Ajit Kumar Saxena^1* 

¹Human Molecular Genetic Laboratory, Department of Pathology/Lab Medicine, All India Institute of Medical Sciences, Patna-801507, India.

*Corresponding Author:Dr.Ajit Kumar Saxena, Ph.D. Professor and Head Department of Pathology/Lab Medicine, All India Institute of Medical Sciences Patna, Phulwarisharif-801507, Bihar, India, Tel: +91-7781012677; Fax: +91-7781012677; E-mail: draksaxena1@rediffmail.com

Citation: Ajit Kumar Saxena (2023)Circulating Tumor Cells - The Future of Medicine as Diagnostic and Therapeutic Biomarker”. Cancer Prog Diagn 7: 136.

Received: March 22, 2023; Accepted: April 17, 2023; Published: April 20, 2023.

Human circulating tumor cells (CTCs) are synonyms of cancer cells, originated from primary tumor sites after activation of oncogenes such as kras and started circulated into the blood stream. These CTCs reach targeted neighbouring tissue or nearby organs, show proliferation and show symptoms of metastasis after invasion. However, identification and characterization of CTCs is challenging task because of very low and heterogeneous population using epithelial mesenchymal transition markers -Sox4, EpCAM and CK 19. Even after technological advancement and use of flowcytometry or RT-qPCR for the study of CTCs required 10-20 ml of blood samples seems to be raise several ethical issues with discordance findings in different types of tumors bearing patient. The author emphasizes the use cell and tissue culture technology and minimal use of sample (0.5ml) from cancer patients, culture for 72 hours under sterile conditions and isolates CTCs by Ficoll’s gradient procedure showing satisfactorily results. The purpose of the CTCs based on cell-culture is to enhance cell population and reduce sample size. The study of CTCs has great clinical relevance as “biological marker” for early diagnosis or before metastasis in breast cancer patients may reduce the incidence of mortality and may help clinicians for better management. However, the biomarkers vary depending upon the cancer. Clinical significance of CTCs is evaluated in breast, lung, ovary, prostate, colorectal, melanoma cancer. Several illustrations had been documented circulating tumor cells may be used as biological marker for monitoring of cancer but still fail for early detection. Hence, a comprehensive review has been written including latest finding of CTCs including clinical significance in diagnostic, prognostic and therapeutic monitoring. Moreover, this review will enhance the knowledge of CTCs and contribute to providing significant knowledge on clinical research for early management.

Circulating Tumor Cell, Cancer Biomarker, Breast, Colorectal, Liver Cancer.

During tumorigenesis, cells undergo epithelial to mesenchymal transition (EMT) and separated from the primary tumor site, travel through the vascular system are called circulating tumor cells (CTCs) (1). The dissemination process of CTCs is still under investigation, however, preliminary studies showed tumor cells migrate via localized invasion of basement membrane during primary tumor progression(2). Further, they intravasate into bloodstream or lymphatic systems (3), during circulation they interact with blood components for survival by competing with immune cells, and shear stress. However, during circulation most of the CTCs died while some of them survive, extravasate, migrate and colonise to form micro metastases followed by angiogenesis and grown into metastatic tumors (4,5). However, during this process only few (> 0.01%) of the CTCs metastasize (6) (Figure 1.).

CTCs intravasate into bloodstream encounter series of hostile environmental changes including immune attacks, shear stress, anoikic, oxidative stress and the lack of cytokines and growth factors (6). During this process only a few of the CTCs survived. CTCs are similar to mesenchymal cells thus, during primary tumorigenesis the EMT process is vital to understand the transition of epithelial cells to mesenchymal cells (7). During the EMT, epithelial cells lose epithelial characteristics such as the expression of EpCAM, keratins, and E-cadherin and upregulate matrix metalloproteinase (MMP) activity, which enables these cells to navigate through the local extracellular matrix (ECM) and enter the microvasculature(8–10). Thus, CTCs can be easily separated from primary tissue, invade the capillaries and possess a significantly improved ability to survive and metastasize. The presence of CTCs in bloods were first reported back in 1869 by Ashworth Thomas Ramsden, revealing the size, shape, and properties similar to the primary tumor (11). Since then, several groups have focused on development of CTCs detection technique and diagnostic potential to assess the tumor heterogeneity (11,12). Several studies reported the presence of CTCs in different cancers including lung, prostate, breast, gall bladder, and ovary (13–15). The CTCs detection, characterization and their potential in diagnostics, prognostics and therapeutics has been evaluated over the past two decades. However, several obstacles are accompanying the isolation, and detection of CTCs. First, CTCs are rarely available in blood stream. Around 1–5 cells are only found in millions of blood cells per 7.5 mL of blood (16,17). Moreover, CTCs cells are heterogenous, therefore, variation in expression of cell surface marker (8,18). Therefore, it is not easy to recognize different types of CTCs by the identical standard (19). Herein, we systematically review CTCs in different cancers, potential as diagnostics, prognostics and therapeutic molecules in different cancers. During this systemic review we used keyword “CTC, CTCs in different cancer, CTCs in diagnosis of cancer, CTCs as prognostic biomarker, CTCs as therapeutic agent” and many more in google search engine, PubMed, Google Scholar to find past and current literature for review. We briefly reviewed CTCs biology, advancement of CTCs in different cancers, and investigated the diagnostic, prognostics and therapeutic potential of CTCs in different cancers. Moreover, the clinical significance of CTCs is extrapolated in terms of significance in monitoring cancer progression. Although the detection of CTCs is a promising technique for precision medicine, notably, there are still many unsolved problems. In this review, we present the existing challenges and offer our own insights into the future development of CTCs.

