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Hepatology. Author manuscript; available in PMC 2022 January 18.
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Published in final edited form as:

Hepatology. 2021 January ; 73(1): 422–436. doi:10.1002/hep.31165.

Detection of circulating tumor cells and their implications as a

novel biomarker for diagnosis, prognostication, and therapeutic
monitoring in hepatocellular carcinoma
Joseph C Ahn1, Pai-Chi Teng2, Pin-Jung Chen5, Edwin Posadas2,3,4, Hsian-Rong Tseng5,
Shelly C. Lu6,8, Ju Dong Yang6,7,8
1Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55904, United States
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2UrologicOncology Program and Uro-Oncology Research Laboratories, Cedars-Sinai Medical

Center, Los Angeles, CA 90048, United States
3Translational Oncology Program, Cedars-Sinai Medical Center, Los Angeles, CA 90048, United
States
4Divisionof Hematology/Oncology, Department of Medicine, Cedars-Sinai Medical Center, Los
Angeles, CA 90048, United States
5Department of Molecular and Medical Pharmacology, California NanoSystems Institute, Crump
Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, CA 90095,
United States
6Division
of Digestive and Liver Diseases, Department of Medicine, Cedars Sinai Medical Center,
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Los Angeles, CA 90048, United States

7Comprehensive Transplant Center, Cedars Sinai Medical Center, Los Angeles, CA 90048, United
States
8Samuel Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, CA
90048, United States

Abstract
Hepatocellular carcinoma (HCC) is among the leading causes of worldwide cancer-related
morbidity and mortality. Poor prognosis of HCC is mainly attributed to tumor presentation at an
advanced stage when there is no effective treatment to achieve the long term survival of patients.
Currently available tests such as alpha-fetoprotein (AFP) have limited accuracy as a diagnostic or
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prognostic biomarker for HCC. Liver biopsy provides tissue that can reveal tumor biology but it is
not used routinely due to its invasiveness and risk of tumor seeding, especially in early stage
patients. Liver biopsy is also limited in revealing comprehensive tumor biology due to intra-
tumoral heterogeneity. There is a clear need for new biomarkers to improve HCC detection,
prognostication, prediction of treatment response, and disease monitoring with treatment. Liquid

Corresponding Author: Ju Dong Yang, MD, MS, Division of Digestive and Liver Diseases, Department of Medicine, Cedars Sinai
Medical Center at 8900 Beverly Blvd, Los Angeles, California 90048, United States. judong.yang@cshs.org, Telephone: +1-
310-4231971, Fax: +1-310-4232356.
Conflict-of-interest statement: All authors have nothing to disclose.
 Ahn et al. Page 2

biopsy could be an effective method of early detection and management of HCC. Circulating
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tumor cells (CTCs) are cancer cells in circulation derived from the original tumor or metastatic
foci, and their measurement by liquid biopsy represents a great potential to facilitate the
implementation of precision medicine in patients with HCC. CTCs can be detected by a simple
peripheral blood draw and potentially show global features of tumor characteristics. Various CTC
detection platforms utilizing immunoaffinity and biophysical properties have been developed in
order to identify and capture CTCs with high efficiency. Quantitative abundance of CTCs, as well
as biological characteristics and genomic heterogeneity among the CTCs, can predict disease
prognosis and response to therapy in patients with HCC. This review article will discuss the
currently available technologies for CTC detection and isolation, their utility in the clinical
management of HCC patients, their limitations, and future directions of research.

Keywords
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Hepatocellular Carcinoma; Circulating tumor cells; Biomarkers; Immunoaffinity; Epithelial-to-

Mesenchymal Transition; CTC Heterogeneity

Introduction
Hepatocellular carcinoma (HCC) is an aggressive primary liver cancer that typically occurs
in the setting of chronic liver disease and cirrhosis, and it is the sixth leading cause of cancer
incidence and the fourth cause of cancer death globally(1). While a select group of patients
with small, localized HCC may undergo curative therapies, those with large tumor burden,
vascular invasion, or metastasis have a poor prognosis and are managed with systemic
treatment and supportive care. There exists an unmet need for HCC biomarkers for early
detection and prognostication, as well as prediction and monitoring for treatment response.
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Currently, alpha-fetoprotein (AFP) is the most widely used biomarker for HCC. Biannual
liver ultrasound with or without serum AFP is the main HCC screening strategy
recommended by major societies(2–4). AFP is used as a prognostic and predictive biomarker
in patients with HCC. Elevated levels of AFP have been associated with increased tumor
size and portal vein thrombosis, as well as increased risks of liver transplant waitlist dropout
and posttransplant recurrence(5, 6). Serum AFP is also a predictor of treatment response in
HCC patients after liver transplant and ramucirumab treatment(7, 8). However, AFP’s role
as a biomarker for early detection of HCC is limited by its poor sensitivity. Alternative
protein-based serum tumor markers such as AFP lectin fraction (AFP-L3) and des-y-carboxy
prothrombin (DCP) have been shown to improve the diagnostic performances when used in
combination with AFP(9). Glypican-3 (GPC3)(10), cytokeratin 19 (CK19)(11), golgi protein
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73 (GP73)(12), midkine(13), osteopontin(14), squamous cell carcinoma antigen (SCCA)

(15), and annexin A2(16) have all been shown to have diagnostic and prognostic roles in
HCC as well, but they have not been adopted for widespread clinical practice.

Liver biopsy allows direct sampling of the tumor tissue and molecular characterization of the
tumor. However, it is an invasive test with a risk of bleeding and concern for possible tumor
seeding. Moreover, as HCCs exhibit significant inter- or intra-tumoral heterogeneity from
genetic aberrations, transcriptional and epigenetic dysregulation, a single biopsy specimen

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containing a small amount of tumor tissue may not be representative of the whole HCC
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tumor(17).

