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High serum IL-6 correlates with reduced clinical benefit of atezolizumab and bevacizumab in unresectable hepatocellular carcinoma

  • Author Footnotes
    # These authors equally contributed to this work.
    Hannah Yang
    Footnotes
    # These authors equally contributed to this work.
    Affiliations
    Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea
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  • Author Footnotes
    # These authors equally contributed to this work.
    Beodeul Kang
    Footnotes
    # These authors equally contributed to this work.
    Affiliations
    Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea

    Yonsei Graduate School, Yonsei University College of Medicine, Seoul, Korea
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  • Author Footnotes
    # These authors equally contributed to this work.
    Yeonjung Ha
    Footnotes
    # These authors equally contributed to this work.
    Affiliations
    Department of Gastroenterology, CHA Bundang Medical Center, Seongnam, Korea
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  • Author Footnotes
    # These authors equally contributed to this work.
    Sung Hwan Lee
    Footnotes
    # These authors equally contributed to this work.
    Affiliations
    Department of Surgery, CHA Bundang Medical Center, Seongnam, Korea
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  • Ilhwan Kim
    Affiliations
    Division of Oncology, Department of Internal Medicine, Inje University College of Medicine, Haeundae Paik Hospital, Busan, Korea
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  • Hyeyeong Kim
    Affiliations
    Department of Internal Medicine, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea
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  • Won Suk Lee
    Affiliations
    Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea
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  • Gwangil Kim
    Affiliations
    Department of Pathology, CHA Bundang Medical Center, Seongnam, Korea
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  • Sanghoon Jung
    Affiliations
    Department of Radiology, CHA Bundang Medical Center, Seongnam, Korea
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  • Sun Young Rha
    Affiliations
    Yonsei Graduate School, Yonsei University College of Medicine, Seoul, Korea

    Division of Medical Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea
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  • Vincent E. Gaillard
    Affiliations
    F. Hoffmann-La Roche Ltd., Basel, Switzerland
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  • Jaekyung Cheon
    Correspondence
    Corresponding author. Jaekyung Cheon, Medical Oncology, CHA Bundang Medical Center, 59 Yatap-ro, Bundang-gu, Seongnam 13496, Republic of Korea, Phone: 82-31-780-7590; Fax: 82-31-780-3929;
    Affiliations
    Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea
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  • Chan Kim
    Correspondence
    Corresponding author. Chan Kim, Medical Oncology, CHA Bundang Medical Center, 59 Yatap-ro, Bundang-gu, Seongnam 13496, Republic of Korea, Phone: 82-31-780-7590; Fax: 82-31-780-3929;
    Affiliations
    Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea
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  • Hong Jae Chon
    Correspondence
    Corresponding author. Hong Jae Chon (Lead contact), Medical Oncology, CHA Bundang Medical Center, 59 Yatap-ro, Bundang-gu, Seongnam 13496, Republic of Korea¸ Phone: 82-31-780-7590; Fax: 82-31-780-3929;
    Affiliations
    Medical Oncology, Department of Internal Medicine, CHA Bundang Medical Center, CHA University, Seongnam, Korea
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  • Author Footnotes
    # These authors equally contributed to this work.
Open AccessPublished:January 16, 2023DOI:https://doi.org/10.1016/j.jhepr.2023.100672

      Highlights

      • Patients with high baseline IL-6 had worse survival outcomes following Ate/Bev.
      • Excess IL-6 impaired cytokine production and proliferation of CD8+ T cells.
      • High serum IL-6 levels were associated with non-T cell-inflamed tumors.

      Abstract

      Background & Aims

      We elucidated the clinical and immunologic implications of serum interleukin (IL)-6 levels in patients with unresectable hepatocellular carcinoma (HCC) treated with atezolizumab and bevacizumab (Ate/Bev).

      Methods

      We prospectively enrolled 165 patients with unresectable HCC (Discovery cohort: 84 patients from three centers; Validation cohort: 81 patients from one center). Baseline blood samples were analyzed using a flow cytometric bead array. The tumor immune microenvironment was analyzed using RNA sequencing.

