Despite several advances in oncological management of colorectal cancer, morbidity and mortality are still high and devastating. The diagnostic evaluation by endoscopy is cumbersome, which is uncomfortable to many. Because of the intra- and inter-tumour heterogeneity and changing tumour dynamics, which is continuous in nature, the diagnostic biopsy and assessment of the pathological sample are difficult and also not adequate. Late manifestation of the disease and delayed diagnosis may lead to relapse or metastases. One of the keys to improving the outcome is early detection of cancer, ease of technology to detect with uniformity, and its therapeutic implications, which are yet to come. "Liquid biopsy" is currently the most recent area of interest in oncology, which may provide important tools regarding the characterization of the primary tumour and its metastasis as cancer cells shed into the bloodstream even at the early stages of the disease. By using this approach, clinicians may be able to find out information about the tumour at a given time. Any of the following three types of sampling of biological material can be used in the "liquid biopsy". These are circulating tumour cells (CTCs), circulating tumour DNA, and exosomes. The most commonly studied amongst the three is CTCs. CTCs with their different applications and prognostic value has been found useful in colorectal cancer detection and therapeutics. In this review, we will discuss various markers for CTCs, the core tools/techniques for detection, and also important findings of clinical studies in colorectal cancer and its clinical implications.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, which stands second and third in women and men, respectively, across the globe with more than 1.2 × 10⁶ new cases and 608700 mortalities annually[1]. It develops due to genetic and epigenetic alterations in human genome and environmental factors. Mode of presentation of CRC can be inherited, familial, and sporadic. Inherited CRC accounts for 5%-10% of all cases, for example, Lynch syndrome, familial adenomatous polyposis, and Peutz-Jeghers syndrome. Among all the CRCs, familial CRC accounts for 20%-30% and sporadic cases approximately 70% of all CRCs which are associated with somatic mutations[2]. There are many invasive and non-invasive diagnostic and prognostic tools with varying sensitivity and specificity, and each has its limitation. There is a need for new tools which may be simpler, non-invasive, cheaper, reproducible, and easily available with high sensitivity and specificity. "Liquid biopsy" is currently the most recent area of interest in oncology, which may provide important tools regarding the characterization of the primary tumour as well as metastasis because tumour cells shed into the bloodstream at the early stages of the disease. In "Liquid biopsy", one of the three types of sampling of biological material can be used, which are-circulating tumour cells (CTCs), circulating tumour DNA, and exosomes. CTCs are one of the main components of liquid biopsy, where subsets of tumour cells can disseminate from the primary tumour and intravasate to the circulatory system. CTCs are non-invasive and safe in comparison to traditional tissue biopsy, and can be used for monitoring of tumour progression and tumour response to therapy in real time. CTCs in peripheral blood serve as a source of valuable tumour markers. The present review will describe the main areas of the ongoing investigation on CTCs with particular emphasis on different tools and techniques used for CTC capturing and analysis, and also currently available data of clinical relevance of CTCs.

A tumour cell contains millions of cells maintaining genetic mutations driving them to grow, divide, and invade the local tissues. Some cells separate from the edges of a tumour and are released into the bloodstream or lymphatic system. These cells are CTCs. CTCs can also be defined as cells spreading into vasculature by a primary tumour and they keep circulating in the bloodstream of cancer patients[3]. It was Ashworth (1869) who reported the CTCs for the first time and described the presence of tumour cells with resemblance to the cells from the primary tumour, in the blood of a patient with metastatic breast carcinoma. Later, in 1955, evidence of the presence of CTCs in the blood of a patient with primary and metastatic carcinoma was found by immunohistochemistry. In 1990, Moss and Sanders in their study found evidence for CTCs in seven out of ten disseminated neuroblastoma patients by immunostaining. In CRC, CTCs were first reported in 1993 with the help of conventional cytology and cytokeratin staining. Tumour cells were isolated from 42 patients who underwent resection with the help of density gradient centrifugation, immune histological evidence for CTCs was reported in 4 out of 42 patients. Above mentioned studies have showed that tumour cells could be detected by traditional immunochemistry techniques; however, their results were based on small sample size and single-center studies.

Some studies have also reported CTC circulation in the body fluids before metastasizing to other parts of the body even in the early stages of the disease[4,5]. Wang et al[6] analysed the prognostic role of CTCs, highlighting the importance of CTC count before and after chemotherapy. They found that the presence of CTCs during chemotherapy is an unfavorable but independent factor and may play a role in deciding overall survival (OS) and survival without disease progression [progression free survival (PFS)] in advanced CRC cases. From this study, it was clear that CTCs in peripheral blood can be used as useful tumour markers. Characterization and early detection of CTCs have been reported to play an important role as a prognostic and predictive factor in different types of solid tumours[7,8]. Many epithelial cancers, including breast, prostate and lung cancers, have also been found to be associated with CTCs[9,10].