Over recent years, a variety of “liquid biopsy” techniques have shown significant promise as
novel biomarkers for HCC. In liquid biopsy, samples of body fluids are collected to obtain
vital pieces of phenotypic, genetic, and transcriptomic information about the primary
tumor(18). The primary forms of liquid biopsy include circulating tumor cells (CTCs),
circulating tumor DNA (ctDNA), microRNA (miRNA), and extracellular vesicles (EVs).
First described in 1869, CTCs are malignant cells derived from either the primary tumor or
metastases that migrate into the systemic circulation(19). CTCs represent a unique
biomarker different from any of the existing cancer biomarkers as they represent a sampling
of the patient’s live tumor cells (20). Analysis of CTCs can help guide treatment plans by
identifying specific mutations in target genes and predicting response or resistance to
specific treatments. This review article will discuss the CTCs and provide an overview of
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their biology, current and emerging techniques, and various studies investigating their roles
as potential diagnostic and prognostic biomarkers for HCC.

Overview of CTC biology and clinical implications

As a malignant tumor proliferates and invades into the adjacent tissue, the tumor cells
secrete matrix metalloproteinase (MMP) which breaks the basement membrane, enabling the
tumor cells to gain direct access to the nearby blood and lymphatic vessels (21) (Figure 1).
Once in the bloodstream, the tumor cells become CTCs and can remain in circulation until
they reach different tissues of the body and invade them. Most of the CTCs introduced into
circulation get rapidly killed by processes such as anoikis, immune attacks, or shear
stress(22). Thus, the CTCs undergo a number of adaptations in order to survive in their
hostile new environment. A key process is epithelial to mesenchymal transition (EMT),
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where CTCs lose epithelial-type surface markers and gain mesenchymal markers, allowing
them to behave like mesenchymal cells. CTCs that have undergone EMT can easily detach
themselves from the primary tumor tissue and invade the capillaries and possess
significantly improved ability to survive and metastasize. Also, CTCs may form aggregates
with fibroblasts, leukocytes, endothelial cells or platelets to form CTC clusters, which
possess a significantly higher metastatic potential and increased ability to survive compared
to individual CTCs(23). It should be noted that CTCs are not identical clones of each other,
but represent a heterogeneous population of cells from different tumor foci, with abilities to
change their phenotypic and molecular characteristics under selective microenvironmental
and therapeutic pressures(24). Studies have shown that profound heterogeneities exist
between tumor cells within the primary tumors and those within sites of metastasis.
Therefore, liquid biopsy using CTCs should not be regarded merely as a less invasive
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alternative to an actual biopsy, but a novel way to obtain a comprehensive understanding of

the heterogeneous tumor cells throughout the body. Detection and isolation of CTCs can
enable the implementation of precision medicine by revealing the molecular characteristics
of the tumor and identify markers for targeted therapy.

CTCs are being extensively studied in a variety of solid organ malignancies. In patients with
metastatic prostate cancer, CTC enumeration has been shown to be a reliable predictor of

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prognosis and treatment response(25). Detection of HER2+ CTCs in patients with breast
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cancer can identify candidates for targeted therapy(26). While CTCs are highly promising as
a form of liquid biopsy, there are technical challenges to be overcome before there can be
widespread utilization of CTCs. Most importantly, CTCs have an extremely rare frequency
in the circulation and the number of CTCs tends to be proportional to tumor volume, which
makes their detection in early-stage disease challenging(27). Therefore, the challenge of
CTC research lies in improving the sensitivity and specificity of CTC detection to the levels
necessary for accurate and comprehensive molecular characterization.

Another cornerstone of liquid biopsy, ctDNA is the fraction of cell-free DNA (cfDNA)
which originates from dying tumor cells or macrophages that have phagocytized tumor
cells(28). As ctDNA contains genetic mutations identical to those of their originating tumor
cells, ctDNA can be used to identify heterogeneous tumor-specific mutations and epigenetic
changes, and to monitor tumor dynamics in patients who are undergoing therapy. ctDNA is
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already used for treatment response monitoring or early detection of relapse, and also to
identify specific mutations to make therapy decisions(29–31). Similar to CTCs, the clinical
application of ctDNA is challenged by the technical difficulty of identifying ctDNA in the
background of a significantly larger amount of cfDNA from other tissues. Also, it is unclear
whether ctDNA accurately represents the genetic make-up of actively proliferating and
metastasizing tumor cells, as ctDNA may disproportionately represent DNA from “weaker”
tumor cells that are more prone to dying and releasing their contents into circulation(32).
Compared to ctDNA, the main advantage of CTCs is that intact, viable tumor cells are being
captured. As long as their cellular integrity is preserved, the captured CTCs can be used for
functional assays and be cultured to evaluate drug resistance(33).

CTC Detection and Isolation

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Several systems have been developed to improve the detection of CTCs, utilizing their
distinct physical and molecular characteristics. The platforms can largely be broken down
into three categories of immunoaffinity-based enrichment, biophysical property-based
enrichment, and enrichment-free methods (Table 1).

Immunoaffinity—Immunoaffinity-based CTC enrichment techniques use antibodies

against cell surface markers tethered to the device surface or a magnetic substance. Positive
enrichment refers to capturing CTCs by using antibodies against specific tumor-associated
antigens expressed on CTC surfaces, while negative enrichment refers to depleting the
hematopoietic cells in the background by using antibodies against CD45(34). Until recent
years, CTCs were defined as nucleated EpCAM+/CK+/CD45- cells. The only FDA-
approved CTC diagnostic technology, the CellSearch™ system, uses an immunomagnetic
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separation system of ferrofluid beads coated with antibody to EpCAM (anti-EpCAM)(35).

