Correlation between peripheral blood circulating tumor cell level and tumor response in advanced hepatocellular carcinoma patients treated with immunotherapy and molecular targeted therapy

doi:10.21203/rs.3.rs-4715651/v1

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Research Article

Correlation between peripheral blood circulating tumor cell level and tumor response in advanced hepatocellular carcinoma patients treated with immunotherapy and molecular targeted therapy

https://doi.org/10.21203/rs.3.rs-4715651/v1

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Background

Circulating tumor cells (CTCs) are closely associated with hepatocellular carcinoma (HCC) progression, but the value of CTCs in systemic treatment remains to be explored.

Methods

One hundred and twenty-five patients with advanced HCC who underwent systemic treatment were included in the study. Five millilitre peripheral blood were drawn before treatment, one month, three months, six months, and one year after systemic treatment. isolation by size of epithelial tumor cells (ISET) is used to capture circulating tumor cells. The tumor response to systemic treatment was assessed by modified response evaluation criteria in solid tumors criteria (mRECIST). The correlation between the dynamic change of peripheral blood CTCs level and tumor response to systemic treatment was analysis.

Results

The objective response rate (ORR) and disease control rate (DCR) were 39.2% (49/125) and 82.4% (103/125) respectively. Dynamic CTCs analysis showed that the CTCs levels of patients with (progressive disease) PD status continued to be at a high level and gradually increased. The CTCs levels of patients in complete response (CR), partial response (PR), and stable disease (SD) status gradually decreased as treatment continued. Patients with higher initial CTCs levels have worse tumor treatment response. Patients with less than 15 initial CTCs, less than 13 CTCs in the first month, and less than 10 CTCs in the third month are more likely to achieve objective response and have a better response to tumor treatment. The PFS and OS were better than those of patients with more than 15 initial CTCs, more than 13 CTCs in the first month, and more than 10 CTCs in the third month.

Conclusions

The number of CTCs in peripheral blood is closely related to tumor treatment response in patients with advanced HCC. Detecting the number of CTCs in patients has important clinical significance in predicting tumor treatment response and evaluating patient prognosis.

circulating tumor cells

hepatocellular carcinoma

systemic treatment

tumor treatment response

modified response evaluation criteria in solid tumors criteria

Hepatocellular carcinoma (HCC) stands out as the primary liver cancer subtype, making up over 90% of cases[1]. Various risk factors contribute to HCC, including chronic hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infection, alcohol misuse, non-alcoholic steatohepatitis (NASH), autoimmune liver conditions, drug-induced liver harm, and exposure to aflatoxin[2]. Despite advancements in treatment, HCC has a grim outlook with a 5-year survival rate of 15–38% in the United States[3, 4] and Asia[5], mainly due to late detection, resistance to chemo, and frequent recurrence and spreading. Processes like surgical removal, radiofrequency ablation, and transarterial chemoembolization work well for localized liver HCC, while drugs targeting the tumor microenvironment (TME) offer options for unresectable HCC[6]. The debut of Sorafenib that prolonged survival for unresectable HCC patients started the evolution of systemic therapies involving molecular-targeted agents (MTAs) as a valuable treatment route for advanced HCC. Broadly used multi-kinase inhibitors like Sorafenib, Lenvatinib, Regorafenib, and Cabozantinib, alongside the vascular endothelial growth factor (VEGF) blocker Ramucirumab, have shown significant success[7–9]. Besides MTAs, novel strategies like immunotherapy based on immune checkpoint inhibitors (ICIs) have made headway recently. Combining ICIs with VEGF inhibitors has proven more effective than sorafenib for advanced HCC treatment[10], with Atezolizumab and Bevacizumab now a primary therapy option for advanced HCC patients.

Currently, we usually use modified response evaluation criteria in solid tumors criteria (RECIST/mRECIST) to evaluate tumor treatment response in advanced liver cancer to evaluate the effect of systemic treatment in patients. In addition, monitoring changes in alpha fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) levels is also crucial for evaluation of disease prognosis.