Early diagnosis, prediction of prognosis, assessment of recurrent risk, individualized treatment, and treatment with curative intent have focused research in the field of CTCs[11]. CTCs have faced difficulties for years because of their very low number (1–10 cells per 10 mL of blood) in many studies, and they have a short half-life which ranges from 1 to 2.4 h in blood[12,13], hence posing difficulty in further study. Their detection, quantification, and characterization of molecular features are also difficult. At present, there are several limitations to available CTC isolation techniques. Moreover, only a very small number of CTCs possess metastatic property[14]. Hence, it is very important to characterize them exactly so as to differentiate the non-metastatic CTCs from metastatic ones. There are several techniques which are described here for isolation and detection of CTCs effectively.

The high sensitivity and specificity of CTC detection methods have a great effect in improving patient outcomes. Politaki et al[28] have compared CTC detection rates and prognostic significance in breast cancer patients by comparing three commonly used methods including CellSearch, qRT-PCR, and double immunofluorescence (IF) microscopy. They analyzed early diagnosed (n = 200) and metastatic (n = 164) breast cancer patients before the start of adjuvant or first-line chemotherapy. They compared CellSearch system, qRT-PCR for CK19 mRNA detection, and double IF microscopy by using A45-B/B3 and CD45 antibodies and concluded that patients were more likely to be CTC-positive using the CellSearch (37%) than qRT–PCR (37% vs 18.0%, P < 0.001) or IF (37% vs 16.9%, P < 0.001). In another study[29], CellSearch was compared with Adna Test and RT-PCR in breast cancer, and it was found that multimarker qRT-PCR showed a superior sensitivity for the detection of CTCs in metastatic breast cancer patients compared with the CellSearch system and the AdnaTest. There is limitation of the assessment by PCR as it provides the number of target transcripts based on the actual number of CTCs present in a sample[30] and does not allow the morphological assessment of cells. Two cell-based detection assays, the CellSearch and Onco-Quick (for density gradient centrifugation), on comparison revealed that the CellSearch was a far more accurate and sensitive method to detect and enumerate CTCs[31].

There is one study by Gervasoni et al[32], in which they compared the capacity of three methods, multimarker RT-PCR assay, standardized CellSearch method, and dHPLC-based gene mutation analysis, to detect CTCs in the blood of 20 CRC patients (stage I = 5, stage II = 8, stage III = 6, and stage IV = 1). They found CTC positivity in 75% of samples by RT-PCR, 20% by CellSearch method, and only 14.3% of samples were found to be gene mutated with the presence of CTCs by HPLC method. These results show that out of these three methods tested, multimarker RT-PCR assay provides the maximum probability of CTC detection. Future studies, by using the above three distinct methods for follow-up, may provide more information about the prognostic significance of CTCs detected through single method assay vs combination of different assays[32].

CTC characterization and number may be useful in several ways where they can be used both as a prognostic marker for survival as well as prediction of response to cancer treatment[33]. A multivariate analysis[34] demonstrated that CTC count is the strongest prognostic biomarker for patient survival. If the CTC number increases or remains static, the treatment can be deemed to be ineffective, whereas, if CTC number decreases, the treatment may be effective. Several studies have shown that the presence of as few as 3 to 5 CTCs in 7.5 mL of blood is associated with poor PFS and OS rates[35]. Studies with the CellSearch system and others have shown that high numbers of CTCs are associated with lower DS and OS rates[36]. In a study of 413 metastatic CRC patients being treated with first, second, or third-line therapy, patients with a baseline CTC number of more than 3/7.5 mL had significantly poor median PFS (4.4 mo vs 7.8 mo, P = 0.004) and OS (9.4 mo vs 20.6 mo, P < 0.0001) compared with patients with less than 3 CTCs/7.5 mL[37,38]. CTC evaluation, during treatment, may be used as a prognostic predictive marker to determine progression-free survival (PFS) and OS. The CellSearch system has its own limitation; the method of isolation utilizes EpCAM expression on the cell surface of the tumour, which is expressed in 75% of cancer types. A study by Fang et al[39] (2016) analyzed the expression of cell surface markers CD133, CD54, and CD44 with the help of flow cytometry to analyze the correlation between cellular subpopulations and colorectal liver metastasis. They observed that the expression of cellular subpopulations (CD133+, CD54+, and CD44+) was higher in the peripheral blood of CRC liver metastasis in comparison with those with no metastasis (P < 0.001). In a study by Lalmahomed et al[40] (2015) on peripheral blood of 151 CRC patients who underwent liver metastasectomy, CTCs were detected by the CellSearch system after a density-gradient-based enrichment step. They found that CTCs were detected in 75 samples (43%), out of which 16% had 3 CTCs/7.5 mL of blood. Patients with or without detectable CTCs have an almost similar 1-year recurrence rate (47% vs 48%, respectively). A similar recurrence rate was also reported with low vs high CTC count (< 3 or 3 CTCs/7.5 mL of blood: 50% vs 47%, respectively). In their report, no difference was found in disease-free survival and OS among patients with or without CTCs. A report by Shimada et al[41] (2012) found that detecting CEA/CK/CD133 mRNA in tumour drainage blood (RT-PCR method) could act as a prognostic marker in patients with Duke's stages B and C CRC. The findings of the CTC isolation techniques and their clinical significance have been given in detail in Table 2. Hendricks et al[42] (2020) used qRT-PCR for indirect CTC detection, which was already applied in previous studies on CRC patients and found to have prognostic value. An earlier study by Sastre et al[43] (2008) reported that the CellSearch system could identify CTCs in CRC patients and that CTC positive cases were correlated with the stage of the disease (P = 0.005) but there was no significant correlation between CEA levels, tumour locations, grade of differentiation, and lactate dehydrogenase (LDH) levels. A meta-analysis by Katsuno et al[44] (2008) of a total of nine studies found that CTC-positive patients (in blood samples by RT-PCR), correlated with lymph node (LN)-positive patients (50%) vs LN-negative patients (21%).