Microfluidic devices such as the CTC-Chip™ are composed of antibody-coated microposts
with a geometric arrangement to optimize cell attachment(36). The CTC-iChip™ combines
microfluidic and immunomagnetic technology and has demonstrated a greater sensitivity of
CTC detection compared to CellSearch™(37). In parallel, Wang et al. pioneered a unique
concept of “NanoVelcro™”, which utilizes antibody-coated nanostructured substrates with

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integrated microfluidic chaotic mixers to facilitate CTC-substrate contact to enhance CTC

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capture(38).

Epithelial markers such as EpCAM are often down-regulated or lost during the epithelial to
mesenchymal transition (EMT)(39). These CTCs with EMT phenotype have highly
metastatic properties and can escape positive enrichment systems that target the epithelial
markers such as EpCAM and CK. Given such limitations of relying on epithelial markers,
positive enrichment strategies targeting stem cell markers (e.g., CD133), mesenchymal
markers (e.g., vimentin), and cancer-specific antigens (e.g., HER2, PSMA) have also been
developed(40).

However, even antibody cocktails targeting a wide variety of antigens may not account for
the heterogeneity of CTC antigens. Negative enrichment strategies address this by targeting
and removing background hematopoietic cells. There are commercialized platforms
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dedicated to negative enrichment, and some of the positive enrichment technologies such as
magnetic activated cell sorting (MACS) and CTC-iChip™ can also be used for negative
enrichment by replacing anti-EpCAM with anti-CD45(34). While they allow for higher
sensitivity compared to positive enrichment technologies, negative enrichment technologies
alone typically have a much lower purity(41).

Biophysical Properties—Compared to blood cells, CTCs are distinguished by their large

size, mechanical plasticity, and dielectric mobility properties. This enables them to be
isolated using techniques such as membrane filtration, density gradient stratification, inertial
focusing, and dielectric mobility. These so-called “label-free” methods are increasingly
popular, as they avoid the challenges of targeting numerous specific antigens. Also, they
make downstream processing easier as the isolated CTCs are not tagged with antibodies(34).
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Microfiltration utilizes the larger, more rigid phenotype observed by the CTCs. Isolation by
Size of Epithelial Tumor Cells (ISET™)(42) and ScreenCell™ (43) use track-etched
membranes, while CellSieve™(44) and Flexible Micro Spring Array (FMSA) ™(45) use
photolithography to construct membranes that minimize captured cell damage. The Cluster-
Chip™ is a unique 3D microfiltration system, specifically designed to capture CTC
clusters(46). The main advantage of microfiltration is its ability to rapidly process blood for
CTC enrichment. However, microfiltration systems are subject to clogging, and size overlap
between leukocytes and CTCs makes it challenging to achieve high purity(34).

Density-based gradient centrifugation utilizes the differences in specific gravities of

leukocytes and CTCs(47). Though not initially developed for CTC isolation, Ficoll-Paque™
has been able to detect CTCs in patients with various cancers(48). OncoQuick™ combines
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centrifugation and filtration and has superior CTC capture ratio compared to Ficoll-Paque™
(49). Finally, the RosetteSep™ CTC Enrichment Cocktail integrates immunoaffinity with
density centrifugation, using antibody complexes targeting an extensive mixture of
antigens(50). Centrifugation is widely used for CTC isolation due to its reliability and
inexpensiveness, but it is best used as an initial step prior to additional enrichment using
other strategies(34).

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Inertial focusing utilizes the differential inertial effect on different sizes of cells to help with
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the isolation of CTCs. It is applicable to CTCs larger than background hematologic cells.
This method has been combined into many microfluidic devices(51). By establishing a
model accounting for wall interaction force, shear gradient lift force and secondary-flow
drag force as well as other effects including particle properties (i.e., cells in CTC isolation
devices), rotation, interparticle spacing, and fluid properties, researchers have recently had
significant improvements predicting the motion of cells according to inertial focusing in
microfluidic devices.(52) These devices have shown promising results with a high sensitivity
of CTC detection and viability of retrieved CTCs(53).

Another novel technology, dielectrophoresis separates cells using distinct electrical

fingerprints between different cell types(54). The dielectric characteristics of cancer cells are
significantly different from blood cells. By applying an alternating electrical field, cells are
electrically polarized, and CTCs can be isolated through differential electric forces. Becker
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et al. has demonstrated differential dielectric parameters in breast cancer cell lines,
lymphocytes and erythrocytes. The dielectric differences between cancer and blood cells
may result from the different morphologic features of the cell membrane including
microvilli, membrane folds and blebbing.(54) DEPArray™ can trap single cells in
dielectrophoresis cages and has been used to recover single CTCs for highly specific genetic
analyses(55).

Enrichment-Free Methods—All enrichment technologies require verification of the

captured cells, which can be significantly time-consuming. Advancements in the field of
high-speed, fluorescence imaging have led to the development of imaging platforms that
enable a direct identification of CTCs from blood samples without the enrichment step(56).
Imaging flow cytometry distinguishes CTCs from leukocytes using parameters such as
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higher karyoplasmic ratio and size(57, 58). Another unique, in-vivo direct imaging method
to detect CTCs is photoacoustic flow cytometry (PAFC), which enables real-time detection
of CTCs in veins using a laser-based technology(59). Lastly, there are functional assays to
detect CTC which exploit aspects of liver cellular activity such as secretion of tumor-
associated proteins and preferential adhesion of CTCs to a specialized matrix(60).

CTCs as a Biomarker in HCC

EpCAM-based CTC detection in HCC—The commonly used, immunoaffinity-based
CTC enrichment techniques such as CellSearch™ have been used to detect and capture
EpCAM+ CTCs in patients with HCC (Table 2). In 2013, Sun et al. used CellSearch™ to
detect EpCAM+ CTCs in HCC patients undergoing tumor resection. The authors
preoperatively detected EpCAM+ CTCs in 67% of HCC patients. A preoperative CTC count
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of two or greater was shown to be a predictor of tumor recurrence after surgery(61).