Circulating Tumor Cells in Cancer

Circulating-Tumor-Cells (CTC) provide a blood biomarker for early carcinogenesis, cancer progression and treatment effectiveness. An increase in CTCs is associated with cancer progression, a CTC decrease with cancer containment or remission (20). The presence of CTC has also been associated with early carcinogenesis and risk of cancer (21). Discovery of CTCs opens new insight in understanding of metastatic cancer and its progression. Several cancers have been reported to disseminate via CTCs including breast, prostate, pancreas, gall bladder, ovary cancer etc. Herein, we briefly describe the recent progress in CTCs based study in different cancers.

Figure 1: Diagrammatic presentation of biogenesis of CTC during course of tumor formation and epithelial mesenchymal transition.

Circulating Tumor Cells in Breast Cancer

In 2020, breast cancer surpassed lung cancer as the most prevalent type of cancer worldwide. The metastatic nature of cancer is of great concern accounting for 90% of cancer-related deaths in breast cancer patients (22). One theory confers that tumor metastasis is triggered by blood-based CTC (23). Cristofanilli et al revealed the prognostic value (PFS and OS) of CTC in metastatic breast cancer patients having cut-off of 5 CTC/7.5 ml of blood using CellSearch system (24). Hence, this study transformed the applicability of CTC in several cancer types. Since, several studies have been conducted on enumeration of CTCs in metastatic breast cancer and correlated with clinical significance (25,26).

A recent study evaluated the clinical efficacy of CTC quantification for the prognostication of patients with metastatic breast cancer in 1944 different patients by the Cell Search method at baseline (before start of new treatment). According to the authors, normalization for CTC levels at baseline in early treatment, the shorter PFS was linked to higher CTC counts between three- and five-weeks providing role in prognosis of breast cancer patients (27). In accordance with the most recent recommendations, hormonal therapeutics and anti-HER-2 treatments are provided for breast cancer patients based on the underlying tumor's hormones (ER/PR expression) and HER-2 status. The hormone receptor and HER-2 status in CTC, particularly during disease recurrence or progression in breast cancer patients, can differ from that in primary tumors and even change over time, according to a growing body of research (28,29). Accordingly, molecular characterization of CTCs hormone receptor and HER-2 status could be used as optional personalized treatment and have potential for clinical use (30,31). In the metastatic cascade, the mechanism of epithelial-mesenchymal transition (EMT) is crucial (32,33). CTC molecular evaluation highlights the significance of EMT, which is essential for enabling malignancies to penetrate and thrive at remote sites from the tumor origin. The evaluation of EMT-inducing TFs expression profiles has been assessed in CTC patients with primary breast carcinoma. (34).

Similarly, CTCs expressing stemness markers have significant effects on treatment resistance. Patients with metastatic and early breast cancer were shown to have a high percentage of CTCs that exhibit EMT and tumor stem cell features as well as CTCs that express TWIST and vimentin (35,36). The existence of a subpopulation of CTCs with putative stem cell progenitor phenotypes in patients with metastatic breast cancer has been shown by using triple-marker immunofluorescence microscopy (37). Currently used detection methods for CTC are not efficient to identify this subtype of CTC which underwent EMT (38). Moreover, studies on the molecular characterization of CTCs have revealed that CTCs even within the same patient are heterogeneous. In non-metastatic breast cancer patients, the expression of estrogen, progesterone and epidermal growth factor receptor (EGFR) by immunofluorescence experiments revealed heterogeneous expression of these hormonal receptors in samples from the same patients.

Recently, Jin, et al. reported CTCs could be used as marker for diagnosis and staging of breast cancer patients with the diagnostic value of 86% with 95.4% specificity and 76.56% of sensitivity (39). Similarly, Zhou et al reported the 81% of preoperative patient’s blood samples were CTCs marker positive, while, after surgery around 67.1% patient’s turnout to have negative status with respect to CTCs marker in blood and could aid in therapeutic and disease progression (40). Recent study by Pang et revealed prognostic value of CTC treated with pre-adjuvant chemotherapy and post adjuvant chemotherapy breast cancer patients undergoing surgery. The count of CTCs was having significant correlation with progression free survival and overall survival of patients (14). Similarly, Krol et al, revealed presence of CTC cluster in early breast cancer are possible risk factors for progression of disease (41). In another recent study, Carlotta et al, revealed CTC positive patients have poor overall survival compared to CTC negative patients. Moreover, NGS sequencing of CTCs showed high genomic instability and mutation (42). Recent, clinical trial-III (NCT01619111) revealed that CTC count could serve as biomarker for metastatic breast cancer. The recent development and clinical significance of CTCs in breast cancer are tabulated in table 1.

