Chang-Can Li , Hua-Yu Yang , Yi-Lei Mao

Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences (CAMS), Beijing 100730, China

Hepatocellular carcinoma (HCC) is one of the most common cancers and is the second leading cause of cancer-related deaths worldwide [ 1 , 2 ]. The main therapies for HCC include surgical resection, local ablation, or liver transplantation, which are only applied to early-stage HCC. However, most patients at the time of initial HCC diagnosis have already progressed to an advanced stage, and survival can only be prolonged finitely via palliative therapies, such as transarterial chemoembolization, systemic therapy with tyrosine kinase inhibitors, and selective internal radiation therapy [ 3 , 4 ]. Moreover, as HCC has a high recurrence rate, the treatment efficacy is not satisfactory [5] . Currently, the diagnosis and monitoring of HCC primarily depend on serum biomarker detection, pathological examination, and imaging analysis. Common serum markers display poor diagnostic performance, and imaging and pathological examinations have limitations in diagnostic accuracy and sensitivity [ 6 , 7 ]. Therefore, we urgently require better approaches for the diagnosis and monitoring of HCC.

The new diagnostic concept of “l(fā)iquid biopsy” has emerged in recent years [ 8 , 9 ]. It can obtain tumor information for tumor diagnosis and dynamic monitoring by assessing circulating tumor cells(CTCs), circulating tumor DNA (ctDNA), microRNA, and extracellular vesicles in the non-solid biological tissues of the body, such as blood, cerebrospinal fluid, saliva, pleural fluid, ascites, and urine,for various analyses. Liquid biopsy allows regular analyses to monitor tumor progression or treatment response in real-time utilizing a non-invasive approach. Therefore, it could be an effective method for the early detection and management of HCC [10] . CTC testing is one of the cornerstones of liquid biopsies. CTCs are cancer cells that have been shed from the original or metastatic tumors and that circulate in the bloodstream; they carry valuable information regarding tumor progression and metastasis. The CellSearch definition of CTCs is accepted as the standard definition: a CTC is a circulating nucleated cell larger than 4μm, expressing epithelial proteins EpCAM and cytokeratins 8, 18, and/or 19, while being negative for the leukocyte-specific antigen CD45 [11] . Here, we briefly introduce CTCs and discuss their clinical applications and future value in the diagnosis and monitoring of HCC.

Numerous studies have shown that CTCs play a crucial role in the initiation of tumor metastasis, suggesting that CTCs may be a biomarker for the early detection of HCC [ 9 , 11 ]. Previous studies [ 12 , 13 ] on the early detection of HCC were primarily based on the assessment of CTC numbers and showed a significant positive correlation between the CTC number and the standard Barcelona Clinic Liver Cancer stage as well as the serum alpha-fetoprotein level. Other studies [ 14 –16 ] have indicated that CTC testing is not an ideal independent diagnostic tool for HCC and that simultaneous detection of CTCs and alpha-fetoprotein might improve the detection accuracy of HCC in patients.

In HCC patients, the testing of CTCs not only contributes to neoplasm staging but also is useful for prognosis [17] . A study [18] showed that after radical resection, the disease-free survival and overall survival of HCC patients were both significantly better in patients with lower CTC counts ( ≤2 cells in 5 mL blood)when compared to those with higher CTC counts ( > 2 cells in 5 mL blood), indicating that a high CTC count in HCC patients predicts a poorer prognosis. The association of increased postoperative CTC numbers with a worse prognosis in HCC patients has also been reported [ 19 , 20 ]. These studies indicated that the CTC phenotypes might serve as a prognostic indicator for HCC patients, and that certain specific CTC phenotypes may be more significant for prognosis than others. Since CTC numbers may change after antitumor treatment, they could be used for predicting or evaluating therapeutic efficacy before or after treatment. Ye et al. [18] found that postoperative CTC counts and pre/postoperative changes in CTC counts were significantly associated with progression-free survival and were a better predictor of performance than absolute counts. In addition, Zhou et al. [21] measured the preoperative levels of EpCAMmRNA + CTCs and CD4+CD25+Foxp3+Treg cells in HCC patients and found that elevated CTC/Treg levels implied a higher risk of postoperative recurrence.

Mutation profiles and drug-resistant molecular expression profiles have been extensively identified and ascribed to multiple solid tumors. CTC analysis also has great potential to identify the patient population most likely to respond to specific treatments, with the determination of therapeutic targets and resistance mechanisms to cancer therapies at the DNA, RNA, and protein levels [22] . Li et al. presented a novel system to provide quantitative information of sorafenib-related targets by simultaneously detecting phosphorylated ERK (pERK) and Akt (pAkt) levels in CTCs in HCC [22] .This study demonstrated that CTCs could substitute tumor tissues in the characterization of pERK/pAkt in overall characteristics, instead of a tiny part of the pathological specimen taken from the entire tissue. The most sorafenib-sensitive cells, pERK + /pAkt- CTCs,could serve as independent progression-free survival indicators in sorafenib-treated HCC patients.

Currently, the information acquired from a single specimen is limited to the site of collection and does not always reflect the overall characteristics of HCC. However, CTC testing can overcome this weakness by providing the genetic profile of integral cancerous lesions and offer the opportunity to systematically and dynamically track genetic changes [23] . Moreover, analyzing therapeutic targets and drug resistance-related gene mutations in CTCs would contribute to a better understanding and clinical management of drug resistance in cancer patients [24] .

There are still challenges in the identification, quantification,and/or characterization of CTCs. Currently, novel and certified tools for rapid isolation and characterization of CTCs are urgently needed [ 25 , 26 ]. For a comprehensive understanding of CTC biology, multiple omics disciplines should be combined for single-cell analysis. Furthermore, as part of liquid biopsy, CTC testing should be used in combination with other liquid biopsies such as analysis of ctDNA and exosomes to increase the efficiency of clinical CTC tests. In addition, more multicenter, larger, and longer-term studies are a pressing need for the clinical implementation of CTC testing, including clinical trials. Moreover, CTC testing is a critical part of precision medicine, and improvement of CTC testing technology would be beneficial for the early diagnosis, effective treatment, and prognostic management of HCC.

Acknowledgments

None.

CRediT authorship contribution statement

Chang-Can Li: Data curation, Investigation, Writing – original draft. Hua-Yu Yang: Data curation, Investigation, Writing – original draft. Yi-Lei Mao: Conceptualization, Resources, Supervision, Writing – review & editing.

Funding

None.

Ethical approval

Not needed.

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.