Additional studies using CellSearch™ demonstrated that the presence of EpCAM+ CTCs in
HCC patients was associated with vascular invasion(62, 63), significantly elevated AFP(63),
more advanced BCLC stage(62), disease progression(64), higher recurrence rate(65), and
shorter overall survival(62–66). In 2014, Guo et al. established an optimized platform based
on negative enrichment and qRT-PCR for the detection of EpCAM+ CTCs and exhibited
76.7% consistency with the CellSearch™ system(67). In 2016, Wang et al. used CTC-

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BioTChip to detect EpCAM+ CTCs in 60% of 42 HCC patients and found significant
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correlations between both the positive rate and number of CTCs with TNM staging(68). In
2016, Zhou et al. studied the prognostic value of EpCAM+ CTCs and regulatory T cells
(Treg) in HCC patients and found that elevated EpCAM+ CTC and Treg levels were
associated with early recurrence and poor clinical outcome(69). Unfortunately, EpCAM-
based CTC enrichment platforms are limited by the loss of epithelial markers in CTCs
undergoing EMT. Furthermore, a study showed that only a small proportion (30–40%) of
HCC cells express EpCAM(70).

Combined Markers based CTCs detection in HCC—To overcome the low sensitivity
of EpCAM-based CTC detection platforms in HCC, alternative markers have been
investigated (Table 2). For example, asialoglycoprotein receptor (ASGPR), a transmembrane
protein exclusively expressed on hepatocyte surfaces, has been used in a variety of
enrichment methods for CTC detection in HCC patients(71–78). In 2011, Xu et al.
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performed immunomagnetic separation using HCC cells bound by biotinylated asialofetuin,

a ligand of ASGPR, and was able to detect CTCs in 81% of HCC patients(71). Both the
presence of CTCs and the quantity of CTCs significantly correlated with tumor extent, portal
vein tumor thrombus, and differentiation status(71). In 2014, the same group used a
synthetic anti-ASGPR antibody instead of ASGPR ligand with successful CTC detection in
89% of HCC patients(73). Another study achieved an even higher CTC detection rate of
91% in HCC patients by using a mixture of antibodies against ASGPR and CPS1, combined
with negative enrichment(75). In 2016, Zhang et al. used a CTC-Chip with antibodies to
ASGPR, P-CK and CPS1 and isolated CTCs from 100% of 36 HCC patients(76). Recently,
Court et al. used the NanoVelcro CTC Assay with a multimarker panel of EpCAM, ASGPR,
and GPC3, and captured CTCs in 97% of patients with HCC(78).
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Label-Free (Biophysical and Enrichment-Free) Methods of CTC detection in

HCC—“Label-free” methods have also been investigated for CTC detection in HCC
patients (Table 2). In 2000, Vona et al. used the ISET™, a 2-D microfiltration system, to
detect CTCs in HCC patients undergoing liver resection, and was able to capture tumor
microemboli during surgery(42). Vona et al. also found that the number of CTCs detected by
ISET™ correlated with the presence of a diffuse tumor, portal tumor thrombosis, more
advanced liver disease, as well as shorter survival(79). A direct comparison of CellSearch™
and ISET™ in their ability to detect CTCs in HCC patients revealed a significantly superior
performance by ISET™ (ISET™: 100%, CellSearch™: 28%)(80). However, one
disadvantage of the ISET™is the difficulty of releasing CTCs for downstream genetic
analysis(27).
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In 2016, Liu et al. used imaging flow cytometry (IFC) to detect CTCs from blood samples of
HCC patients, utilizing a higher karyoplasmic ratio (HKR) seen with CTCs. When
compared to the traditional CTC detection method based on CD45- EpCAM+ cells, IFC
using HKR cells showed significantly greater AUROC of 0.82 vs. 0.73(57). Ogle et al.
studied IFC focusing on cell size and compared its effectiveness to a multi-marker panel
consisting of CK, EpCAM, AFP, GPC-3, and DNA-K. IFC with the inclusion of size
criteria, combined with the depletion of CD45 cells, led to the capture of all positive

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biomarker CTCs, as well as an additional 28% of CTCs which did not express any of the
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biomarkers tested(58). The advantages of IFC are its ease and cost-effectiveness without
requiring antibodies or complex handling of blood samples. However, its specificity could be
limited by the presence of immune cells which also have large size and high karyoplasmic
ratios(57).

Biological Characterization and Heterogeneity of CTCs—Multi-marker

combination panels and downstream molecular analysis can reveal biological characteristics
and heterogeneities of HCC CTCs (Table 2). Identification of CTCs expressing specific
tumor markers and drug targets allows providers to predict response to therapy. Shi et al.
used three CTC markers MAGE3, survivin, and CEA for evaluating the efficacy of
cryosurgery on unresectable HCC, and found that they were reliable in predicting treatment
response(81). In 2016, Li et al. used the expression status of pERK and pAkt to detect CTCs
in 93% of patients with advanced HCC undergoing sorafenib treatment(82). Among
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different patterns of pERK/pAkt expression, presence of pERK+/pAkt- CTCs was