Table 1: Clinical Significance of CTCs Detection in Breast Cancer Patients.

Circulating Tumor Cells in Prostate Cancer

The FDA has approved CTC enumeration for use in monitoring advanced disease in prostate cancer after it was thoroughly examined and verified as a prognostic tool. We searched published articles Google Scholar, PubMed, and Science Direct with specific keywords (CTCs in prostate cancer, CTCs in prostate cancer therapeutics, CTC as biomarker of prostate cancer), in return more than 150 studies were observed. The FDA has approved CTC enumeration utilizing the Veridex CellSearch TM system for estimation of burden of tumor cell spread in baseline and post therapeutic patients advance stage prostate cancer patients. This test is being used as standard clinical practice to predict the survival, and effectiveness of treatment (51).

In addition, the innovative anti-androgen treatments abiraterone acetate and MDV3100 are currently being investigated in major phase III trials to determine the clinical value of tracking CTC changes with treatment as an efficacy-response surrogate biomarker of survival (52). It is possible to identify and define molecular factors in CTC as predictive biomarkers of tumor sensitivity to a treatment approach (53). Latest studies have gone into greater depth on the major CTC research in metastatic and localised prostate cancer, showing the significant benefits as well as the difficulties presented by diverse techniques, especially potential implications for improving prostate cancer care (30).

In 2001, Moreno, et al. were the first to reveal the blood CTC level of patients with advanced prostate cancer and correlated the CTC levels with disease development with no diurnal variation over time (54).Later, in 2007, Danila and colleagues revealed that CTC count before therapy offers specific information on prognosis as well as the release of cells into the bloodstream is a characteristic of the  tumor that is distinct from the severity of the disease (55). In 2008, de Bono and colleagues reported CellSearchTM system based enumeration of CTC in patients with metastatic castration- resistant prostate cancer (CRPC) has prognostic and predictive value and could be used as independent predictor of OS in CRPC, enabling the FDA to approval for the assessment of CRPC (56).CTC counts are able to track the status of the disease and, when examined as a continuous variable, can determine OS and provide independent prognostic information of disease development (57). Resel et al, examined the relationships among CTC count and PSA level, Gleason scores, and TNM stages of patients with malignant hormone-sensitive prostate cancer revealed the CTC from blood could accurately stage the prostate cancer and have prognostic value assessment of metastatic hormone-sensitive prostate cancer (58)

Combining CTC and PSA kinetics may provide knowledge on management and diagnosis of advanced prostate cancer (59,60). Recently, detection of CTCs in early prostate cancer and may be utilized as a novel surrogate marker for systemic and local treatment (61). Another study revealed the presence of CTCs in early stage of prostate cancer could be used as surrogate biomarker, moreover, reduction of CTC number in post treatment could be indicator of therapy response (62). Prostate cancer development is dependent upon the androgen receptor (AR) signaling. The enhanced expression of splice variant of androgen receptor helps in disease progression and is induced by androgen ablation therapy. Intervention of Taxane based CRPC therapy gold standard, a recent finding suggests the relationship of spliced androgen receptors (ARv567 and Arv7) with microtubules and dynein motor protein resulting in differential sensitivity toward taxane in cell line as well as in vivo system (63). In addition, discovery of ligand mediated AR signaling in CRPC enhances the use of abiraterone acetate (AA), an androgen biosynthesis inhibitor, which increases the patient’s survival rate. Miyamoto et al, demonstrated that assessment of AR signaling in blood derived CTC may aid to guide therapy in metastatic prostate cancer (64,65). In recent study by Ried et al, revealed diagnostic value of CTC in detection of prostate cancer by combining prostate specific marker. The blood based ISET® CTC assay showed 99% of positive predictive value and 97% of negative predictive value in diagnosis of prostate cancer (66). In another study, Yang et al reported telomerase-based CTC detection (TBCD) method of prostate cancer. The diagnostic value based on ROC analysis was 78.9% (67). The recent development in field CTCs based clinical application in prostate cancer is depicted in table 2.

Table2: Clinical Significance of CTCs in Diagnosis and Prognosis of Prostate Cancer.