Guadagni et al[45] (2020) have published a couple of studies about the role of CTC based therapeutic decision making in CRC. In the first study[45], they included 62 patients with advanced unresectable rectal cancer and reported that where the patients were selected for the treatment based on CTCs (HPP/target-therapy group, n = 43); the disease control rate was significantly higher (PFS = 8 mo, OS = 20 mo) as compared to those given systemic chemotherapy (n = 19) based on age, co-morbidity, and performance status (PFS = 4 mo, OS = 8 mo). The second study[46] was performed on 106 advanced unresectable CRC patients. The therapy was decided based on CTCs (HAI/targeted, n = 44), age, and co-morbidity performance status (systemic chemotherapy, n = 62). The authors found that the group where treatment was given based on CTCs had longer PFS and median survival (MS) (PFS = 5 mo, MS = 14 mo) as compared to those given therapy based on age and co-morbidity performance status (PFS = 3 mo, MS = 8.5). Finally, they concluded that CTCs can be used to choose therapeutic options in unresectable CRC.

Inherited or acquired resistance in response to specific treatment can be assessed with CTCs which may also work as pharmacodynamic markers. CTCs have enhanced our knowledge and understanding about the primary mechanisms of cancer metastasis. This understanding may be useful in therapeutic manipulation with the help of new targets. CTCs were evaluated in phase I trial based on their count and the expression of insulin-like growth factor-1 receptor (IGF-1R) to find out their therapeutic applications. The CellSearch system was used, either alone or in combination with docetaxel, to count CTCs in patients treated with monoclonal antibodies against IGF-1R. Positive IGF-1R and CTC response was seen in 23 out of 26 patients. These patients responded better in case of combined treatment than in case of the remaining three patients who were negative for IGF-1R. From these findings, it was concluded that CTCs can be used as a potential marker for the selection of chemotherapy[47].

CTC interpretation is quite promising but has limitations such as factors like requirement of large volume of blood, small size of the cancer patient population, and the standard value for comparison (i.e., CellSearch, blood sample, other micro-devices, etc.). Till now, many reports have enlightened the prospects for cancer patient monitoring, and for few years researchers have focused on CTCs to explore their biological metastatic property and role in cancer treatment monitoring. Among the several important clinical applications for CTC technology is the correlation of CTC count with OS and PFS as a measure of clinical outcome.

The presence of CTCs in the blood sample is also a major challenge. If they are present, their heterogeneity of unknown extent is also present. Because of this nature, it demands an ongoing diversity in the detection and characterization of CTCs using the present available and upcoming methods in the future.

CTCs have become a hot pursuit and in recent years many new CTC detection technologies have emerged. Discoveries of these technologies from laboratory to clinical practice are non-trivial. Only a few systems are available for routine use in the clinical setting, but not freely available. CTC detection is challenging because of the small number of circulating cells but has been found both in metastatic and non-metastatic cancer (Table 3). It has been well correlated with the stage of the disease, prognosis, and survival but has a limited role in therapeutic decision-making. There is a need for the development of newer, cheaper techniques of CTC detection which can be used as an alternative to invasive diagnosis and treatment monitoring. Future research is required as the current literature has limited information on its use in routine clinical practice but the future is promising.