significantly associated with longer progression free survival and higher rate of response to
sorafenib(82). Winograd et al. evaluated for CTCs expressing programmed death-ligand 1
(PD-L1) in HCC patients, and found that presence of PD-L1+ CTCs discriminated HCC
patients with early stage and advanced/metastatic disease. Of six patients receiving anti-PD1
therapy, all three patients demonstrating response had PD-L1+ CTCs, compared to only one
of three non-responders, suggesting the possibility that PD-L1+ CTCs might be predictive of
treatment response to immunotherapy(83). In 2017, Kalinich M et al. also devised a unique
strategy of using CTC-iChip™ to isolate CTCs, then applying RNA-based digital PCR to
detect a panel of liver-specific transcripts which were combined into a single metric CTC
score. The CTC score was highly correlated with BCLC staging and declined in patients
receiving therapy(84).
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Expression of EMT markers such as twist and vimentin in CTCs of HCC patients have been
studied as markers for predicting recurrence and prognosis. The majority of these studies
used the CanPatrol™ CTC analysis platform, a two-step technique including microfiltration
and subsequent characterization of CTCs using a variety of epithelial (EpCAM, CK8/9/19)
and mesenchymal markers (Vimentin and Twist)(85). Using this method, Chen et al.
detected CTCs in 95% of 195 HCC patients(86). The proportion of hybrid and mesenchymal
CTCs was associated with increased age, BCLC stages, metastasis, and AFP levels(86).
Wang et al. studied 62 HCC patients undergoing surgical resection and found that patients
with a higher number of mesenchymal CTCs had increased risk of recurrence and shortened
disease-free survival(85). Other studies using CanPatrol™ showed a consistent association
between mesenchymal CTCs and poor clinical outcomes(87–91).
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An emerging body of literature suggests profound heterogeneity among the HCC CTCs.
D’Avola et al. developed a novel analytical technique that combines IFC and single-cell
mRNA sequencing (92). Genome-wide expression profiling of CTCs using this approach
demonstrated significant transcriptome heterogeneity, even amongst CTCs from the same
HCC patient(92). Such marked heterogeneity among CTCs was also reported by Sun et al.,
who found significant heterogeneity in the EMT status of CTCs across different vascular
compartments, with predominantly epithelial phenotype at release but switching to EMT

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phenotype during transit(93). A recent study demonstrated even more heterogeneity among
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CTCs, both in size and karyotype. Wang et al. used subtraction enrichment and
immunostaining-fluorescence in situ hybridization (SE-iFISH), which employs in situ
characterization of phenotypes and karyotypes to examine both chromosomal aneuploidy
and tumor marker expressions. Using this novel strategy, the authors discovered the
existence of small HCC CTCs smaller than WBCs, with a very high prevalence of
chromosome 8 aneuploidy. The postsurgical quantity of triploid or multiploid CTCs
significantly correlated to poor prognosis(94).

Conclusion and Future Directions

Decades of research have led to significant advancements in the clinical utility of CTCs in
patients with HCC (Figure 1). As a form of liquid biopsy, CTCs hold great potential to
facilitate the implementation of precision medicine in patients with HCC. CTCs are already
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FDA-approved for disease monitoring and prognostication in patients with metastatic breast
and colorectal cancer, and there is an ongoing NIH-sponsored clinical trial (NCT02973204)
to investigate CTCs and ctDNA as clinical support tools in HCC. However, significant
challenges remain before CTCs can be adopted for widespread clinical use in HCC. In
addition to the technical challengs with CTC detection and isolation, there are numerous
CTC detection methods, each with its own protocols for sample preparation, enrichment,
and analysis. Most studies are small, single-center, case-control studies with widely varying
patient demographics such as ethnicity, etiology of liver disease, and stage of HCC, which
makes validation studies very difficult. Therefore, there needs to be a standardized assay
protocol with high sensitivity and specificity that can capture the full spectrum of CTCs.
This can potentially be achieved by multicenter, prospective studies with a larger sample size
using a uniform CTC detection platform, which can provide effective validation of the
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findings(95).

Acknowledgments
Financial Support: NIH grant R01CA172086 (SC Lu)

List of Abbreviations
AFP alpha-fetoprotein

AUROC area under the receiver operating characteristic curve

BCLC Barcelona Clinic Liver Cancer

CTC circulating tumor cell

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DEP dielectrophoresis

EMT epithelial to mesenchymal transition

FDA Food and Drug Administration

FMSA flexible micro spring array

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HCC hepatocellular carcinoma

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HCV hepatitis c virus

HKR higher karyoplasmic ratio

IFC imaging flow cytometry

ISET isolation by size of epithelial tumor cells

NAFLD non-alcoholic fatty liver disease

NPV negative predictive value

PACF photoacoustic flow cytometry

PPV positive predictive value

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RT-PCR reverse transcriptase polymerase chain reaction

scRNA-seq single-cell mRNA sequencing

SE-iFISH subtraction enrichment and immunostaining-fluorescence in situ

hybridization

TNM tumor, node, and metastases

Treg regulatory T cells

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Figure 1.
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Circulating Tumor Cells in Hepatocellular Carcinoma

HCC exhibits epithelial characteristics with surface expression of epithelial markers such as
EpCAM and CK, as well as hepatocyte-specific markers such as ASGPR and GPC3. HCC
cells enter the bloodstream to become CTCs in different ways - (1) maintaining the epithelial
characteristic, (2) undergoing EMT with the expression of mesenchymal markers such as
Twist and Vimentin, and (3) as CTC clusters consisting of multiple tumor cells as well as
RBCs, platelets, stromal cells and fibroblasts. Upon entering the circulation, most CTCs get
destroyed by anoikis, shear stress or immune attack. Some epithelial CTCs undergo EMT in
transit. Eventually, CTCs that survive have acquired a more metastatic molecular profile
with increased survival and decreased apoptosis. CTC clusters are protected from the
processes that would kill isolated CTCs. Both the individual CTCs and CTC clusters that
survive go on to metastasize and develop highly heterogeneous genetic, epigenetic and
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transcriptomic heterogeneity as well as polyploidy. The CTCs and CTC clusters can be
collected and isolated from the peripheral blood via a variety of different enrichment
techniques and can help with early detection, prognostication, and molecular
characterization of HCC.
Abbreviations: CTC, circulating tumor cells; EMT, epithelial to mesenchymal transition;
HCC, hepatocellular carcinoma

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Table 1.

CTC Detection Technologies

Subcategory Key Features Ref.

Ahn et al.