Circulating Tumor Cells in Lung Cancer

According to the World health organization (WHO)-2020 report, around 2.21 million lung cancer cases were reported worldwide. Online data curation on NIH official website, and other online resources (Google Scholar, Science Direct, PubMed, and Research Gate) showed 350 studies based on CTC in Lung cancer when we used keyword “CTC in Lung Cancer, CTC based diagnosis of Lung cancer, Prognostic and therapeutic role of CTC in lung cancer”. In lung cancer, CTCs frequently display non-epithelial features. Moreover, the majority of detection techniques rely on epithelial markers to identify the CTCs and loss of these markers during EMT in some metastatic tumors can make these techniques useless. Hence, CTC detection in lung cancer has proven a challenging task. There are no established biomarkers for predicting therapeutic efficacy in non-small-cell lung cancer (NSCLC). Zhu et al revealed the EpCAM/MUC1 positive CTC patients have significantly lower DFS and OS in pre-surgery and post-surgery patients (68). Recently, existence of hybrid CTCs (epithelial/mesenchymal) in NSCLC patients was discovered using EpCAM independent blood filtration system (isolation by size of epithelial tumor cells (ISET) and immunofluorescence). Moreover, further characterization can provide significant insight on role of CTCs in EMT and metastases process in NSCLC patients (69). Similarly, another study revealed the prognostic value of CTCs and could tell us about the early therapeutic relevance in NSCLC patients (Stage III-IV) (70). Hou, et al. revealed the clinical and molecular significance of CTC and clustered CTC in SCLC patients. The variation in baseline CTC count of prechemotherapy and post chemotherapy could be used as prognostic factor in SCLC patients (71). In another study by Haber et al showed the EGFR mutation in CTCs from lung have progressive disease than the non-mutated EGFR CTCs in lung cancer patients (72). In another study by, et al. revealed diagnostic potential of CTCs in cohort of 302 lung cancer patients (73). Li, et al. reported the diagnostic value of CTC in detection of lung cancer with AUC of 0.975, sensitivity of 87.5% and specificity of 95.7% (74). Recent study evaluated clinical significance of CTC in prostate cancer are listed in table 3.

Table 3: Study involving CTCs for Evaluation of Diagnostic and Prognostic value in Lung Cancer.

Circulating Tumor Cells in Colorectal Cancer

Enumeration of CTC by the CellSearch system are promising indicators for the therapy and have a substantial prognostic impact on CRC. The identification of CTCs in localized CRC may assist in determining patients who are at risk for metastasis and categorize patients for adjuvant therapeutic treatment. The counting of CTCs in advanced metastatic CRC can assist risk evaluation, overall therapeutic monitoring, and identification of therapeutic resistance.

A contextual trial has convincingly demonstrated the predictive significance of CTC in patients with colorectal liver metastases or widely disseminated metastatic CRC (92). Several studies reported the importance of CTCs in postoperative blood samples of CRC patients. In preoperative patients the enumeration of CTC acts as an independent prognostic factor indicating recurrence of cancer (93). In a retrospective study, the CellSearch technology was used to evaluate the CTC in 430 metastatic colorectal cancer patients at baseline and after beginning first-, second-, or third-line therapy. This analysis revealed that PFS and OS in patients having mCRC were independently predicted by the CTC count before and during treatment leading to the FDA approval of CellSearch assay for mCRC (94). Furthermore, it was demonstrated that evaluating CTC count before and during course of treatment could reliably reflects PFS and OS in late-stage CRC patients undergone chemotherapy plus targeted drugs, moreover, they add on more insight to CT imaging (95). Further research demonstrated the clinical efficacy of CTC count in enhancing the clinician's capacity to precisely assess the outcomes of oxaliplatin-based therapeutic treatment and in accelerating the identification of efficient treatment regimens for specific patients (96).

Similarly, another study on patients undergoing chemotherapy revealed a significant relationship between both the CTCs count and the advancement of the cancer as seen on radiographs (97). Barbazan, et al. evaluated the therapeutic usefulness of six CTC markers (tissue specific and EMT transcripts) as predictive and prognostic markers in patients with mCRC. Patients who were therapy-refractory but were missed by conventional imaging approaches were found using CTC-markers. Patients identified by computerized tomography as respondents but with elevated CTC markers throughout therapy had considerably shorter life durations (98).

Compared to mCRC, the predictive value of CTC in non-metastatic CRC is less evident. Due to the lower CTC count in non-metastatic CRC, particularly precise and intuitive detection techniques are needed. Recent study, focusing on detection procedures and prognostic value, investigated the potential clinical importance of CTC in non-metastatic CRC (TNM-stage I–III). Based on the observations, patients with non-metastatic colorectal cancer who have CTC in their bloodstream may have a worst DFS (99). Enumeration of CTC using FDA approved CellSearch technology may aid in the identification of high-risk stage II CRC patient candidate for adjuvant treatment (100). Iinuma, et al. assessed the clinical importance of blood-based CTCs of post-surgical CRC patients as a predictive factor for overall survival and disease-free survival using CEA, CK, and CD133 as genetic markers. Patients with positive results for these markers in the training sets had significantly worse overall survival and disease-free survival than patients with negative results (101). In order to determine the effectiveness of anti-EGFR therapy in treating metastatic CRC, initial tumors are examined for the existence of KRAS and BRAF mutations. However, differences in KRAS and BRAF mutation level across primary, CTC, and metastatic tumors in patients with advanced colorectal cancer have significant ramifications (29).

