic metastasis was lower than that of the healthy persons, while the TIMP-1 thereof was higher than the healthy persons (P < 0.001); the difference between the groups with or without hepatic metastasis was not obvious at all; after the resection of hepatic metastasis, MMP-2 still remained at the lower level, which might be related to relapse. Actually, the diagnoses and prognosis value of MMP-2, MMP-9, and TIMP-1 are inferior to CEA, so they are not ideal markers. Ishida et al.  examined the level of MMP-9 in the peripheral and portal vein plasma of the patients with colorectal cancer and discovered that the level of MMP-9 in peripheral blood was irrelevant to various clinical pathogeneses; on the contrary, the level of MMP-9 in portal vein blood was associated with tumor pathogenesis type, Dukes stages, hepatic metastasis, and lymphatic metastasis. The level of MMP-9 in the portal vein blood of the patients with hepatic metastasis was apparently higher than that with no hepatic metastasis (P < 0.01). Moreover, the ratio of MMP-9 in portal vein blood to peripheral blood was meaningful to anticipate the relapse of hepatic metastasis. The higher the ratio, the higher the probability of relapse would be. If the critical ratio was set at 1.6, the ratios of sensitivity, specificity, and accuracy can, respectively, reach 77.8 %, 81.3 %, and 80.8 %.
Theoretically speaking, the change of dynamic equilibrium between MMP and TIMP, higher MMP and lower TIMP, is essential to the successful tumor cell invasion and metastasis, but this phenomenon cannot be verified in quite a number of studies on hepatic metastasis from colorectal cancer. Some findings are even the opposite. This may be associated with the complexity of MMP-TIMP system, the selection of samples, sampling time and caseload, etc., so further studies shall be made available.
6.1.5 Vascular Endothelial Growth Factor (VEGF)
Vascular endothelial growth factor (VEGF) is a kind of heparin-binding growth factor related to the specificity of vascular endothelial cell, which can stimulate the splitting, reproduction, and movement of endothelial cells and enhance vascular permeability, thus helping the growth of vasculogenesis. So, VEGF plays a vital role in the growth of angiogenesis.
Alabi et al. reported that higher level of VEGF-A in preoperative serum was related to relapse after the operation of colorectal cancer ; VEGF-C was quite lower in the patients with distant metastasis . Ding Wei et al.  examined the level of VEGF in peripheral and portal vein blood of 101 cases with colorectal cancer and discovered that the level of VEGF was apparently higher than that of the healthy persons; the level of VEGF in portal vein blood with synchronous hepatic metastasis was apparently higher than that with no hepatic metastasis; the level of VEGF in portal vein blood on all the patients with hepatic metastasis was higher than 250 μg/L, and the proportion of hepatic metastasis on the patients with VEGF higher than 250 μg/L was 67.6 %. Also, sensitivity was 100 % and specificity was quite higher. But some scholars  propose objection, the level of preoperative VEGF on 18 cases with postoperative relapse, 21 cases with hepatic metastasis, and 40 cases with the survival time over 5 years free from diseases, and the level of VEGF on 28 healthy persons was divided into four sets. No significant difference was found on any two sets.
E-cadherin (E-cad) is the hypotype of transmembrane protein in the cadherin protein molecular family. The expression of E-cad is on the epithelial surface of normal large intestinal mucosae, and the major functions thereof are to mediate adherence among epithelia and maintain normal morphological structure of tissue. The hepatic metastasis from colorectal cancer originates from the breakdown of complete epithelium, and then tumor cells pass through epithelium, blood, or lymphatic system and finally arrive at the liver. E-cad is essential for the completeness of epithelium, thus preventing the occurrence of metastasis.
Elzagheid et al. conducted tissue-related research  and discovered that the expression of E-cad on the cell membrane of primary tumor was apparently higher than that of metastasis; the patients with metastasis had higher E-cad and the expression of E-cad in hepatic metastasis was higher than other metastases. Moreover, higher E-cad in the cytoplasm of primary tumor was associated with shorter DFS. Upon a small sample-based research on the soluble E-cad in serum , the soluble E-cad in serum in the patients with colorectal cancer and benign diseases was higher than healthy persons; the concentration thereof was related to T stages rather than N and M stages. Among the patients with hepatic metastasis, the concentration of E-cad was related to the level of serum CEA. Currently, there is no report on large sample-based research on soluble E-cad.