4. Extracellular Vesicles (EVs): Exosomes

Exosomes are a type of EV, nanoparticles encapsulating a variety of cargo including DNA and RNA fragments in a lipidic double-layer, which protects them from enzymatic degradation. With these unique features, circulating exosomes allow RNAs to circulate without being degraded plasma. Their nature and roles remain largely unknown but exosomes may not only be passively released from apoptotic cells into the bloodstream. Data suggested they may be actively secreted, acting as messengers in the cell-to-cell communication network, conferring them priceless values like accuracy and tissue-specificity [68,69,70,71].

In HCC, the data exploring the contribution of exosomes remain limited, particularly for prognosis. However, these analytes have demonstrated promising and polyvalent performance in other cancer types both for diagnosis and prognosis [72,73].

Several projects analyzed exosomal miRNAs. In a cohort of 59 HCC patients, authors found a correlation between tumor recurrence after liver transplantation and a higher level of miR-718 [60]. Similar signals were detected after liver resection and other exosomal miRNAs: high levels of miR-665 or low levels of miR-638 and miR-320a were identified as predictors of poor survival [61,63,66]. In a cohort of 79 HCC patients of different stages receiving various treatments, Lee et al. focused on two candidates: a miRNA (miR-21) and a long non-coding RNA (lncRNA) (lncRNA-ATB). On multivariable analysis, both markers were independently associated with disease progression [62]. A recent study profiled 57 plasma cell-free RNA transcriptomes and 20 exosomal RNA transcriptomes to test their diagnostic and prognostic performance. RN7SL1 and its S fragment were promising, showing a high diagnostic accuracy (AUC = 0.87). Furthermore, a higher concentration of RN7SL1 S fragment was an independent factor of worse survival [64]. The analyses of blood samples from 124 HCC patients treated with surgical resection and 100 healthy controls identified an exosomal circular RNA (circAKT3) as a prognostic factor; a high level of circAKT3 predicted both higher recurrence and lower survival [65].

5. Circulating Tumor Cells (CTCs)

CTCs play a pivotal role during the hematogenous dissemination of cancers. Most technologies to analyze CTCs include two steps: enrichment (isolation) and detection (identification). The development of sensitive and specific technologies is challenging. Estimated to be released by cancers of intermediate and advanced stages, CTCs are probably more useful for prognostication than for early cancer detection. In this context, studies have demonstrated the prognostic value of CTC enumeration in different cancers including HCC [74,75]. More sophisticated technologies, like single-cell RNA sequencing, have allowed further characterization of CTC subtypes [76]. Studies exploring the value of CTCs for HCC prognostication are summarized in Table 3.

Table 3. Circulating tumor cells (CTCs).

Most studies investigating CTCs included HCC patients undergoing surgical resection. In 2004, Vona G et al. isolated and enumerated CTCs based on their size and morphology, showing that the presence and number of detected CTCs were associated with shorter survival [77]. CellSearch^® is an isolation system that targets EpCAM positive cells. It was approved by the Food and Drug Administration (FDA) and became the most commonly used technique for CTC enumeration. Its use remains debated in HCC as only around 30% of HCC cells express EpCAM [102]. Nonetheless, several studies utilized this approach in HCC, demonstrating that the detection of EpCAM positive CTCs was associated with higher tumor recurrence [75] or lower survival [82,87]. Thereafter, more sophisticated technologies for CTC isolation have been reported, like ImageStream flow cytometry. A study has provided the proof-of-concept of this technology, demonstrating its capacity to detect CTCs using a panel of markers. This technology also generates high-resolution images of isolated CTCs [103]. Its value in detecting CTCs was confirmed and the CTC count was further confirmed as an independent prognostic factor [86]. Other reports have aimed at exploring the impact of subgroups of CTCs, clustered based on cell surface markers, RNA expression or genomic aberrations. Studies using surface markers to detect CTCs with cancer stem cell-like [79,80,81] or mesenchymal [92] features, revealed their clinical value to predict tumor recurrence. CTCs expressing AFP were also associated with an increased risk of metastasis [85], whereas CTCs harboring CNV (chr 8) predicted worse survival [88]. Ha et al. used a simple isolation technique but introduced the concept of ΔCTC, referring to the perioperative fluctuation of detected CTCs, which appeared as an independent factor of lower survival and higher recurrence rates after partial hepatectomy [95]. Besides their intrinsic biological traits, CTC dissemination seemed to be impacted by treatment. Data suggested that surgery-induced manipulation of the liver is associated with a release of CTCs [94]. A comparison between anterior and conventional surgical approaches suggested that the latter was associated with a higher release of CTCs as well as poorer outcomes [89]. Toso et al. described five steps during orthotopic liver transplant (OLT) in HCC patients to minimize CTC dissemination and thereby the risk of recurrence [104].