Similarly, Gasch C, et al. used the CellSearch method to evaluate expression of EGFR expression, EGFR gene amplification, and KRAS, BRAF, and PIK3CA mutations in single CTCs from patients with metastatic (102). Similarly, another study on single CTC isolated from CRC patient revealed the KRAS mutational correlation among CTCs and primary tumor in around 50% of matched instances (103). Similarly, evaluation of primary and metastatic samples of matched patient revealed presence of mutation of APC, KRAS and PIK3CA at sub clonal level (104). Compared to the traditional tumor marker CEA, CTCs are more promising markers for the assessment and forecasting of treatment outcomes in patients with rectal cancer. Evaluation of clinical relevance of CTCs in comparison to CEA shows strong association among CTC counts and patient response, while serum CEA doesn’t establish any correlation (105). Recent study signifying clinical significance of CTC in colorectal cancer are tabulated in table 4.

Table 4: Study involving CTCs for evaluation of Diagnostic and Prognostic value in Colorectal Cancer.

Circulating Tumor Cells in Melanoma Cases

Cutaneous melanoma develops when epidermal melanocytes change into melanoma cells, a process that is commonly linked with harmful prolonged ultra-violate exposure (118). CTCs from cutaneous and acral melanomas are almost certain to metastasize to other organs (118,119). Metastasis and migration of melanoma CTCs in different organs of body has unique properties and make them special as compared to other solid tumors (120). In recent years, qRT-PCR based single plex or multiplex biomarkers has emerged as a popular tool for melanoma CTC evaluation. The melanoma genes should be constitutively expressed in melanoma tumors and CTCs, compared to PBMCs healthy patients. To overcome heterogeneity of tumor and CTC, numerous biomarkers have already been evaluated. The expression of MMA specific gene is heterogeneous and major challenge. Diagnostic and treatment of melanoma is highly dependent upon the heterogeneity of tumor and variable expression of MAA biomarker. Therefore, evaluation of multiplex-gene expression in CTCs has been a crucial strategy for increasing test sensitivity, especially in patients with lower CTCs count. Enhanced sensitivity and specificity are key to anticipating the possible spread of metastases and therapeutic response. In addition, comprehensive analysis of transcriptome, mutation-based expression analysis of genes or related pathways become highly evident in Cancer Genome Atlas (TCGA). Additionally, the FDA-approved monoclonal Antibody-based checkpoint inhibitor immunotherapies (CII) and several small molecule inhibitors (SMI) for MMA patients provide increased relevance on CTC measurement in monitoring outcomes (121).

CTC presence in patient’s blood is often related with advance-stage metastatic MMA with poor PFS. qRT-PCR based expression analysis of MART-1, MAGE-A3, and PAX3 gene in CTC shown to have higher expression in stage-IV MMA 54%), and the CTC in blood was substantially linked with DFS and OS (122). Kiyohara et al, evaluated a panel of marker using qRT-PCR technique for direct CTC identification, instead of CTC fractionation from leukocytes. The assay constitutes MART-1/Melan-A, MAGE-A3, PAX3, and GalNAc-T gene and shown to have higher sensitivity, specificity toward detection of CTC with crucial clinical application (123). In addition, uveal melanoma termed as most deadly ophthalmological illnesses. Therefore, it requires biomarkers that can foretell the advent of metastases. In recent study, with lengthy follow-up duration, revealed CTC as negative prognostic biomarker in uveal MMA patients. Moreover, CTCs molecular characterization will certainly add in interpretation of metastasis and could enhance patients care (124). Using CTC profiling, this might be possible to identify MMA patients who could benefit from intensive adjuvant therapy or to group individuals for adjuvant clinical trials. Sentinel lymph node (SLN) metastases in MMA patients may result in a wide variety of conditions, rendering it difficult to reach agreement on their course of care. Identification of high-risk individuals from clinical context may aid with prognostic and treatment planning. Hoshimoto et al, assessed the clinical significance of multiplexed biomarker panel (MART-1, MAGE-A3, and GalNAc-T) using qRT-PCR for identification of CTC from SLN metastasized MMA after complete lymphadenectomy. Result revealed 13.4% to 17.5% of detection range for single CTC biomarker without any disease correlation. However, detection of more than one CTC biomarker was substantially linked to lower recurrence-free survival, distant metastasis, and disease-free survival (122). Assessment CTC level in blood of melanoma patients could be used to evaluate progression and therapy response (125). Moreover, advancement in molecular characterization of CTC may provide new personalized therapeutic approaches for management of disease (126). Recent published study providing clinical significance in melanoma cancer are listed in table 5.

Table 5: Represent study involving CTCs for evaluation of Diagnostic value in Melanoma cancer.

CTC: circulating tumor cell, DFS: disease-free survival, OS: overall survival, DDFS: distant-disease free survival, PFS: progression-free survival, RFS: recurrence-free survival.