Liquid biopsy has emerged as a promising source of novel biomarkers in recent years and has been extensively utilized in advanced HCC for monitoring tumor recurrence and evaluating the effect of tumor treatment response and surveilling treatment resistance[11]. Consequently, there has been a growing interest in liquid biopsy studies evaluating the predictors of response to systemic treatment, including immunotherapy, molecular-targeted agent therapy. Components of liquid biopsy include circulating tumor DNA (ctDNA), CTCs, various lymphocyte subpopulations, exosomes, and metabolites, among others. Liquid biopsy offers numerous advantages over traditional single-site tissue sampling or serum protein markers, such as increased sensitivity and specificity, non-invasiveness, dynamic monitoring, and most importantly, overcoming spatial and temporal heterogeneity limitations[12]. For patients with HCC, obtaining tissue samples can be challenging, and a single biopsy may not accurately represent the entire genetic profile, especially in cases of multiple or large tumors. During the course of treatment (transarterial chemoembolization, targeted therapy, and immunotherapy), clonal evolution under selection pressure can lead to changes in the tumor genome, resulting in drug resistance and treatment failure. However, genetic information obtained from tissue biopsies prior to treatment does not always inform treatment decisions. Despite the invasiveness and impracticality of repeated tumor biopsies in clinical settings, liquid biopsy offers a non-invasive alternative, with ctDNA and CTCs genetic information found to be representative of the tumor genome[13]. Therefore, utilizing liquid biopsy can provide a more comprehensive view of the tumor genome and enable real-time monitoring of genetic changes during therapy[14]. Overall, liquid biopsy can assist in identifying eligible HCC patients for immunotherapy based on various criteria.

Therefore, a prospective observational study was conducted to explore the relationship between changes in peripheral blood CTCs levels and tumor treatment response and long-term prognosis.

Patients

A total of 125 patients undergoing liver resection were enrolled consecutively from June 2019 to March 2022, at the Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. The inclusion criteria encompassed the following: (1) individuals aged over 18, (2) a clinical diagnosis of HCC confirmed through AFP and imageological examination, (3) initial immunotherapy and molecular targeted therapy (I-O and MTT), (4) initial detection of the tumor, (5) being classified as Child-Pugh class A or B, and (6) having an Eastern Cooperative Oncology Group (ECOG) performance status between 0 and 1. On the other hand, patients were excluded if they met any of the following criteria: (1) a history of prior anticancer treatment, (2) incomplete records for follow-up, or (3) failure to comply with drug therapy. The study was approved by the Ethical Committee of Tongji Medical College of Huazhong University of Science and Technology [TJ-IRB20191101], and all procedures followed the Declaration of Helsinki. Informed consent was obtained from all the patients included in the study.

Trial design

This study was an observational clinical study. One hundred and twenty-five patients received conventional immunotherapy and molecular targeted therapy (I-O and MTT). In addition, transcatheter arterial chemoembolization (TACE), radiotherapy and other treatments were individually administered according to standard for diagnosis and treatment of primary liver cancer (2019 edition). The time of blood collection were before treatment, first month, third month, sixth month and first year after treatment. Five-millilitre blood samples were drawn to detect CTCs by the isolation by size of epithelial tumor cells (ISET) method. This involves blood filtration and analysis by microscopy using standard histopathological/cytomorphological criteria[15, 16]. The ISET technique—membrane filtration and separation of tumour cells—is based on differences in size and deformability between tumour cells and blood cells. The specific tumour cell stain also identifies the captured CTCs. In this study, the ISET technique combined with blood filtration was applied to isolate CTCs, and histopathological criteria were used to analyse the CTCs by microscopy. The ISET instrument filtered the blood to capture CTCs by a polycarbonate membrane with an 8 µm pore. An experienced cancer cytologist identified the degree of malignancy using histopathological criteria. CTCs were defined with respect to the following six characteristics: a) abnormal karyotypes, such as lobulated nuclei; b) cell diameter larger than 15 µm; c) irregular, dented or shrivelled nuclear borders; d) nucleus-to-cytoplasmic ratio > 0.8; e) giant nucleoli and f) non-homogeneous nuclear staining. Cells meeting at least 4 of these criteria were identified as CTCs. In addition, if giant nucleoli or abnormal karyotypes appeared and at least 2 other criteria were met, the cells were also identified as CTCs. In our study, a total of 461 blood samples were detected. Blood samples were not collected from 75 patients at the twelfth months, 42 patients at the sixth months and 20 patients at third months due to death and loss of follow-up. In addition, 27 blood samples were substandard due to poor preservation condition. Therefore, the test rates of blood samples at initial treatment and first month, third month, sixth month and twelfth month were 100% (125/125), 97.6% (122/125), 76.0% (95/125), 61.6% (77/125) and 32.8% (42/125) respectively.