Capture yield*
Immunoaffinity
CellSearch Immunomagnetic Ferrofluid beads functionalized with anti-EpCAM. The only FDA-approved platform for CTC enumeration in
≥85% 35
metastatic prostate, breast and colorectal cancer.

MACS Immunomagnetic MNPs conjugated to various antibodies. Large surface area to volume ratio. Can be used for both positive/negative
40–90% 34
enrichment.

SERS Immunomagnetic MNPs used as the CTC enrich platform and the SERS signal amplification substrate. Dual-selectivity using anti-
> 90% 77
ASGPR nanoparticles and anti-GPC3 nanorods for HCC CTC isolation.

SE-iFISH Immunomagnetic, in situ Combines subtraction enrichment followed by in situ phenotypic and karyotypic characterization, especially useful for
70–87% 94
karyotyping identifying chromosome aneuploidy.

CTC-Chip Microfluidic Microposts (μpCTC-Chip) with geometric arrangement to generate laminar flow to optimize cell attachment.
Herringbone-shaped grooves (HBCTC-Chip) and microvortices to increase cell contact toward antibody-coated > 60% 36
surfaces. High purity, process whole blood.

CTC-iChip** Microfluidic, Sequential steps of micropillar array, inertial focusing and magnetophoresis (isolation of nucleated cells including
Immunomagnetic, Inertial CTCs and WBCs using deterministic lateral displacement, alignment of nucleated cells in a microfluific channel and 97% 37
Focusing collection of magnetically tagged cells). Combining strengths of microfluidics and magnetic-based cell sorting.

NanoVelcro** Microfluidic Anti-EpCAM/anti-ASGPR/anti-GPC-3 coated nanosubstrates, with integrated microfluidic chaotic mixers to facilitate
CTC-substrate contact to achieve enhanced HCC CTC capture. Vimentin(+) CTCs identified as a poor prognostic 80–94% 38
subset in HCC.

Biophysical property
ISET Microfiltration Size-based isolation of CTCs that are usually larger than hematologic cells. Track-etched membranes with nano to
N/A 42
micron-sized pores in thin polycarbonate films.

ScreenCell Microfiltration Size-based isolation of CTCs that are usually larger than hematologic cells. Track-etched membranes with nano to
74–91% 43
micron-sized pores in thin polycarbonate films.

CellSieve Microfiltration Precision pores arranged in arrayed patterns to enable CTC capture under low-pressure state and preserve intracellular

Hepatology. Author manuscript; available in PMC 2022 January 18.

83–91% 44
architecture.

FMSA Microfiltration Flexible polymer micro springs minimize cell damage. Allows rapid enrichment directly from whole blood. 90% 45

CanPatrol Microfiltration, Microfiltration followed by detection of EMT markers using RNA in situ hybridization.
80–89% 85
Immunomagnetic

Ficoll-Paque DGC Inexpensive, easy-to-use in combination with other techniques. The ratio of CTCs to PBMCs remain unchanged. 84% 48

OncoQuick DGC Microfiltration Porous membrane above separation media for additional separation by filtration. Superior CTC capture ratio
87% 49
compared to Ficoll-Paque.

RosetteSep CTC DGC Immunomagnetic Antibody complexes targeting an extensive mixture of CTC antigens. Can be used with centrifugation platforms such
36–60% 50
Enrichment Cocktail as Ficoll-Paque to enhance capture efficiency.

DEPArray Dielectrophoresis Traps single cells in dielectrophoresis cages generated via an array of electrodes. Can recover single CTCs. N/A 55
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Subcategory Key Features Capture yield* Ref.

Enrichment-free
ImageStream IFC Utilizes size and karyoplasmic ratios to detect CTCs, without requiring antibodies or complex handling of blood
N/A 57
samples.
Ahn et al.

PAFC In vivo direct imaging Absorbtion of laser by nanoparticles which are tagged on targeted cells via antibodies. Enables real-time detection of
N/A 59
CTCs in veins using a laser-based technology.

EPISPOT Functional assay Detects viable CTCs at the single-cell level by identifying proteins secreted/released/shed (antibody-based) from
N/A 60
single epithelial cancer cells. Theoretically can be combined to any CTC enrichment step with live tumor cells.

CTC, circulating tumor cells; DEP, dielectrophoresis; DGC, density gradient centrifugation; IFC, imaging flow cytometry; MNP, magnetic nanoparticle; PAFC, photoacoustic flow cytometry; PBMC,
peripheral blood mononuclear cell; SE-iFISH, subtraction enrichment and immunostaining-fluorescence in situ hybridization; SERS, surface-enhanced Raman scattering
*
From cell line spiking study (the capture yield may vary in different cancer types and different generations of the technology)
**
In each processed patient sample, background 500 and 200–1,000 WBCs were non-specifically captured in the CTC-iChip and NanoVelcro platform, respectively. Other platforms did not report a reliable
purity.

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Table 2.

CTC Studies in HCC

Study Patients Method CTC Marker Main Findings

Ahn et al.

EpCAM-based CTC detection in HCC

Sun YF et al[61]., 2013 123 HCC; 5 BLD; 10 HV CellSearch EpCAM CTCs identified in 67% of preop pts, and 28% 1 month after resection. ≥2 CTCs/
7.5mL predicted recurrence.

Schulze K et al[62]., 59 HCC; 19 BLD CellSearch EpCAM CTCs identified in 31% of HCC pts, associated with advanced stage, vascular
2013 invasion and shorter OS.

Guo W et al[67]., 2014 299 HCC; 24 BT; 25 CLD; RosetteSep, EpCAM Negative enrichment and qRT-PCR-based platform had AUC 0.70, sensitivity
71 HV MACS, qRT-PCR 42.6% and specificity 96.7%. Combined with AFP level, AUC improved to 0.86
with sensitivity 73.0% and specificity 93.4%.

