Early release of circulating tumor cells after transarterial chemoembolization hinders therapeutic response in patients with hepatocellular carcinoma

Abstract

Background

Transarterial chemoembolization (TACE) is the first-line therapeutic option for patients with intermediate-stage hepatocellular carcinoma (HCC). Tumor neovascularization allows tumor growth and may facilitate the release of circulating tumor cells (CTCs) to the bloodstream after TACE. We investigated the relationship between early release of CTCs and radiological response after TACE.

Methods

Prospective, single-center study including patients with HCC undergoing a first TACE from January 2019 to June 2023. The IsoFlux® system was used to evaluate EpCAM+ CTC counts before TACE, at day 1 (D1), and at day 30 after TACE. Radiological response to TACE was assessed according to the mRECIST criteria one month after the procedure. Tumor vascularity was assessed by an interventional radiologist.

Results

In all, 48 patients with HCC undergoing TACE were included (age 64.2 ± 7.6 years, 14.6% women). CTC levels increased at D1 (114.0% [IQR 76.5%-178.0%], p = 0.019) and normalized to baseline levels in the first month after TACE (76.5% [IQR 41.3%-131.8%], p = 0.263). Higher CTC counts at baseline (p = 0.009) and at D1 (p = 0.026) were associated with tumor hypervascularity. Larger tumor size [OR: 1.9 (95% CI: 1.1–3.3), p = 0.020] and CTC increase at D1 [OR: 5.3 (95% CI: 1.3–21.0), p = 0.017] were independent predictors of non-response to TACE, especially for those patients with hypervascular lesions.

Conclusions

A meaningful release of CTCs 24 h after TACE was associated with suboptimal tumor response one month after the procedure. Future studies should evaluate the role of CTC dynamics to select candidates for adjuvant therapy after TACE and to analyze their impact on long-term outcomes.

Background

Transarterial chemoembolization (TACE) plays a pivotal role in the treatment of hepatocellular carcinoma (HCC). It consists in a selective catheterization of the arterial blood supply of the tumor to administer a chemotherapeutic agent followed by embolization to induce tumor necrosis. According to the Barcelona Clinic Liver Cancer (BCLC) staging system, TACE is the first line therapy in most patients with intermediate stage HCC (ie. BCLC stage B), which comprises unresectable multifocal HCC with preserved liver function, and absence of cancer-related symptoms, vascular invasion or extrahepatic spread. In addition, TACE may be used as a bridging therapy for patients waiting for a liver transplant or to downstage tumors beyond transplant criteria. Thus, TACE is recommended in a heterogeneous population of patients with HCC involving different tumor stages [1]. In addition, with the upcoming of novel systemic therapies, several randomized trials have explored the efficacy and safety of combining TACE with immunotherapy and/or antiangiogenics [2]. The LEAP012 trial is a phase III study that evaluates progression free survival and overall survival in HCC patients receiving TACE alone or combined with the anti-VEGFR inhibitor lenvatinib and the anti-PD-1 inhibitor pembrolizumab [3]. Another phase III study, EMERALD-3, evaluated the efficacy and safety of tremelimumab and durvalumab (a dual checkpoint blockade) with or without lenvatinib, administered in combination with TACE versus TACE alone [4]. Although further trials are needed, it seems that adjuvant therapy after TACE could improve outcomes, especially in selected patients with predictors of poor response to TACE alone.

Tumor vascularity is a critical factor that could influence the success of TACE, with poorly vascularized lesions responding worse to the treatment [5]. Tumor hypervascularity could also promote the release of cancer cells to the bloodstream [6], where they are known as circulating tumor cells (CTCs) [7, 8]. The role of CTCs to produce metastases is a complex process involving molecular changes, homotypic cell clustering and heterotypic interactions with immune and stromal cells [9], promoting invasion, intravasation-extravasation, and colonization.