CTCs detection

The ISET procedure (CTCBIOPSY® detection system, YZY-CTC-D100, Wuhan Youzhiyou Medical Technology Co., LTD. (China))[17] utilized a filtration module, generously provided by Youzhiyou company, along with a polycarbonate membrane of Track-Etch type. The membrane featured cylindrical pores calibrated to 8 µm in diameter. There were 12 wells in the filtration module, enabling the simultaneous filtration of 12 distinct samples. Each sample underwent filtration over a surface area of 0.6 cm in the membrane. Five milliliters of peripheral blood from carcinoma patients or healthy volunteers were obtained in EDTA buffer. The blood was then diluted at a ratio of 1:10 with filtration buffer consisting of 0.175% saponin, 0.2% paraformaldehyde, 0.0372% EDTA, and 0.1% bovine serum albumin. After resting for 10 minutes at room temperature, the blood was subjected to filtration. Each well received ten milliliters of the diluted solution, equivalent to 1 ml of undiluted blood, and underwent gentle vacuum filtration using a vacuum pump. Following filtration, the membrane was rinsed once with phosphate-buffered saline (PBS), detached from the filtration module, and air-dried. Subsequently, one or more areas, each representing 1 ml of filtered blood, were stained with hematoxylin and eosin (H&E) or May-Grumwald-Giemsa, washed with PBS, and dried. For microscopic analysis, the membrane was positioned on a specific stage 8 without applying vacuum pressure.

Usage of I-O and MTT

Sorafenib (Nexavar®, Bayer, Germany) orally at a dose of 400 mg twice daily[18], Lenvatinib (Levima®, Eisai, Tokyo, Japan) orally at a dose of either 8 mg/day for patients < 60 kg or 12 mg/day for those ≥ 60 kg[7]. ICIs included Camrelizumab [19](SHR-1210, Jiangsu HengRui Medicine Co., Ltd.), Pembrolizumab[20](KEYTRUDA, Merck Sharp & Dohme Co., Inc.), and Tislelizumab[21] (BGB-A317, BeiGene). Dose and duration of ICIs were according to the guidelines or expert consensus. All patients receiving I-O and MTT treatments had controllable adverse reactions and treatment was not stopped for controllable adverse events.

Outcome

The primary endpoint of the trial was the proportion of patients achieving a tumor treatment response (partial response [PR] or complete response [CR]) in all eligible patients. Secondary endpoints included proportion of patients achieving an objective response rate (ORR) defined as the percentage of patients who experienced a PR or CR. modified response evaluation criteria in solid tumors criteria (mRECIST) were used to determine tumor treatment response. Progression-free survival (PFS) defined as the time from start of systemic therapy to progression or death, and overall survival (OS) defined as the time from start of systemic therapy to death, with survival outcomes censored at the date of last follow-up. Tumor progression was assessed by contrast-enhanced CT or contrast-enhanced MRI every 3 months. Follow-up was terminated on June, 2023. Governmental death registration and telephone follow-ups were used to determine patient survival status.

Statistics analysis

Statistical analysis was performed using IBM SPSS Statistics version 22 (SPSS Inc., Chicago, USA). The statistical graphs were drawn using GraphPad Prism version 10. Continuous variables were presented as the median and interquartile range (IQR) and categorical data as number and percentage. χ² test or Mann-Whitney U test was used for comparison between groups where appropriate. The receiver operator characteristic (ROC) curve is used to evaluate the ability of CTCs to predict prognosis and confirm the optimal cut-off value. The relationship between the prognosis and different groups was analyzed using Kaplan-Meier curves. A two-sided P < 0.05 was considered statistically significant.

Demographics

The large number of patients are male (80.8%). 80.8% of patients were infected chronic hepatitis B virus infection. One hundred and sixteen (92.8%) patients were classified as Child–Pugh class A, indicating well-preserved liver function, and 84 patients had an ECOG performance status of 0 at baseline. More than half of the patients had PVTT. BCLC C stage accounted for 71.2% of patients. Distant metastases were detected at the beginning of treatment in 17.6% (22/125) of patients including lung metastases (8%), bone metastases (1.6%), peritoneal cavity metastases (0.8%) and multiple metastases (7.2%). All patients were treated with local therapy combined with systemic therapy. Of these, 92 (73.6%) patients received TACE, 7 received microwave ablation, 25 received radiation, and the rest received multiple local treatments. The mean follow-up time was 26 months (median, 24 months, range, 15–48 months). By the end of follow-up, 81 people had died, accounting for 61.8%.