      Results

      In the Discovery cohort, clinical benefit 6 months (CB6m) was defined as complete or partial response, or stable disease for ≥ 6 months. Among various blood-based biomarkers, serum IL-6 levels were significantly higher in patients without CB6m than in those with CB6m (mean 11.56 vs. 5.05 pg/mL, P=0.02). Using maximally selected rank statistics, the optimal cut-off value for high IL-6 was determined as 18.49 pg/mL, and 15.2% of patients were found to have high IL-6 levels at baseline. In both the Discovery and Validation cohorts, patients with high baseline IL-6 levels had a reduced response rate and worse progression-free and overall survival after Ate/Bev treatment compared with those with low baseline IL-6 levels. In multivariable Cox regression analysis, the clinical implications of high IL-6 levels persisted, even after adjusting for various confounding factors. Patients with high IL-6 levels showed reduced interferon-γ and tumor necrosis factor-α secretion from CD8+ T cells. Moreover, excess IL-6 suppressed cytokine production and proliferation of CD8+ T cells. Finally, patients with high IL-6 levels exhibited a non-T cell-inflamed immunosuppressive tumor microenvironment.

      Conclusions

      High baseline IL-6 levels can be associated with poor clinical outcomes and impaired T cell function in patients with unresectable HCC after Ate/Bev treatment.

      Lay summary

      Although patients with hepatocellular carcinoma who respond to treatment with atezolizumab and bevacizumab exhibit favorable clinical outcomes, a fraction of patients still experience primary resistance. We found that high baseline serum levels of interleukin-6 correlate with poor clinical outcomes and impaired T cell response in patients with hepatocellular carcinoma treated with atezolizumab and bevacizumab.

      Graphical abstract

      Keywords

      Abbreviations:

      AFP (Alpha-fetoprotein), Ate/Bev (Atezolizumab and bevacizumab), CB6m (Clinical benefit 6 month), CR (Complete response), CTLA-4 (Cytotoxic T-lymphocyte associated protein 4), DC (Dendritic cell), ECOG (Eastern Cooperative Oncology Group), HCC (Hepatocellular carcinoma), IL (Interleukin), MDSC (Myeloid-derived suppressor cell), MSI (Microsatellite instability), MVI (Macrovascular invasion), OS (Overall survival), ORR (Objective response rate), PFS (Progression-free survival), PR (Partial response), PD (Progressive disease), PD-1Programmed-death-1 (PD-L1¸Programmed-death ligand-1), TME (Tumor microenvironment)

      Introduction

      Recently, combined treatment with atezolizumab, an anti-PD-L1 antibody, and bevacizumab, an anti-VEGF neutralizing antibody, has demonstrated superior survival outcomes compared to those associated with sorafenib in the IMbrave150 trial, and became the standard of care in the new first-line systemic therapy for unresectable hepatocellular carcinoma (HCC).
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      Considering these hurdles, circulating biomarkers can be clinically more feasible alternatives to tissue-based biomarkers for predicting the efficacy of systemic therapy in unresectable HCC.
      Cytokines are immunological messengers that act locally in an autocrine and paracrine manner, but also systemically on distant target organs.
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      Bioanalytical chemistry of cytokines–a review.
      Interleukin (IL)-6 is a multifunctional cytokine produced by a variety of cell types, including immune cells, fibroblasts, endothelial cells, and tumor cells.
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      IL-6 is upregulated in various malignancies and plays a key role in tumorigenesis by affecting the survival, proliferation, angiogenesis, invasiveness, and metastasis of tumor cells.
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      Serum levels of IL-6, IL-8, and IL-10 are indicators of prognosis in pancreatic cancer.
      In the liver, IL-6 levels increase in a stepwise manner from a healthy status to hepatitis, cirrhosis, and HCC.
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      Furthermore, several recent studies have reported mechanisms of resistance to immunotherapy through IL-6 signaling.
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      High Serum Levels of IL-6 Predict Poor Responses in Patients Treated with Pembrolizumab plus Axitinib for Advanced Renal Cell Carcinoma.
      Excessive activation of IL-6 signaling may attenuate Th1 responses and impair T cell recruitment in lymph nodes and tumor microenvironment (TME), thus dampening antitumor T cell immunity.
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      • Nishimura Y.
      Immune‐suppressive effects of interleukin‐6 on T‐cell‐mediated anti‐tumor immunity.
      Although the Ate/Bev combination therapy has emerged as a new standard of care for HCC, the role of serum cytokines in patients treated with Ate/Bev has not been comprehensively addressed. Here, we elucidated the clinical and immunological implications of serum IL-6 levels in patients with unresectable HCC treated with Ate/Bev.