Liquid biopsy refers to the detection of cancer byproducts such as circulating tumor DNA, circulating miRNA, and CTCs in biological fluids, mainly blood. Liquid biopsy may be a source of biomarkers for diagnosis, prognosis, and personalized therapy in cancer [10]. The PETAL phase Ib study, which included patients with HCC receiving a combination of TACE and immunotherapy, showed that the levels of circulating tumor DNA mirrored the radiological response of the tumor, thus confirming its monitoring potential [11]. Regarding CTCs, they can be detected in the bloodstream as single cells or as clusters, which have greater metastatic potential and survivability than single CTCs [12, 13]. Recent studies have suggested the potential of CTCs to predict outcomes in patients with early-stage HCC (BCLC 0-A) undergoing potentially curative therapies such as liver transplantation or liver resection [14, 15]. The assessment of CTCs in patients with HCC treated with TACE would allow individualizing therapeutic decisions including an earlier prescription of systemic therapies in patients with persistent CTCs after treatment, thus preventing tumor progression and prolonging survival.

Among the main methodologies used for CTC enumeration is the immunomagnetic enrichment of EpCAM⁺ cells and subsequent immunolabeling and detection of CD45⁻ cytokeratins (CK) 8⁺, 18⁺, and/or 19⁺ CTCs in whole blood. CTC counts > 2 and ≥ 6 measured by the CellSearch system before TACE were independent predictors of shorter overall survival (OS) [16] and progression-free survival in patients with HCC [17], respectively. Moreover, the mere presence of CTCs in the analysis of blood samples (i.e., CTC positivity) was proposed as candidate biomarker for adjuvant TACE administration in patients previously treated for liver resection of HCC lesions [18]. In addition to immunomagnetic enrichment, several other strategies have been used with different results to determine the role of CTCs in HCC patients undergoing TACE. These methodologies combined positive or negative cell enrichment strategies with different techniques such as immunolabeling [19], qRT-PCR [20], fluorescence in situ hybridization [21, 22], RNA in situ analysis [23], or flow cytometry [24]. IsoFlux® is an immunomagnetic bead based system that offers a much better harvest rate and purity than other methodologies for CTC isolation using EpCAM antibody [25, 26]. In general, the most used methodologies for the analysis of CTCs described above detect a low number of cells compared to the IsoFlux system.

The primary aims of the study were to describe the dynamics of CTCs after TACE in patients with HCC and to explore its impact on the therapeutic success. We also aimed to investigate whether hypervascular tumors are more prone to release CTCs after TACE and produce worse clinical outcomes.

Methods

Study design, population and TACE procedure

This prospective observational study included a consecutive cohort of adult patients diagnosed with liver cirrhosis and HCC, including patients classified as BCLC stage 0 (n = 2), A (n = 32), B (n = 11) and C (n = 3), who underwent TACE as a first line therapy from January 2019 to June 2023. The eligibility for TACE was determined in a multidisciplinary meeting as per routine clinical practice after ruling out the possibility of local ablation, resection, or liver transplantation. Patients were followed until death or March 2024. Exclusion criteria were as follows: portal vein thrombosis, severe impairment of liver function defined as Child–Pugh class C, moderate-severe ascites, prior therapies for HCC, and being admitted in the waiting list for liver transplantation at baseline.

Patients were hospitalized 24 h before TACE according to local clinical practice to verify the absence of analytical contraindications. The locoregional therapy was performed as we previously described in [27]. Under conscious sedation using intravenous midazolam, fentanyl, and local anesthesia, a 4–6 Fr vascular sheath was inserted through the femoral artery or radial artery at the discretion of the interventional radiologist. The vascular sheath was placed in the mesenteric artery to perform an indirect portography, which allowed ensuring portal vein patency. Afterwards, the sheath was advanced through the intrahepatic arterial branches to locate the typical aberrant tributaries of the tumor. Then, 100–500 micron-sized drug-eluting beads with doxorubicin were infused in the main arterial supplier of the tumor. The process was repeated in each tumor location with a supra-selective approach. Patients remained hospitalized for 24 h after the procedure and were discharged afterwards in the absence of complications. The radiological response to TACE was assessed either by dynamic computed tomography or magnetic resonance one month after the procedure using the Modified Response Evaluation Criteria In Solid Tumors (mRECIST) [28], which assess changes in tumor size and vascularity compared to baseline, stratifying patients in the following categories: complete response, partial response, stable disease and progressive disease. For the present study, we defined the group of responders as those patients who achieved complete radiological response after TACE as this is the primary aim of the procedure. The remaining categories (partial response, stable disease, and progressive disease) were grouped as non-responders. Clinical follow-up visits were scheduled at 3, 6, 9, and 12 months after TACE and then every 6 months until the end of the study. Liver function tests, kidney function and serum alpha-fetoprotein levels were determined at each visit. Patients underwent dynamic imaging techniques every 3 to 6 months to assess tumor recurrence/progression in accordance with our usual clinical practice.