Circulating Tumor Cells in Pancreases

According to Cancer Research Statistics-2022, pancreatic carcinoma ranked fourth in terms of morbidity in the USA and Europe. The incident rate and mortality are equal with overall five-year survival rate of <6% (134) and will become second morbidity causing cancer by 2030 (135). The poor recovery of pancreatic carcinoma patients is often associated with hyper-metastasis of tumor at early stage without symptom. The frequent dissemination of pancreatic cancer to other organs revealed their potential of migrating through the circulating system. Therefore, evaluation of CTC count in blood could be used as liquid based diagnostic and prognostic biomarker for pancreatic cancer. In recent decades CTCs clinical significance has been extensively explored in several cancers along with pancreatic cancer, however, complete significance of CTCs has not been explored in pancreatic cancer. However, the presence of CTCs in benign pancreatic neoplasm stimulated researchers to identify the role of CTCs in diagnosis, prognosis and metastasis extensively studied in pancreatic tumor (136,137). Several studies reported the prognostic role of CTCs in different pancreatic cancer (Table 1). Conventionally, tumor size, nodal status, and perineural invasion were associated with prognostic factor and require surgical resection. Hereby, CTCs shows difference as they are minimally invasive and look into the developmental as cancer progression. Martini, et al., suggested the CTC presence in pancreatic cancer patients have clinical relevance and have unfavorable outcome (138). In another study, enumeration of CTC in pancreatic carcinoma patients using CellSearch® system revealed to have positive association with poor tumor differentiation and smaller had a significantly lower PFS, moreover, CTC-positive group had a lower OS than patients in the CTC-negative group (140). Bidard, et al. evaluated CTCs in locally advanced PDAC patients and reported 11% of CTC-positive patients with worse overall survival (141). Similarly, another study reported the MTHFR C677T and CK19 mutation in CTCs isolated from the blood of pancreatic tumor patients (142). However, variation in result is more often due to CTC enumeration technique and patients. To gain a deeper understanding of the clinical consequences of CTC discovery in pancreatic cancer patients, further investigations and characterization of the CTC population are needed. Recent study involving CTC in pancreatic cancer are depicted in table 6.

Table 6: Represent study involving CTCs for evaluation of diagnostic and prognostic value in pancreatic cancer.

Circulating Tumor Cells in Ovarian Cancer

According to the world cancer research foundation (WCRF-2020), the ovarian cancer (OC) burden annually increases by 6.6%, accounting for 313,959 new cases a year. While morbidity rate was 4.2, accounting for 207,252 deaths. The early diagnosis of OC is still compromised, and the majority of patients are detected at advanced stages. Early diagnosis and prognosis of OC is critical for management of disease, however, the current biomarker regime failed to do so. Liquid biopsy-based biomarker such as cell free DNA, Methylation based biomarker, miRNA, and circulating tumor cells bring new possibility of early diagnosis of OC. Circulating tumor cell-based studies are flourishing in recent decade for evaluation of their potential in diagnosis, prognosis and management of cancers. Several studies reported the clinical relevance of CTC in diagnosis, prognosis and therapeutic monitoring in ovarian cancer (156)

. Lou et al, revealed the predictive value of CTC in ovarian cancer patients, CTC count was significantly elevated in metastatic OC patients than in primary ovarian cancer patients (157). In another study, Yang, et al. reported the clinical significance of CTC count in ovarian cancer, where increase in CTC was associated with higher recurrence rate as compared to patients with lower CTC count (158). Study by Perl, et al. revealed the CTC enrichment based on cell adhesion matrix (CAM) has sensitivity of 83% and specificity of 95% for detecting OC (159). Similarly, Zhang, et al. reported early diagnostic value of CTC, moreover, they revealed the EpCAM-positive CTC could help in prognosis and treatment monitoring (15). Obermayr, et al. reported RT-PCR based detection of six genes isolated from CTC could detect 19 % of the ovarian cancer patients.

In recent study by Zhang, et al. reported the EpCAM positive CTC has diagnostic, and prognostic value along with therapeutic progression monitoring (15). Po et al reported that detection of EpCAM and N-cadherin embedded CTC can enhance the management of ovarian cancer patients (160). Guo, et al. evaluated the diagnostic potential of HE4+ CTC cell in ovarian cancer patients, the AUC, sensitivity, and specificity of HE4+ CTC assay was 71%, 76.7%, and 63% respectively (161). In another recent study, folic acid receptor-based capture of CTC showed 80% sensitivity in ovarian cancer (162). Similarly, Obermayr et al reported the multi-protein marker-based detection of CTC in debulked OC patients for therapeutic stratification (163). Rao, et al. reported microfluidic method conjugated with EpCAM antibody for enumeration of CTC have higher sensitivity in detection EOC patients compared to healthy patients (164). Similarly, lee, et al. evaluated EpCAM+, DAPI+ and CD45 negative feature-based CTC enrichment in EOC patients, result showed to have higher diagnostic and prognostic value (165). Several recent studies have evaluated the diagnostic, prognostic and clinical significance in diagnosis of OC patients based on CTC count. However, conclusive study is still lacking behind their use as biomarkers. Some of the recent research progression in CTC based detection of EOC and their clinical significance are tabulated in table 7.