Tumor response

The waterfall plot in Fig. 1 shows the tumor treatment response of all patients. six patients achieved CR. 33.6% (43/125) patients were PR and 43.2% (54/125) patients were SD. The DCR was 82.4% (103/125) and the PD was 17.6% (22/125). None of the patients discontinued or died because of adverse effects, but 14 patients (11.3%) were hospitalized for serious complications, including hand-foot reactions, hypertension, and liver function damage.

CTCs and tumor response

CTCs counts for HCC and healthy controls are shown in Fig. 2A. ten healthy controls had 2 CTCs (P < 0.001). A comparison of CTCs counts between ORR and SD, PD patients is shown in Fig. 2B (17 versus 25, P = 0.021). All patients were divided into three groups: CR and PR group, SD group and PD group. Figure 2C showed that the dynamic level of CTCs counts for the three groups. We found that the number of CTCs in patients with PD status continued to increase after treatment, while the number of CTCs in patients with CR, PR and SD status decreased significantly after the third month of treatment. Before that, the number of CTCs remained high and did not decrease significantly.

The clinical value of CTCs in predicting tumor response

The analysis of optimal cut-off value of initial CTCs and initial AFP were shown in the Fig. 3A. When initial CTCs = 15, the sensitivity and specificity were 69%, 84% respectively (P = 0.028). The ROC of the combination index showed a sensitivity of 95% and a specificity of 84% (P = 0.015) (Fig. 3A). Similarly, third month CTCs = 10, as an optimal cut-off value the sensitivity and specificity were 80%, 88% respectively (P = 0.021). The ROC of the combination index showed a sensitivity of 95% and a specificity of 91% (P = 0.003) (Fig. 3B). However, the number of CTCs in the first month had no predictive value for tumor response, the sensitivity and specificity were 59%, 64% respectively (P = 0.074) (Fig. 3C).

CTCs and prognosis

The patients were divided into two groups with the optimal cut-off, and the PFS and OS were compared between the two groups respectively. Patients were divided into two groups with CTCs = 15 as the optimal cut-off value. We found that there were statistical differences in both PFS and OS between the two groups. Patients with initial CTCs < 15 had much longer PFS (not reached versus 7 months, P = 0.027) and OS (not reached versus 12 months, P = 0.014) than those with initial CTCs > = 15 (Fig. 4A and 4B). Patients with first month CTCs < 13 had longer PFS (14 months versus 6 months, P = 0.038) and OS (not reached versus 13 months, P = 0.021) than those with first month CTCs > = 13(Fig. 4C and 4D). Patients with third month CTCs < 10 had longer PFS (not reached months versus 8 months, P = 0.041) and OS (not reached versus 15 months, P = 0.021) than those with third month CTCs > = 10(Fig. 4E and 4F).

Cases

Figure 5 showed the initial contrast-enhanced CT images in a 61-year-old man with multifocal HCC in the left inner lobe. At the beginning of treatment, we gave the patient a TACE and RFA treatment and sequentially gave an I-O and MTT. Figure 5A showed the images of arterial, vein and delay phases and a total of 5 CTCs in the peripheral blood of the patient at the beginning of treatment were detected. Figure 5B shows the contrast-enhanced CT images of the patient at the 3rd month after treatment. A CTCs was detected in peripheral blood, which was significantly lower than the level at the beginning of treatment. Figure 5C shows the contrast-enhanced CT images of the patient at the 6th month after treatment. The tumor progressed in the 6th month. The lesion of left outer lobe showed obvious enhancement in arterial phase. In addition, more CTCs were detected.