All patients were required to sign an informed consent document in order to participate in the study. The research study was conducted according to the Declaration of Helsinki and the study protocol was approved by the Andalusian Research Ethics Committee (code 0117-N-18).

Evaluation of tumor vascularity

Tumor vascularity was assessed by the enhancement effect on the arterial phase of angiography during the TACE procedure by the interventional radiologist, as described in [29]. Easily visible tumors by celiac and selective angiography were classified as hypervascular lesions while tumors minimally visible by celiac angiography but evident by selective angiography were assessed as mildly vascular. Finally, those tumors described in the computed tomography or magnetic resonance at baseline, which were not detected by celiac angiography or selective angiography were assessed as hypovascular.

Sample collection and data management

Peripheral venous blood samples were obtained by phlebotomy within 24 h prior to TACE. Samples were collected in a Vacutainer tube (K2-EDTA) (BD Bioscience, Franklin Lakes, NJ, USA) and kept at room temperature until analysis (less than 24 h). Clinical characteristics and outcomes were recorded in a dedicated electronic datasheet, which was anonymized. Any missing value was retrieved from the patient electronical records. The investigators evaluating CTC enumeration were blinded to the patient clinical outcomes.

Isolation and enumeration of CTCs

CTC isolation was performed by positive-enrichment using the IsoFlux system (Fluxion Biosciences, Oakland, CA, USA), as we previously described in [27]. Briefly, the fraction of PBMCs containing the CTCs was isolated by density gradient centrifugation with Ficoll (GE Healthcare, Chicago, IL, USA) and Leucosep tubes (Greiner Bio-One, Kremsmünster, Austria), from 7 ml of peripheral blood. The resulting cellular pellet was used for the magnetic immunocapture of EpCAM⁺ CTCs, using the CTC Enrichment Kit (Fluxion Biosciences) and the IsoFlux system. Recovered CTCs were immediately fixed for subsequent immunolabeling with the CTC Enumeration Kit (Fluxion Biosciences). Fixed cells were blocked and consecutively incubated with anti-human CD45 primary antibody and anti-rabbit IgG-Cy3 secondary antibody. After permeabilizing, cells were labeled with anti-human pan-CK-FITC and stained with Hoechst 33342 dye. Finally, individual CTCs were identified as CD45⁻ CK⁺ nucleated cells in a spectral confocal microscope LSM710 (Carl ZEISS, Oberkochen, Germany). CTC clusters were defined as a group of 3 or more CTCs aggregated in tight contact. The CTC counting was performed manually from a single fluorescence image obtained by the automatic scanning of the sample, and with the ZEN Lite Blue/Black Edition free software (ZEISS). The image analysis was independently performed by two trained members of the research group, who were blinded to the clinical data. Any disagreement was resolved by a third investigator.

Genomic analysis of CTCs

The purification and amplification of genomic DNA from CTCs was carried out using the IsoFlux system and the IsoFlux NGS DNA Kit, following the manufacturer's recommendations (Fluxion Biosciences). The subsequent analysis of the presence of DNA variants (including single nucleotide variants -SNVs- and indels) across the hotspot regions of 65 oncogenes and tumor suppressor genes, with a total size of 55,199 bp, was carried out by generating sequencing-ready libraries with amplicon enrichment technology (CleanPlex OncoZoom Cancer Hotspot panel; Paragon Genomics, Fremont, CA, USA), which allows reliable detection of somatic variants present in the sample with frequencies of up to 1%. Spectrophotometry, fluorimetry and microelectrophoresis were used to determine the quality of the starting genomic DNA and the libraries at the different stages of their preparation. Sequencing was performed on an Illumina NextSeq 2000 system (sequencing by synthesis technology), using paired-end reads of 150 bp and P3 reagents of 300 cycles (sequencing kit Flow cell P3 300c). We obtained more than 100M total reads per sample and ≥ 10,000 × coverage in more than 97% of the regions analyzed with the panel. The CE-IVD certified Varsome Clinical platform was used for the analysis of the variants, thanks to a bioinformatics pipeline developed by the company specifically for the cancer hotspot panel and that allows rapid and reliable detection, annotation and interpretation of the NGS variants analyzed. For the final analysis, we set the frequency of the gene variant in the sample (allelic balance) above 1.5% and its frequency in the general population below 2%.