Table 7: Diagnostic and prognostic value of CTC in diagnosis of ovarian cancer patients.

Circulating Tumor Cells in Hepatocellular Carcinoma

According to cancer statistics-2018 hepatocellular carcinoma (HCC) ranked as second in terms of morbidity causing cancer and 7th as common cancer (180). Surgical removal, local ablation or transplantation of liver are the most effective way to treat hepatocellular carcinoma, depending upon early diagnosis. However, the current therapeutic approaches have downflow due to higher rate of recurrence (181). The current diagnostic approaches depend upon changes in serum protein marker, histopathological analysis and imaging techniques, however, the sensitivity and specificity is under the question mark. Recent technological advancement in field of liquid biopsy, provided plethora of biomarker (cfDNA, miRNA, circulating tumor cells) having potential in diagnosis of hepatocellular carcinoma at early stage with higher sensitivity and specificity compared to conventional biomarker (182).

Several recent reports evaluated the potential of circulating tumor cells in diagnosis, prognosis and therapeutic monitoring of hepatocellular carcinoma. A recent meta-analysis by Cui, et al. on 1191 patients showed the specificity and sensitivity toward diagnosis of HCC. Moreover, presence higher CTC count in blood increases the risk of hepatocellular recurrence with higher Hazard Ration (183). Winograd, et al. revealed the therapeutic relevance and diagnostic accuracy of CTC in hepatocellular carcinoma (184). Similarly, Saxena, et al. revealed the presence of MTHFR-C677T gene polymorphism and KRAS mutation in CTC isolated from Hepatocellular carcinoma patients increases the risk factor of disease (185).

The clinical implication of CTC has been assessed in several solid carcinomas. CTC based enumeration in breast, colorectal and prostate cancer has been approved by FDA for clinical diagnosis. CTC based studies proved to have role in diagnosis, prognosis, screening, therapeutic monitoring, and therapeutic alignment in several cancers (Figure 2). Cristofanilli et al remarked CTC as prognostic biomarker that predicts the PFS and OS of breast cancer patients. This study got FDA approval for clinical application of CTC in breast cancer and further revolutionized the clinical implication of CTC in many cancers (24,186).

Since a CTC test is less costly and non-invasive than a traditional biopsy, it can be repeated frequently throughout treatment. These factors combine to make CTC analysis a potent tool for tracking the progression of cancer. Early diagnosis of breast cancer has great impact on increasing survival of patients, Early diagnosis plays a key role in decreasing mortality of breast cancer, although the grades and breast cancer types have their own importance. For instance, grade I breast cancer progresses considerably more slowly than grades II and III, therefore early detection when tumor diameter is less than two centimeters can improve prognosis and OR (overall survival) of breast cancer patients (187). A recent study evaluated the diagnostic value of CTC in predicting breast cancer with 86.0% of AUC, 95.4% specificity and 76.56% sensitivity (39). Similarly, Yun, et al. revealed the diagnostic potential of CTC in predicting lung cancer with AUC of 93% with 75.0% sensitivity and 89.0% specificity (188). The diagnostic value of CTC at cut-off value of 2 in metastatic colorectal cancer was 90.4% AUC, 83.05% sensitivity and 100% specificity (106). Recently, clinical study by Yang, et al., revealed early diagnostic potential CTC using CellMax biomimetic platform in early colorectal cancer. A total of 620 samples were enrolled with different stages and clinicopathological features. The CTC showed higher early diagnostics accuracy (88%) than routine clinical diagnostics method (189). Similarly, Yasmine, et al. performed LSC-MS analysis (Living single-cell mass spectrometry) and revealed the CTCs complex heterogeneity in single cancer (190). Li, et al. revealed diagnostic potential of CTCs derived genes (CEA, C-Met, MUC1, CK19, EGFR and EpCAM) from 50 CRC patients with 87% sensitivity and 85% accuracy (191). Since now, colonoscopy and biopsy are diagnostic benchmarks, and addition of CTC-positive biopsy could improve the specificity.

Figure 2: Significance of Circulating Tumor Cells in Cancer.

The evaluation of disease progression has been crucial for cancer treatment and prognosis. There have been efforts to incorporate CTC evaluation with well-established diagnostic methods for lung and breast. colorectal, prostate and other cancers. He, et al. concentrated on enhancing the reduced CT screening program's specificity by incorporating CTC examination. CTC numbers were assessed in patients and healthy individuals with detected lung "ground-glass" nodules. CTCs were only found in bloodstream of a small percentage of patients having nodules. Following molecular research, it was shown that these CTCs had a "cancerous predisposition" (192). In another study by Ilie et al evaluated CTC numbers in patients with chronic obstructive pulmonary disease (COPD). Out of 168 patients, only 5 (3%) of them were CTC positive and more interestingly these all five patients got lung cancer in under four years. In addition, no malignancy was discovered in any of the CTC-negative patients during the observational period. These results have been supported by some investigations (193,194). Apart from diagnostics, CTC load could be used in prognosis to predict the recurrence and metastasis.  François, et al. revealed the prognostic role of CTC load (higher count) in liver metastatic patients compared to post treatment patients CTC count (195). The CTC count (pre- and post-surgery) plays a vital role in assessment of recurrence and metastasis (196). Similarly, another study revealed the recurrence CRC in postoperative CTC-positive patients (110,197). The ratio of mesenchymal phenotype CTCs to total CTCs can be used to evaluate prognosis and metastasis (198). In addition, the recurrence of breast cancer is a major problem in that increases the burden of cancer and leads to lower survival rate. It is widely acknowledged that early-stage breast cancer malignant tumors have a potent capacity for invasion into distant organs including the bones and lungs.