Recent studies also underscored the relevance of CTC analysis in patients undergoing OLT for HCC, highlighting an association between CTC detection and recurrence [97,101].

Guiding decision-making would be another application of CTCs. For example, selection of patients who would benefit from adjuvant transarterial chemoembolization (TACE) after surgery or to predict response to systemic therapies like tyrosine kinase inhibitors or immunotherapy [84,99,100]. An interesting study analyzed CTCs using samples collected from different vessels. By doing so, they were able to demonstrate spatial heterogeneity in the distribution of CTCs, with a predominance of epithelial status at release, which gradually switched to EMT-activated phenotype during hematogenous transit [90]. Overall, data consistently identified that the number of CTCs was a surrogate of poor prognosis, predicting higher recurrence and/or lower survival. A recent meta-analysis and data from experimental models corroborated these findings [105,106].

2. Challenges and Future Perspectives
The field of liquid biopsy has numerous challenges. Besides those related to cost and technology, there is a limited understanding of the fundamental mechanisms responsible for the release of tumor molecular components to the bloodstream. A better understanding of these mechanisms would provide new tools and targets to improve the diagnostic and prognostic performance of liquid biopsy analytes. Gasparello et al. performed one of the few studies of liquid biopsies in animal models, identifying potential gateways regulating the detection of ctDNA [107]. Experimental models will be instrumental to better understand these mechanisms. ITH has emerged as a major drawback for singlebiopsy biomarker development. The clinical impact of ITH is progressively recognized, even at early tumor stages [108]. Liquid biopsy can help address the clinical issues posed by ITH as it likely includes a molecular composite of tumor components released by any potential tumor area. Thus, it is not restricted by the specific tumor section sampled by a needle-biopsy. There are few data of integrative analysis of different analytes within the liquid biopsy space (e.g., simultaneous evaluation of ctDNA and CTCs). Finally, it is key to have prospective data to determine the exact role of liquid biopsy as a prognostic biomarker in HCC, and which is the clinical niche that will be better suited for this transformative technology.

3. Conclusions
While data on liquid biopsy in HCC remain scanter than for other malignancies, there has been numerous recent publications demonstrating its prognostic value in HCC patients. Potential contributions in HCC prognostication were detected for each of the tumor byproducts (e.g., DNA, RNA, exosomes and cells). The next step will be to determine the optimal way of integrating liquid biopsy in the clinical management of HCC patients and to modify current clinical practice guidelines accordingly.

Author Contributions: Study concept and design: I.L., A.V., O.D., N.D., E.M.; Acquisition of data:
I.L.; Analysis and interpretation of data: I.L., A.V., E.M.; Drafting of the manuscript: I.L.; Critical
revision of the manuscript for important intellectual content: I.L., A.V., O.D., N.D., E.M. All authors
have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Acknowledgments: The authors would like to thank Jill Gregory for the design of the figures.
Conflicts of Interest: AV has received consulting fees from Guidepoint, Fujifilm, Boehringer Ingelheim, FirstWord, and MHLife Sciences; advisory board fees from Exact Sciences, Nucleix, Gilead and
NGM Pharmaceuticals; and research support from Eisai.

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