Statistical analysis

Statistical analyses were performed by using SPSS version 25.0 software (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as means ± standard deviations (for variables with normal distribution) or with medians and interquartile ranges (for skewed distributions). Categorical variables were expressed as frequencies (%). For comparisons involving a continuous variable, the Student T test or the Mann–Whitney U test was used depending on the distribution of the variable. The non-parametric Wilcoxon and Friedman tests were used for paired analyses. Correlation between continuous variables was assessed by the Spearman test. Regarding categorical variables the Chi-squared test was used, excepting for comparisons involving forecasted frequencies < 5, in which the Fisher test was indicated. The independent predictors of complete radiological response were assessed using multivariate logistic regression. ROC curves were derived from logistic regression, which allowed calculating sensitivity, specificity, and positive/negative predictive values for complete radiological response. Cumulative tumor-free and OS rates were evaluated using Kaplan–Meier curves (long-rank test). For the OS analysis after TACE, we right-censored patients who underwent liver resection or liver transplantation after the procedure, as these therapeutic options are potentially curative and may change the natural history of the disease. All statistical analyses were two-tailed and a p-value < 0.05 was considered statistically significant.

Results

Characteristics of HCC patients

The baseline characteristics of 48 patients with HCC who underwent TACE are listed in Table 1. The average age was 64.2 ± 7.6 years and 85.4% (n = 41) were men. The main etiology was alcohol-related liver disease (75.0%, n = 36), followed by chronic hepatitis C (39.6%, n = 19), chronic hepatitis B (12.5%, n = 6), and non-alcoholic fatty liver disease (10.4%, n = 5). Most of patients had Child–Pugh class A cirrhosis (83.3%, n = 40). The median model for end-stage liver disease score was 9 (interquartile range (IQR) 7–11). The median serum concentration of alpha-fetoprotein was 10.7 ng/mL (IQR 3.6–40.0). A single HCC nodule was observed in 52.1% (n = 25) of patients. Patients with multinodular disease had two nodules (29.2%, n = 14), three nodules (10.4%, n = 5), or four nodules (8.3%, n = 4). The total tumor diameter (i.e., the sum of the diameter of all nodules) was 3.6 cm (IQR 3.2–5.2), being less than 5 cm in 66.7% (n = 32) of patients. The median diameter of the largest tumor nodule was 3 cm (IQR 2.1–3.8), being less than 3 cm in 41.7% (n = 20) of patients. Tumor hypervascularity was observed in 64.6% of patients (n = 31). Therapies received thereafter were as follows: liver transplantation (n = 13, 27.1%), liver resection (n = 2, 4.2%), locoregional therapy (n = 15, 31.3%), or systemic therapy with sorafenib/lenvatinib (n = 9, 18.8%). Five patients (10.4%) were referred directly to palliative care after tumor progression without receiving other treatments. The remaining patients (n = 4, 8.3%) did not require additional therapies after the first TACE.

Table 1 Clinicopathological characteristics of patients. Data from 48 prospectively enrolled patients with hepatocellular carcinoma (HCC) undergoing transarterial chemoembolization (TACE) as a first-line therapy are included