Figure 6 showed the contrast-enhanced CT images at the beginning of treatment in a 67-year-old man with multifocal HCC in the liver. Most of the liver was occupied by the tumor and the patient had no extrahepatic metastasis. At the beginning of treatment, we gave the patient a TACE treatment and sequentially gave an I-O and MTT. Figure 6A showed the images of arterial, vein and delay phases and a total of 5 CTCs in the peripheral blood of the patient at the beginning of treatment were detected. Figure 6B shows the contrast-enhanced CT images of the patient at the 3rd month after treatment. The tumor progressed in the 3rd month. The lesion area of liver was not necrotic and expanded. In addition, the patient developed lung metastases. The lesion area of liver expanded and developed lung metastases. By detecting CTCs in patients, we were surprised to find that the number of CTCs in peripheral blood increased significantly, and even circulating tumor microemboli (CTM) appeared. We defined that three or more tumor cells were considered to be a CTM.

The study presents a novel technique, known as ISET, for isolating circulating tumor cells based on their size in individuals with various types of cancer. Examination of tumor cell lines from liver, prostate, breast, and cervix cancers has revealed the notably larger size of these malignant cells compared to normal white blood cells. This approach is simple, quick, cost-effective, and enables direct isolation and quantification of tumor cells in cancer patients. Previous research[22, 23] has utilized filtration methods to isolate tumor and epithelial cells from peripheral blood using immunomagnetic selection. Filtration has been employed to remove unwanted white blood cells and beads. Unlike traditional methods, ISET allows direct filtration of peripheral blood without the need for immunomagnetic or flow cytometric cell separation. In the study, we found that CTCs can be detected in the blood of most patients with hepatocellular carcinoma, while the number of CTCs detected in the blood of healthy volunteers is very small, indicating that the sensitivity and specificity of the system is high. In addition, we also found that there was a significant correlation between tumor treatment response (TTR) and CTCs level, and the more significant the patient's TTR, the more significant the decrease in CTCs level. Ye et al.[24] have demonstrated that higher CTCs counts post-surgery (> 2 and > 5) and the variance in CTCs counts before and after the operation were closely linked to PFS, presenting a more reliable indicator of treatment outcomes than just the raw numbers themselves. Consistent with the conclusions of previous studies, by dynamically detecting CTCs levels in the blood of patients, we found that the level of CTCs is synchronously present in time with the response to tumor therapy. When the disease is in remission, the number of CTCs decreases; conversely, when the tumor progresses, the level of CTCs increases.

In order to further explore the relationship between CTCs and TTR, we included AFP and CTCs indicators, and evaluated the predictive value of the above indicators for ORR. In the study, we included CTCs in the initial state, first month and third months after treatment, and explored the value of CTCs in predicting ORR by establishing a ROC model. We found that initial CTCs and third month CTCs have excellent predictive value for ORR, but first month CTCs counts after treatment has no significant predictive value for ORR. Patients with less than 15 initial CTCs were more likely to achieve ORR after treatment. Previous studies have explored the relationship between preoperative CTCs and recurrence and metastasis after hepatectomy. They found that CTCs count ≥ 16 before resection were significantly associated with early recurrence, multi-intrahepatic recurrence, and lung metastasis. Previous studies have shown that CTCs are closely related to tumor staging and tumor recurrence. The more advanced the tumor stage, the higher the CTCs level. The earlier the recurrence occurs, the more obvious the CTCs level rises in patients[17, 25]. The greater the tumor burden, the lower the response efficiency of the tumor in the early stage of treatment, and the more difficult it is to achieve objective remission of the tumor. Therefore, initial CTCs can be used as a predictor of tumor response to treatment. In addition, the response time of most tumors to systemic therapy is generally from 1 to 2 months[26]. During this period, most patients have not achieved objective remission, but the CTCs level in patients has decreased significantly. Therefore, the CTCs level is not synchronized with the tumor treatment response 1 month after treatment. On the contrary, CTCs and tumor treatment response at 3 months after treatment had better synchronization.

There were several limitations to this study. Firstly, the data we collected originated solely from one study center and a multi-centers study should be conducted. Secondly, as Asian HCC patients have a higher prevalence of hepatitis B virus infection, the use of antiviral therapy after surgery can greatly affect patient outcomes. Thirdly, HBV was the major etiology. The effect of I-O for non-HBV non-HCV hepatocellular carcinoma (NBNC-HCC) might be decreased. HBV was the major etiology. The effect of I-O for NBNC-HCC might be decreased. Previous studies have shown that compared with non-viral-related HCC, the tumor immune microenvironment of HBV–HCC had a stronger immunosuppressive effect, which was reversed by PD-1 inhibitors[27]. Finally, at present, the mainstream method of CTC capture is the immunomagnetic bead method (CellSearch™), and we only use the ISET method to capture CTCs, which are not the same in the principle of capturing CTCs. Therefore, it is necessary to include different methods of CTC capture to verify the relationship between the number of CTC and the therapeutic effect of HCC.