Basic yet reliable indications of cancer development include CTC count and count fluctuations throughout therapy in many cancer types, thus, CTC count has been shown to be a prognostic predictor (86,199,200). Moreover, evaluation of CTC counts while receiving treatment is a tool that enables evaluation of cancer progression in real time, even before blatant clinical manifestation of relapse. Following surgery and/or chemotherapy, a declining CTC number is most likely an indication of remission. Conversely, a rise in CTC counts signals the recurrence of the disease and should cause the therapy to be revalidated (193,201). According to a recent study, the load of CTCs in blood of pre-therapeutic breast cancer patients has a significant impact on both OR (overall survival) and progression-free survival (24). Rack, et al. study on continuous existence of the CTCs in in pre and post chemotherapeutic patients revealed that the continuous existence of the CTCs in in pre and post chemotherapeutic patients were related with poor overall survival and at risk of recurrence (202). Similarly, Madic, et al. revealed the higher CTC counts in TNBC patients have worst prognosis and lower survival rate (203).

Moreover, post-therapeutic observation of patients is needed for the assessment of disease progression. The CTC count based monitoring of therapy for evaluation of condition has been evidently reported in several studies. During chemotherapy, the clinical utility of CTCs count was evaluated in real-time in breast cancer patients’ data (204). Moreover, a phase-III trials (S0421) validated the prognostic role of CTC count with docetaxel plus prednisone with or without atrasentan for overall survival and diseases response. The baseline CTCs number is prognostic while increasing CTCs at early three weeks signifies worst overall survival, revealing the potential utilization of CTC in therapeutic response measurement (205). Similarly, Sinoquet et al, revealed the PD-L1(+) CTCs from NSCLC patients have poor survival and could be used to predict the anti-PD-1/PD-L1 therapeutic response and resistance (91). In another study Lu et al revealed that CTC could be used as diagnostic marker and predictor metastasis in lung cancer (73). Li et al reported the therapeutic relevance of CTC in lung cancer, post chemotherapy significant decrease in CTC count was associated with enhanced PFS and OS (74).

In the last decade, the increase in cancer burden and morbidity has become a major challenge to the scientists. The early diagnosis of cancer has an impeccable effect on the survival of the patients. Several biomarker regimes have been advocated and approved by FDA for diagnosis of cancer, however the early diagnosis of majority of cancers is still a major concern for clinical scientists. Several cancers are being diagnosed at late stage of disease leading increased morbidity. Circulating tumor cells have been studied enormously in the last two decades for exploitation of their role in metastasis, early diagnosis, prognosis, real-time disease/therapy monitoring. The CTC enumeration method has revealed distinct patterns of expression in different cancers, specifically stages, pre- and post-therapy. The CTC account based approached significantly diagnose the disease at early stage, moreover, they have potential to be used as prognostic marker and could assess the therapy monitoring in real-time. These properties make CTC as a possible biomarker assessment of cancer and disease progression. However, rigorous exploration of CTCs is warranted to validate the clinical application as well as to stand with clinical decision-making compared with other available biomarkers that have been proven and broadly accepted. Nevertheless, CTCs harbor the potential for earlier tumor detection and future, large-scale trials are needed.

Abbreviations:

CTC: Circulating Tumor Cell

EMT: Epithelial Mesenchymal Transition

OC: Ovarian Cancer

HCC: Hepatocellular Carcinoma

PFS: Progression Free Survival

OS: Overall Survival

DFS: Disease Free Survival

HR: Hazard Ration

TIC: Tumor Initiating Cell

TCGA: The Cancer Genome Atlas

AUC: Area Under Curve

EOC: Epithelial Ovarian Cancer

CRC: Colorectal Cancer

PSA: Prostate-Specific Antigen

CRPC: Castration Resistant Prostate Cancer

PBMC: Peripheral Blood Mononuclear Cells

FDA: Food Drug Administration

EGFR: Epidermal Growth Factor Receptor

mCRC: Metastatic Colorectal Cancer

Acknowledgement

We Acknowledge to the Honorable Director, AIIMS Patna, and Department of Science and Technology (Government of India), New Delhi, for providing financial assistance. 

Ethics approval and consent to participate:

Ethical approval and consent are not required in the present study.

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