**5. Traditional and novel tumour markers in the diagnostics of colorectal cancer**

The patients with metastatic colorectal cancer nowadays survive longer, thus they need prolonged follow-up. CT is a sensitive method but some authors have expressed fears that the patient is subjected to radiation exposure [41]. MRI benefits from high sensitivity and lack of ionising radiation, but it is expensive. Blood test for surveillance thus seems to be an attractive, patient-friendly and radiation-free option. Although the follow-up of colorectal cancer patients after resection of the primary tumour is controversial, increased blood level of the carcinoem‐ bryonic antigen (CEA) can disclose cancer recurrence and is used traditionally. In a recent study, 25% increase of CEA level in comparison with the previous value detected 23% of recurrences while 46% of recurrences were evident both by radiology and CEA and 31%—only by radiology data. The radiologic imaging in this study comprised US after surgical treatment and CT after thermal ablation as well as in difficult cases. The resectability of the recurrent cancer did not differ in patients who were identified through CEA or by imaging [41]. Thus, CEA alone is not sensitive enough to identify the recurrence but can be helpful in complex diagnostic protocol. In contrast, CEA alone did not identify any additional case of curable recurrence after liver resection for metastatic colorectal cancer in comparison with CT [42].

CEA has several benefits, including cheapness and availability. In addition, prognostic information can be obtained. High perioperative CEA levels indicate worse survival after liver resection for CRC metastases [43].

CEA has been explored in association with other biological markers both for comparison and in order to create wider diagnostic protocol. Regarding circulating tumour cells, the findings along with CEA level added prognostic information in patients with metastatic colorectal cancer undergoing chemotherapy. In a multivariate analysis, circulating tumour cells but not CEA at the baseline predicted the survival, but both parameters predicted survival at 6–12 weeks after the initiation of treatment. There was no correlation between CEA and circulating tumour cells [10]. The levels of circulating tumour cells in colorectal cancer are reported to be lower than in other cancers due to homing within the liver [44]. The complex mechanism of metastasis involving epithelial–mesenchymal and mesenchymal–epithelial transformation as well as blood clearing in the liver and secondary spread from liver metastasis to systemic circulation hypothetically can influence the results and interpretation of circulating tumour cell tests.

Plasma levels of the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) have also been explored in parallel with CEA in patients undergoing chemotherapy for metastatic colorectal cancer. High plasma TIMP-1 and CEA levels both before and during treatment were related to poor response. Worse survival was predicted by high TIMP-1 level before or during chemotherapy, and by high CEA values before treatment [45]. However, chemotherapy and radiation treatment itself influenced serum levels of these markers, decreasing CEA and increasing TIMP-1 [46]. The treatment-induced switches in the biomarker levels would limit their application in the surveillance.

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally modulate the expression of the target genes [47, 48]. These endogenous molecules are evolutionarily highly conserved, suggesting an important functional role in cell biology [48]. MiRNAs are located either between protein-coding genes, or in the introns of protein-coding genes. Transcription of miRNAs results in primary miRNAs that undergo processing within the nucleus. The processing yields miRNA precursors that are transported to the cytoplasm and transformed into mature miRNAs. These molecules perform their regulatory function by complementary binding to mRNA [11]. miRNAs regulate such crucial steps in cancer development (Table 3) as cell proliferation, invasion, angiogenesis, epithelial-mesenchymal transformation and the reverse process [47]. The value of miRNAs is the ability to function as large genomic switches.


**Table 3.** MiRNAs involved in different steps of carcinogenesis

From the practical standpoint, miRNAs at present are explored as diagnostic markers and therapeutic targets [11]. In contrast to mRNA, miRNAs are stable in formalin-fixed, paraplast embedded tissues [48–50]. In the blood and plasma, MiRNA also circulate in persistent form, suitable for testing [51, 52]. The stability might be ensured by development of extracellular microvesicles [52]. The specificity and sensitivity issues still must be finalised, but promising

results have already been reported. Thus, 6 serum miRNA-based biomarker signature, including miR-21, let-7g, miR-31, miR-92a, miR-181b and miR-203, had high sensitivity (93%) and specificity (91%) in the diagnosis of colorectal cancer. The sensitivity of such traditional serum markers as CEA and CA19-9 was significantly lower: 23% and 35%, respectively. The tested panel could discriminate stage I and II colorectal cancer from healthy controls [12], thus showing appropriate sensitivity for low tumour burden. Moreover, miR-92a, miR-21 and miR-29a serum levels could discriminate healthy controls from patients affected by colorectal cancer or advanced adenomas, the well-established precursor lesion of colorectal cancer [53, 54]. The levels of miR-17-3p, miR-92 and miR-221 also differed in plasma of colorectal cancer patients and healthy controls [55, 56].

metastasis involving epithelial–mesenchymal and mesenchymal–epithelial transformation as well as blood clearing in the liver and secondary spread from liver metastasis to systemic circulation hypothetically can influence the results and interpretation of circulating tumour