Molecular characterization of CTCs

Before starting to evaluate the role of CTCs in the response of HCC patients to TACE treatment, we analyzed the genomic profile of 8 samples of EpCAM⁺ CTCs in order to confirm the presence of tumor cells in the population of isolated cells. We identified a total of 59 genomic variants in 27 genes. Most of variants (45.8%, n = 27) were found only once in any one patient and affected genes AKT1 (variant with rsID number rs146875699), APC (rs35031194, rs78919815, and rs2149778544), APC/ENSG00000258864 (rs143927847), ATM (rs3218674 and rs747750958), DERL3/SMARCB1 (rs367593276), ERBB2 (rs779727777), FGFR2 (chr10:123310878_9 insC), FIP1L1/PDGFRA (rs139913632), GNAS (rs1004902), HNF1A (chr12:121,437,046 C⇒T), KIT (chr4:55,592,213 delA), NF1 (rs1490145865), NOTCH1 (rs2133333012 and rs1377515876), PTCH1 (rs149398794 and rs528001004), PTCH1/ENSG00000271155 (rs1805153), PTCH1/LOC100507346 (rs758487789 and rs202007968), PTEN (rs755953294), RB1 (rs774525913), SMAD4/ENSG00000267699 (rs377767334) and TERT (rs35033501 and rs762113246). The variants that were represented in ≥ 50% of total patient samples are shown in the Supplementary Table 1. Among the recurrent mutations, 17 hotspots were identified in all 8 samples and located in 9 different genes, including ATM, BRCA1, CSF1R, MSH6 and SMAD4, and the most represented genes BRCA2, NF1, PTEN and RB1, with 3 positions each one. We also show those variants affecting exclusively ≥ 50% patients from a single response group. Genomic variants rs1135402852 and rs757338143 in the NF1 gene were identified in 3/5 CTC samples from the non-response group and none from the response group. In contrast, the variants rs751132262 and rs1838016977 affecting to the RET gene were exclusively identified in 2/3 and 3/3 patients from the response group, respectively.

CTC count and clinicopathological features of the patients

All patients included in the study had detectable CTCs at baseline, at post-TACE day 1 (D1), and at post-TACE day 30 (D30). The median CTC count at baseline, at D1, and at D30 after TACE were 44.0 (IQR 22.0–105.0), 44.0 (IQR 26.3–112.8) and 28.5 (IQR 11.5–47.8), respectively. When we analyzed the CTC count according to clinical parameters (Supplementary Table 2), we found that hypervascular tumors showed higher CTC counts at baseline (hypervascular: 53.0 [IQR 28.0–119.0] vs. medium/hypo-vascular: 22.0 [IQR 9.5–54.5], p = 0.009) and at D1 after TACE (46.0 [IQR 36.0–143.0] vs. 27.0 [IQR 13.5–91.0], p = 0.026), but not at D30 (27.0 [IQR 16.0–47.3] vs. 30.0 [IQR 10.8–49.5], p = 1.000). Younger patients had increased CTC counts at D1 (< 65 years: 56.0 [IQR 36.5–377.5] vs. ≥ 65 years: 40.0 [IQR 22.0–64.0], p = 0.046), but not at baseline and at D30. CTC counts did not correlate with any other clinical characteristic including tumor burden and liver function. Regarding clusters of CTCs, these were detected more frequently in patients with a single nodule (68.4%) compared to patients with multinodular disease (23.5%) (p = 0.007) at D30 (Supplementary Table 3).

Impact of TACE on CTC count

When we expressed the CTC count as a percentage with respect to the basal situation, we found an increase of 14% at D1 (114.0% [IQR 76.5%−178.0%], p = 0.019; n = 48) that decreased to baseline levels at D30 after TACE (76.5% [IQR 41.3%−131.8%], p = 0.263; n = 36). Compared to D1, the drop in CTC count at D30 was of 43% (57.0% [IQR 28.0%−117.3%], p = 0.033; n = 36) (Fig. 1). We did not observe differences in the count of CTC clusters at any study time (data not shown).

Fig. 1

Differences in circulating tumor cell (CTC) count. CTC kinetics is expressed as a percentage referred to baseline CTC count (left panel) or to day 1 post-TACE (D1) CTC count (right panel). Data represented as the median and 95% confidence interval. Statistically significant differences with *p ≤ 0.050

Patient characteristics and response to TACE

Among the 48 patients receiving TACE, 43.7% of patients (n = 21) obtained complete radiological response at D30 (referred as responders henceforth). The remaining 27 patients obtained either partial response (n = 14, 29.2%), stable disease (n = 4, 8.3%), or had disease progression (n = 9, 18.8%), and they are referred as non-responders. When comparing the patient characteristics between responders and non-responders, we found significant differences in the baseline diameter of the largest HCC nodule (2.5 cm [IQR 2.0–3.2] vs. 3.4 cm [IQR 2.2–4.3], p = 0.044) and the total tumor diameter (3.5 ± 1.0 cm vs. 4.9 ± 2.2 cm, p = 0.005).