Kelley RK et al[63]., 20 HCC; 10 BLD Cell Search EpCAM CTCs detected in 40% metastatic HCC pts. ≥ 1/7.5mL associated with vascular
2015 invasion and decreased OS.

Wang S et al[68]., 2016 42 HCC CTC-BioTChip EpCAM CTCs detected in 60% HCC pts. Positive rate and number of CTCs highly
correlated with TNM staging.

Zhou Y et al[69]., 2016 49 HCC undergoing curative RosetteSep qRT- EpCAM CD4+CD25+Foxp3+ Pts with high EpCAM mRNA+ CTCs and CD4+CD25+Foxp3+ Treg cells had
resection; 50 HV PCR higher risk of postoperative recurrence (67% vs. 10%) and 1-year recurrence (50%
vs. 10%).

von Felden J et al[65]., 57 HCC undergoing resection Cell Search EpCAM CTCs detected in 15% HCC pts. CTC positivity associated with higher recurrence
2017 and shorter median RFS.

Shen J et al[64]., 2018 89 HCC undergoing TACE Cell Search EpCAM CTCs detected in 56% HCC pts. Higher number of CTCs associated with
mortality and progression.

Yu JJ et al[66]., 2018 139 HCC; 23 BHT Cell Search EpCAM Pts with CTC ≥2 had shorter DFS and OS compared to pts with CTC < 2.

Combined Markers-Based CTC Detection in HCC

Xu W et al[71]., 2011 85 HCC; 37 BLD; 14 OT; 20 AutoMACS ASGPR, HER2, TP53 CTCs identified in 81% of HCC pts. Positivity and number of CTCs correlated
HV with tumor size, portal vein tumor thrombus, differentiation status, and disease
extent.

Hepatology. Author manuscript; available in PMC 2022 January 18.

Li J et al[73]., 2014 27 HCC; 12 OT; 13 LC; 11 AutoMACS ASGPR; CPS1 P-CK CTCs detected in 89% of HCC pts. Anti-ASGPR specific and efficient for HCC
BLT; 7 CH; 15HV CTC enrichment. Combining anti-P-CK and anti-CPS1 was superior (96.7%) to
using one antibody alone (CPS1: 62.1%, P-CK 85.2%).

Mu H et al[74]., 2014 62 HCC; 7 CH; 15 HV MidiMACS ASGPR, GPC3, CK GPC3, ASGPR, and CK expression increased in HCC pts. PPV and NPV: 90%
and 71% for GPC3; 93% and 75% for ASGPR; 83% and 29% for CK. GPC3 and
ASGPR expression associated with decreased OS.

Liu HY et al[75]., 2015 32 HCC; 17 OT; 12 BLT; 15 Ficoll-Paque, ASGPR CPS1 CD45 depletion of leukocytes recovered more CTCs compared to ASGPR+
LC; 10 CH; 3 AH; 20 HV RosetteSep selection. Combining anti-ASGPR and anti-CPS1 improved CTC detection vs.
either antibody alone, and detected CTCs in 91% of HCC pts.

Zhang Y et al[76]., 2016 36 HCC; 14 BLD CTC-Chip ASGPR, P-CK, CPS1 CTCs detected in 100% of HCC pts. Captured CTCs readily released from CTC-
chip and could subsequently be expanded to form a spheroid-like structure in a 3D
cell culture assay.
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Study Patients Method CTC Marker Main Findings

Pang Y et al[77]., 2018 8 HCC; 5 OT; 5HV SERS ASGPR, GPC3 SERS nanoplatform detected CTCs in 100% of HCC pts, and enabled detection
with small volume of blood.

Label-Free (Biophysical and Enrichment-Free) Methods to Detect CTCs in HCC

Ahn et al.

Vona G et al[42]., 2000 7 HCC undergoing ISET RT-PCR AFP PSA CTCs detected in 43% and 86% of HCC pts before and after surgery. ISET
hepatectomy; 8 CH; 8 HV allowed subsequent analysis of cell morphology, enumeration of CTCs, and
demonstration of tumor microemboli during surgery.

Vona G et al[79]., 2004 44 HCC; 30 CH; 39 LC; 38 ISET N/A CTCs found in 52% of HCC pts, associated with portal tumor thrombosis and
HV shorter survival.

Morris KL et al[80]., 52 HCC ISET Cell EpCAM GPC3 CellSearch detected CTCs in 28%, while ISET detected CTCs in 100% of HCC
2014 Search™ pts. Presence of GPC3-positive CTCs by ISET was 100% concordant with GPC3-
positive cells in the original tumor.

Liu Z et al[57]., 2016 52 HCC; 12 HV Imaging Flow Karyoplasmic Ratio Using high karyotype ratio (HKR), imaging flow cytometry had AUROC of 0.82.
Cytometry HKR pts had significantly higher presence of microvascular thrombosis, as well as
higher risk of recurrence and mortality.

Ogle LF et al[58]., 2016 69 HCC; 16 LC; 15 HV Imaging Flow Cell Size CK, EpCAM, AFP, Size criteria + absence of CD45 led to capture of all positive biomarker CTCs, and
Cytometry GPC-3, DNA-K 28% of biomarker negative CTCs. Increased CTCs associated with advanced
tumor stage, portal vein thrombosis and poorer survival.

Biologic Characterization and Heterogeneity of CTCs in HCC

Li YM et al[72]., 2013 60 HCC; 10 BLD; 10 OT; 10 MiniMACS Flow ASGPR, Vimentin, twist, CTCs detected in 77% of HCC pts. CTC positivity higher in pts with portal vein
HV Cytometry ZEB1/2, snail, slug, cadherin thrombus and advanced stages. EMT markers highly correlated with portal vein
thrombus, TNM classification and tumor size.