Plasma levels of the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) have also been explored in parallel with CEA in patients undergoing chemotherapy for metastatic colorectal cancer. High plasma TIMP-1 and CEA levels both before and during treatment were related to poor response. Worse survival was predicted by high TIMP-1 level before or during chemotherapy, and by high CEA values before treatment [45]. However, chemotherapy and radiation treatment itself influenced serum levels of these markers, decreasing CEA and increasing TIMP-1 [46]. The treatment-induced switches in the biomarker levels would limit

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally modulate the expression of the target genes [47, 48]. These endogenous molecules are evolutionarily highly conserved, suggesting an important functional role in cell biology [48]. MiRNAs are located either between protein-coding genes, or in the introns of protein-coding genes. Transcription of miRNAs results in primary miRNAs that undergo processing within the nucleus. The processing yields miRNA precursors that are transported to the cytoplasm and transformed into mature miRNAs. These molecules perform their regulatory function by complementary binding to mRNA [11]. miRNAs regulate such crucial steps in cancer development (Table 3) as cell proliferation, invasion, angiogenesis, epithelial-mesenchymal transformation and the reverse process [47]. The value of miRNAs is the ability to function as large genomic switches.

Angiogenesis Activation miR-194; miR-17-92; miR-126; miR-210; miR-424 Suppression miR-221; miR-222; miR-497 Invasion Activation miR-31; miR-122; miR-200; miR-145; miR-103; miR-107;

Suppression miR-122; miR-328; miR-143

Colonisation miR-328; miR-103; miR-107

From the practical standpoint, miRNAs at present are explored as diagnostic markers and therapeutic targets [11]. In contrast to mRNA, miRNAs are stable in formalin-fixed, paraplast embedded tissues [48–50]. In the blood and plasma, MiRNA also circulate in persistent form, suitable for testing [51, 52]. The stability might be ensured by development of extracellular microvesicles [52]. The specificity and sensitivity issues still must be finalised, but promising

Loss of cell adhesion miR-126

Immune regulation miR-155; miR-17-92

miR-29a; miR-21; miR-17; miR-19a

cell tests.

their application in the surveillance.

176 Recent Advances in Liver Diseases and Surgery

**Target process Result MicroRNAs**

Metastasis Vascular invasion miR-21

Apoptosis Induction miR-26b

**Table 3.** MiRNAs involved in different steps of carcinogenesis

Early relapse of colorectal cancer is associated with increased plasma levels of miR-29c [48, 57]. More intense surveillance or postoperative treatment could be offered to these patients.

Patients with liver metastasis exhibit significantly higher miR-21 level in colorectal cancer tissues. MiR-29a serum level is increased in colorectal cancer patients affected by liver metastasis and is considered a promising novel marker for early detection of liver metastasis [58]. In more recent studies, increased serum levels of miR-141 and miR-21 as well as downregulation of miR-126 were advised for early diagnosis of liver metastasis of colorectal cancer while let7a up-regulation was associated with extrahepatic metastases [59]. The applicability of this or similar biomarker signature for metastatic cancer remains to be subjected to deeper analysis as at least few controversies can be expected. It has been shown in gastric and hepatocellular carcinoma that serum and tissue levels of miRNAs can change in opposite directions [60–62], possibly because cancer cells can selectively retain certain miRNAs [63]. In colorectal cancer, liver metastasis exhibits higher levels of miR-29c, although miR-29c is significantly down-regulated in primary colorectal cancers giving rise to distant metastasis. The seeming controversy can be explained by epithelial–mesenchymal and mesenchymal– epithelial transition [64]. In addition, surgical treatment can influence the miRNA level; thus, in hepatocellular carcinoma, miR-92a levels are high in tumour tissue, low in plasma before the treatment and high in plasma after the operation [61]. In colorectal cancer with liver metastases, tissue levels of 28 miRNAs were different (Table 4) from nonmetastatic cancers [65]. The tissue miRNA profile hypothetically could also discriminate between colorectal cancer metastases in liver and lymph nodes [66].



**Table 4.** MiRNAs in colorectal cancer1

Prognostic value has been reported regarding has been reported. In colorectal cancer, shorter disease-free interval was found in patients who exhibited higher miR-21 and higher miR-143 levels in tumour tissues. Notably, in this study, higher miR-21 and lower miR-143 was found in cancer and liver metastases in comparison to normal colonic and liver tissues [67]. The seeming logic discrepancy between the prognostic levels and the differences in normal and neoplastic tissues suggests multiple mechanisms of a single miRNA. These findings are warning about high complexity in the elaboration of diagnostic tests. MiRNAs have also been explored to predict the response to treatment. Thus, increased plasma concentrations of miR-106a, miR-484 and miR-130 are associated with lack of response to oxaliplatin-based treatment [48, 70]. Similar markers would be valuable to identify patients that would benefit from preoperative tumour burden reduction by chemotherapy. The predicted nonresponders could be treated by ablation techniques. As miRNAs function as large genomic switches, they are also attractive potential targets of therapy [11].