TACE responders had a non-significant trend towards a progressive clearance of CTCs whereas non-responders showed a pronounced and early release of CTCs 24 h after the procedure (Fig. 2). Indeed, non-responders to TACE showed an increase in the percentage of CTCs by 38% after the procedure (138.0% [IQR 100.0%−196.0%], p = 0.007; n = 27). After 30 days, the CTC count in the non-response group fell to baseline levels (74.0% [44.0%−119.0%], p = 0.199, n = 19), and to 49.0% (IQR 28.0%−103.0%) compared to D1 (p = 0.009; n = 19). No differences were observed in the CTC cluster distribution at any study time (data not shown).

Fig. 2

Differences in CTC count between the groups of responders and non-responders to TACE. CTC kinetics is expressed as a percentage referred to baseline CTC count (left panel) or to D1 CTC count (right panel). Data represented as the median and 95% confidence interval. Statistically significant differences are marked with **p ≤ 0.010

Based on the CTC kinetics for the entire cohort at D1 after TACE (Fig. 1), we set 15% as the threshold for a clinically meaningful CTC increase. CTC count increase > 15% at D1 was strongly associated with the absence of complete radiological response one month after TACE (p = 0.009): 62.5% of patients (n = 15) with stable CTC count at D1 had complete radiological response to TACE while 75.0% of patients (n = 18) showing an early increase of CTCs after TACE were non-responders. CTC increase by 15% or more at D1 predicted the absence of radiological response at day 30 in 18 out of 27 patients [OR: 5.0 (95% CI: 1.4–17.3), p = 0.011] (Table 2). The decline of CTC count at D30 was not associated with the likelihood of complete radiological response within the first month after TACE.

Table 2 Univariate and multivariate logistic regression comparing patients with and without complete radiological response to TACE. Significance levels, odds ratios (OR), and 95% Confidence Intervals (CI) are shown for predictors of non-response

The absence of CTC clusters at baseline (p = 0.027) or at D30 (p = 0.047) was more frequent among non-responders to TACE. Specifically, the absence of CTC clusters at baseline predicted the non-response to TACE in 15 out of 27 patients [OR: 4.0 (95% CI: 1.1–14.1), p = 0.031)]. In the multivariate analysis, a larger tumor diameter [OR: 1.9 (95% CI: 1.1–3.3), p = 0.020] and CTC increase at D1 by ≥ 15% [OR: 5.3 (95% CI: 1.3–21.0), p = 0.017] were independent predictors of poor response to TACE (Table 2). The multivariate model composed by total tumor diameter and early release of CTCs had an area under receiver operating characteristic curve (AUROC) of 0.796 (95% CI: 0.7–0.9, p < 0.001), with sensitivity of 70.0% (95% CI: 47.4–92.3), specificity of 75.0% (95% CI: 57.2–92.8), positive predictive value of 66.7% (95% CI: 44.1–89.2), and negative predictive value of 77.8 (95% CI: 60.2–95.3) to predict suboptimal response to TACE (Fig. 3).

Fig. 3

ROC curves showing the best diagnostic ability to predict patient response to TACE. A Individual ROC curves for the predictors total tumor diameter (blue), absence of CTC clusters at baseline (red), and CTC increase ≥ 15% at D1 (green). B ROC curve for the combination of total tumor diameter and CTC increase ≥ 15% at D1 by logistic regression. Data for AUROC (95% confidence interval) are shown

When only patients with hypervascular tumors were considered in the analysis (n = 31), larger tumor diameter [OR: 2.5 (95% CI: 1.1–5.6), p = 0.022] and CTC increase at D1 by ≥ 15% [OR: 7.6 (95% CI: 1.1–54.4), p = 0.044] remained independent predictors of poor response to TACE. In patients with non-hypervascular lesions these variables had no predictive capacity for TACE response.