The number of CTC is closely related to the therapeutic effect of HCC and detecting the number of CTCs in patients has important clinical significance in predicting tumor treatment response and evaluating patient prognosis.

HCC hepatocellular carcinoma, RFS recurrence-free survival, OS overall survival, I-O and MTT immunotherapy and molecular targeted therapy, TACE transcatheter arterial chemoembolization, ROC receiver operating characteristic curve, CTC circulating tumor cell, ISET isolation by size of epithelial tumor cells, CR complete response, PR partial response, SD stable disease, PD progressive disease, ORR objective response rate, mRECIST modified response evaluation criteria in solid tumor

Ethics approval and consent to participate

The study was approved by the Ethical Committee of Tongji Medical College of Huazhong University of Science and Technology. All procedures performed in this study abided by the Declaration of Helsinki. All study participants or their legal guardian provided informed written consent about personal and medical data collection prior to study enrolment.

Consent for publication

Not applicable.

Availability of data and materials

The dataset(s) supporting the conclusions of this article is available in the [figure] repository, [10.6084/m9.figshare.20499261]

Competing interests

All the authors report no relevant conflicts of interest for this article.

Availability of data and materials

The dataset(s) supporting the conclusions of this article is available in the [figure] repository, [10.6084/m9.figshare.20499261].

Funding

This work was supported by the National Key Research and Development Program of China, No. 2016YFC0106004.

Authors’ contributions

ZWZ and WW managed patients which included recruiting patients, performing operations and making follow-up. JMZ, HFX and DC analyzed data and wrote the manuscript. BW designed the research route and modified the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Chang Shu (Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology) for her assistance of statistic.

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Table 1 Clinical characteristics of 125 patients with advanced HCC receiving I-O and MTT treatment

Clinical characteristics	No. of patients
Age years, M(IQR)	56(44-63)
Sex
Male	101(80.8%)
Female	24(19.2%)
Child-Pugh score
A	116(92.8%)
B	9(7.2%)
ECOG score
0	84(67.2%)
1	41(32.8%)
Etiology
HBV	101(80.8%)
HCV	2(1.6%)
Others	22(17.6%)
AFP (ng/mL)
Positive	113(90.4%)
Negative	12(9.6%)
PIVKA-II
Positive	118(94.4%)
Negative	7(5.6%)
Tumor diameter(cm)	7.1(6.2-9.4)
Number of tumors
Singe	19
Multiple	106
Portal vein tumor thrombosis
No	58(46.4%)
Yes	67(53.6%)
BCLC stage
B	36(28.8%)
C	89(71.2%)
CNLC stage
IIb	36(28.8%)
IIIa	67(53.6%)
IIIb	22(17.6%)
Extrahepatic metastasis
Lung	10(8%)
Bone	2(1.6%)
Peritoneal cavity	1(0.8%)
Multiple metastases	9(7.2%)
No	103(82.4%)
Combination treatments
TACE alone	78(62.4%)
TACE and RFA/MWA	12(9.6%)
TACE and radiotherapy	35 (28.0)

HCC, hepatocellular carcinoma, I-O and MTT immunotherapy and molecular targeted therapy, M median value, IQR interquartile range, ECOG, eastern cooperative oncology group, HBV hepatitis B virus, HVC hepatitis C virus, AFP alpha fetoprotein, PIVKA, protein induced by vitamin K absence or antagonist-II, BCLC Barcelona clinical liver cancer, CNLC China liver cancer staging, TACE transcatheter arterial chemoembolization, RFA radiofrequency ablation, MWA microwave ablation

No competing interests reported.

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Correlation between peripheral blood circulating tumor cell level and tumor response in advanced hepatocellular carcinoma patients treated with immunotherapy and molecular targeted therapy

Status:

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Methods

Patients

Trial design

CTCs detection

Usage of I-O and MTT

Outcome

Statistics analysis

Results

Demographics

Tumor response

CTCs and tumor response

The clinical value of CTCs in predicting tumor response

CTCs and prognosis

Cases

Discussion

Conclusions

Abbreviations

Declarations

References

Table

Additional Declarations

Status:

Version 1