Li J et al[82]., 2016 109 HCC on sorafenib Ficoll-Paque pERK pAkt pERK/pAkt expression in CTC and tissue concordant in 90%. pERK+/pAkt-
treatment RosetteSep CTCs associated with PFS and predicted good prognosis. In vitro, pERK+/pAkt-
HCC cells showed the greatest response to sorafenib.

Shi J et al[81]., 2016 47 HCC undergoing MACS RT-qPCR MAGE-3, Survivin CEA Average CTCs decreased significantly following cryosurgery. Gene expression for
cryoablation tumor markers MAGE-3, survivin and CEA all significantly decreased following
cryosurgery as well.

Kalinich M et al[84]., 63 HCC; 31 CLD; 6 LM; 38 CTC-iChip Digital AFP, AHSG, ALB, APOH, 10 liver-specific transcripts identified CTCs in 56% of untreated HCC pts vs. 3%
2017 OT; 26 HV PCR FABP1, FGB, FGG, GPC3, of pts with nonmalignant liver disease at risk of developing HCC. CTC positivity

Hepatology. Author manuscript; available in PMC 2022 January 18.

RBP4, TF declined in HCC pts receiving therapy.

Chen J et al[86]., 2017 195 HCC CanPatrol™ CK, EpCAM, Twist, CTCs detected in 95% HCC pts, able to discriminate metastatic HCC with AUC
Cadherin, Snail, Vimentin, 0.86, 86% sensitivity, 81% specificity. Mesenchymal CTCs associated with age,
AKT2 BCLC stages, metastasis, AFP levels, recurrence.

Court CM et al[78]., 61 HCC; 11 BLD; 8 HV NanoVelcro CTC EpCAM, ASGPR GPC3, CTCs detected in 97% HCC pts. Panel identified HCC with sensitivity 84.2%,
2018 Assay Vimentin specificity 88.5%, PPV 69.6%, NPV 94.7%, and AUC 0.92. Vimentin-positive
CTCs associated with aggressive disease and metastasis.

Qi LN et al[87]., 2018 112 HCC treated with R0 CanPatrol™ EpCAM, CK, Vimentin, CTCs detected in 90% HCC pts. CTC count ≥16 and mesenchymal–CTC
resection; 12 HBV; 20 HV Twist percentage ≥2% associated with recurrence and metastasis. BCAT1 gene identified
as a potential trigger of EMT.
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Study Patients Method CTC Marker Main Findings

Ou H et al[88]., 2018 165 HCC undergoing radical CanPatrol™ EpCAM, CK, Vimentin, CTCs detected in 71% HCC pts. Increased CTCs associated with higher AFP,
resection Twist multiple tumors, advanced staging, and tumor embolus. Mesenchymal CTCs
predicted earlier recurrence and shortest RFS.
Ahn et al.

Yin LC et al[89]., 2018 80 HCC; 10 HV CanPatrol™ Twist Twist+ correlated with tumor burden/aggressiveness, staging, as well as postop
recurrence and mortality.

Ye X et al[90]., 2018 42 HCC CanPatrol™ TP53 Postop CTC counts and changes in CTC counts were independent prognostic
indicators for PFS.

Cheng Y et al[91]., 2018 113 HCC; 57 BLD; 6 LM CanPatrol™ CK, EpCAM Vimentin, Twist Total number of CTCs had AUC 0.774, which improved to 0.821 when combined
with serum AFP. Mesenchymal CTCs were increased in late-stage HCC pts.

Wang Z et al[85]., 2018 62 HCC undergoing radical CanPatrol™ CK, EpCAM, Vimentin, CTCs detected in 84% of HCC pts. Pts with postop recurrence had significantly
resection Twist higher number of CTCs, mesenchymal CTCs, and mixed CTCs. Mesenchymal
CTCs associated with shortened postoperative DFS.

D'Avola D et al[92]., 6 HCC; 1 HV Single cell RNA CK, EpCAM GPC3, Single cell RNA sequencing of CTCs showed significant transcriptome
2018 sequencing ASGPR1 heterogeneity. Non-hepatic expression of ASGPR1 was detected in a significant
proportion of non-CTCs, mainly monocytes.

Sun YF et al[93]., 2018 73 HCC undergoing curative Cell Search™ qRT- EpCAM, CK, Cadherin, EMT status of CTCs was heterogeneous across different vascular compartments
resection PCR Slug, Vimentin, Snail (peripheral vein, peripheral artery, hepatic vein, portal vein, IVC).

Wang L et al[94]., 2018 14 HCC; 16 CCA; 4 GBC SE-iFISH C8 Aneuploidy Among CTCs detected, 8% were EpCAM+, and 86% EpCAM−. Small aneuploid
undergoing resection HCC CTCs were discovered. Postsurgical quantity of triploid CTCs, multiploid
CTCs or CTMs correlated to poor prognosis.

Winograd P et al[83]., 73 HCC; 8 HV; 11 BLD CTC-iChip PD-L1 Presence of PD-L1+ CTCs predicted response to anti-PD1 therapy.
2018

AH, acute hepatitis; AUC, area under the curve; BHT, benign hepatic tumor; BLD, benign liver disease; CCA, cholangiocarcinoma; CH, chronic hepatitis; CLD, chronic liver disease; CTC, circulating
tumor cell; CTM, circulating tumor microemboli; DFS, disease-free survival; EMT, epithelial to mesenchymal transition; GBC, gallbladder cancer; HBV, hepatitis B virus; HCC, hepatocellular carcinoma;
HD, hepatic disease without HCC; HV, healthy volunteers; IVC, inferior vena cava; LC, liver cirrhosis; LM, liver metastasis; NPV, negative predictive value; OS, overall survival; OT, other malignant
tumors; PFS, progress-free survival; PPV, positive predictive value; RFS, recurrence-free survival

Hepatology. Author manuscript; available in PMC 2022 January 18.

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