CTC dynamics and patient survival after TACE

The median follow-up after TACE was 22 months (95% CI: 11.7–32.3 months) during which 9 patients (23.1%) underwent liver transplantation and 17 patients (43.6%) died, 15 of them (88.2%) due to tumor progression. We excluded 9 patients from the OS analysis after TACE because they underwent liver resection or liver transplantation within the first 6 months after the procedure. The transplant-free survival rate was not associated with the radiological response to TACE (p = 0.451), nor was the CTC increase ≥ 15% at D1 (p = 0.150) or the presence of CTC clusters at baseline (p = 0.084).

Discussion

The CTC count as a predictive marker for TACE response in HCC has previously been reported with different results (Table 3). Different methodologies have been used to this end, being many of them based on the ‘positivity’ of the samples, that is, the presence/absence of CTCs. Here we use the IsoFlux system for this purpose for the first time. Although similar to the CellSearch system in its basic principles of cell enrichment and detection, the IsoFlux detects a much higher number of CTCs per volume of blood analyzed. The Isoflux system may detect a meaningful number of CTCs even in patients not showing CTCs in the CellSearch system analysis [25]. To date, we have not found CTC-negative samples in the different studies we have carried out using the IsoFlux system in HCC patients undergoing liver transplant, liver resection [14] or locoregional therapy [27]. In this study we continue without having found a patient negative for CTCs. The CTC count was evaluated prior to the start of the treatment and subsequently, on D1 and D30 after therapy. There are only a few studies analyzing the CTC kinetics after TACE.

Table 3 Research studies analyzing the role of CTCs as biomarkers of response to TACE in HCC

First, genomic analysis of isolated CTCs confirmed the presence of cancer cells carrying genomic variants in tumor suppressor genes. Although alteration of these genes is common in HCC [30], most of the variants identified have not previously been linked to this type of liver cancer and deserve further investigation. In particular, variants affecting genes BRCA2 (rsID number rs397507419) [31], MSH6 (rs397507419) and NF1 (rs587781807 and rs1060500295) were classified as pathogenic (source: ClinVar). The only identified variant affecting the tyrosine kinase transmembrane receptor CSFR1 gene (rs386693509) was found in all samples and was the only one that could be considered a germline variant. Although this result should be confirmed, this gene variant has been rarely discussed and its pathogenic impact is unknown. However, it was sequenced in 88.2% of patients (n = 17) with prostate cancer in both, healthy tissue and primary tumor or metastatic lymph node [32]. The affected gene, CSF1R, encodes a transmembrane receptor that control cellular growth, proliferation and differentiation by binding the CSF1 cytokine, and is overexpressed in many tumor types, including HCC [33], and associated with the promotion of an immune-permissive tumor microenvironment and poor prognosis.

Although the interest in analyzing the genetic profile of a small number of samples was to confirm the presence of tumor cells, we compared the presence of mutations between the two established groups of TACE response. We identified two variants affecting the RET gene in the group of responder patients, exclusively. Somatic RET gene mutation in hotspot region at p.L629Q (the same reported here affecting 3/3 patients) was notified in a small cohort of sporadic medullary thyroid carcinoma patients and was not related to the survival outcome [34]. Although in-silico analysis predicted p.L629Q as possibly damaging, its clinical significance is unknown. Regarding the other RET variant, rs751132262, identified in 2/3 responder patients, it was predicted as benign in in-silico analysis. On the other hand, we identified two additional variants of the NF1 gene (rs757338143 and rs1135402852) in 3/5 patients in the non-response group, exclusively. Interestingly, the gene variant rs1135402852 was also classified as pathogenic but not previously reported in patients with HCC (source: ClinVar).

The clinical significance of basal levels of CTCs in HCC patients undergoing TACE has not been fully clarified. Increased levels of CTCs and/or CTC positivity before TACE have been proposed as a prognostic marker, associated with tumor progression [20] and poor OS [16] and progression-free survival [17, 21]. However, other studies have not found such a relationship [24] or even reported contradictory results. Thus, CTC positivity before TACE as rescue therapy after liver resection [18, 23] or TACE before liver resection [22] has been related to improved clinical outcomes and proposed as a biomarker for TACE indication. In the present study, CTC count at baseline was not associated with TACE response or with parameters such as the number and size of tumor nodules. However, we were able to associate the absence of CTC clusters at baseline with the non-response to TACE. As it is known, CTC clusters have higher metastatic potential compared to solitary CTCs and their detection have been associated with poor survival [35]. In line with those works mentioned above, CTC cluster positivity at baseline was a predictive marker for favorable TACE response.

CTC decrease evaluated by qRT-PCR or fluorescence in situ hybridization after 4 weeks of TACE treatment has been previously associated with remission or stable disease [20] and with partial or complete response [21], respectively. However, no differences were observed between patients with high or low CTC count at this study time when they were analyzed by immunomagnetic enrichment and subsequent immunolabeling [16]. In our study, TACE treatment caused a non-significant decrease on CTC levels at D30 when we considered all patients, regardless of their response to therapy. We obtained the same result when we analyzed the two groups of patients separately. Therefore, the response to TACE was not affected by CTC levels at D30.

The immediate detection of CTCs after TACE has been performed at 3 min and 10 min, by using magnetic columns and immunolabeling [19] or by density-based gradient centrifugation and filtration and flow cytometry [24], respectively. None of these studies found differences in the number of CTCs after TACE or related the increase in CTCs with the prognosis of the HCC patients. As far as we know, this is the first study evaluating the number of CTCs after 24 h of TACE. Locoregional therapy caused a significant increase in CTCs by 14% on average at D1. The early CTC release phenomenon could be explained by the TACE procedure itself. Ideally, an effective TACE would block all arterial vessels of the tumor and would induce tumor necrosis, thus precluding CTC release. If any vascular tributaries remained patent after TACE, the viable tumor area could release CTCs to the bloodstream due to the surrounding inflammatory component and vascular damage. These released CTCs could then access other regions of the liver, or even systemic circulation, to create new lesions. This hypothesis would explain why CTC release occurred exclusively in the group of non-responders to TACE. Moreover, hypervascular tumors are easier to identify during TACE and therefore respond better to TACE and have a favorable outcome [36, 37]. The release of CTCs may not always motivate the development of metastases [8] since many other additional critical steps (such as circulatory dissemination, immune evasion, extravasation and infiltration) are necessary for the tumor to spread effectively.

Clinical characteristics of HCC patients related to the tumor burden, such as the tumor size [18, 21, 22] or the number of lesions [16,17,18, 23], have frequently been associated with the prognosis of HCC patients undergoing TACE. In this sense, multivariate analyses showed that larger tumor size along with increased CTC count at D1 were independent predictors of non-response to TACE. The combination of these two variables demonstrated better performance when only patients with hypervascular tumors were considered in the analysis.

It should be noted that we were not able to correlate the response to TACE with patient overall survival. Furthermore, parameters such as the CTC increase at D1 and the absence of CTC clusters at baseline, identified as prognostic factors for non-response to therapy, were also not related to the survival of patients. In this sense, the response of HCC patients to TACE, evaluated one month after the radiological intervention, does not necessarily imply greater survival. The indications for TACE are very heterogeneous and include patients who present different risk factors for poor prognosis, such as alpha-fetoprotein levels, tumor burden, tumor thrombosis, liver function or nutritional status [38]. In addition, the use of subsequent therapies for patients either responding to TACE (resection or liver transplantation) or not responding (subsequent TACE procedures or systemic therapies) make it challenging to evaluate effects on long term survival [39, 40].

The present study has some inherent limitations related to a relatively reduced sample size and absence of long-term follow-up in the most recently included patients. The different response groups to TACE have a heterogeneous number of patients and their grouping into new categories may further decrease statistical power. Future studies with larger and more balanced patient cohorts, and with long-term follow-up, would allow a better understanding of the impact of CTC kinetics and their genetic profile on long term outcomes.

Conclusions

The combination of tumor burden and dynamics of CTCs in patients with HCC undergoing TACE as first-line therapy may predict radiological response one month after the procedure, particularly in patients with hypervascular lesions. The release of CTCs was detected as early as 24 h after the procedure in a subset of patients with incomplete response to TACE, thus offering the opportunity to make clinical decisions regarding initiation of adjuvant therapies such as checkpoint inhibitors and/or antiangiogenics. Future clinical trials with larger sample size, sufficiently prolonged follow-up, and incorporating genomic profiling of CTCs, should evaluate the role of adjuvant therapies particularly in patients showing an early release of CTCs. The prognostic impact of this strategy in terms of recurrence-free and overall survival remains to be elucidated.