      Patients and methods

      Study design and patients

      This study prospectively enrolled patients with unresectable or metastatic HCC treated with Ate/Bev in multiple tertiary cancer centers in Korea. The study was conducted in two stages. In the first stage we aimed to identify clinically relevant biomarkers, including various cytokines, in HCC patients treated with Ate/Bev at three independent cancer centers in Korea, namely CHA Bundang Medical Center, Ulsan University Hospital, and Haeundae Paik Hospital (Discovery cohort). In the second stage we aimed to validate the clinical impact of biomarkers in an independent cohort of patients with HCC treated with Ate/Bev at the CHA Bundang Medical Center (Validation cohort). The eligibility criteria were age ≥ 20 years, unresectable or metastatic HCC confirmed by histologic or radiologic diagnosis according to the American Association for the Study of Liver Diseases criteria,
      • Heimbach J.K.
      • Kulik L.M.
      • Finn R.S.
      • Sirlin C.B.
      • Abecassis M.M.
      • Roberts L.R.
      • et al.
      AASLD guidelines for the treatment of hepatocellular carcinoma.
      no prior systemic anti-cancer therapy, Child-Pugh class A or B7, and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1. The study adhered to the ethical guidelines of the Declaration of Helsinki, and was approved by the participating hospitals’ Institutional Review Boards (CHA Bundang Medical Center, CHA-2017-11-052, CHA-2017-11-054; Ulsan University Hospital, 2020-12-006; Haeundae Paik Hospital, 2020-12-019-001). Written informed consent was obtained from all patients.

      Treatments and outcome evaluation

      Patients were treated with atezolizumab (1200 mg fixed dose) and bevacizumab (15 mg/kg) every three weeks as first-line systemic therapy. Dose interruptions or reductions were determined according to the IMbrave150 protocol.
      • Finn R.S.
      • Qin S.
      • Ikeda M.
      • Galle P.R.
      • Ducreux M.
      • Kim T.-Y.
      • et al.
      Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma.
      Treatment was continued until intolerable toxicity, progressive disease, or withdrawal of consent was observed. Response evaluation was performed every 6 or 9 weeks using computed tomography or magnetic resonance imaging, according to RECIST version 1.1. Overall survival (OS) was defined as the time from treatment initiation to the date of death. Progression-free survival (PFS) was defined as the time between treatment initiation and the date of progressive disease (PD) or death. The objective response rate (ORR) was defined as the sum of complete response (CR) and partial response (PR).

      Sample collection and measurement of serum cytokines

      Intravenous blood samples were obtained immediately before the first administration of Ate/Bev. To obtain serum, blood was centrifuged at 1000 g for 5 min; serum samples were subsequently stored in a deep freezer at -80°C. Serum cytokine concentrations were quantified using a cytometric bead array (560484, BD Biosciences) according to the manufacturer’s instructions. In brief, the capture beads were incubated with serum samples and the detection reagent for 3 h at room temperature. After several washes, cytokine-bead complexes were measured using a flow cytometer (Beckman Coulter), and the data were analyzed using the FlowJo software (Tree Star Inc.).

      Flow cytometry

      Before antibody staining, cells were stained with Fixable Viability Dye eFluorTM 780 (eBioscience) on ice for 30 min to exclude dead cells, followed by treatment with a human Fc receptor-binding inhibitor (eBioscience) for 15 min at room temperature. Surface proteins were stained on ice for 30 min with the following fluorochrome-conjugated antibodies: anti-human CD3 (clone SK7, eBioscience), anti-human CD4 (clone RPA-T4, BioLegend), and anti-human CD8 (clone RPA-T8, eBioscience). For intracellular staining, cells were fixed and permeabilized with a FoxP3 staining buffer kit (Thermo Fisher Scientific) and stained with the following fluorochrome-conjugated antibodies: anti-human interferon-γ (IFN-γ) (clone B27, BD Biosciences), and anti-human tumor necrosis factor-α (TNF-α) (clone Mab11, BD Biosciences). Flow cytometry was performed using a CytoFLEX flow cytometer (Beckman Coulter) and the results were analyzed using the FlowJo software (Tree Star Inc.).

      Cytokine and proliferation assays

      Peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation with Ficoll-Paque PLUS (GE Healthcare). To assess cytokine secretion, PBMCs were activated with a plate-bound anti-CD3 antibody (1 μg/mL), with or without recombinant IL-6 (100 pg/mL). After 4 h, cells were treated with brefeldin A (3 μg/mL, eBioscience) and monensin (2 μM, eBioscience). After 20 h, the stimulated cells were harvested and cytokine production was evaluated by flow cytometry. To assess cellular proliferation, we stained the cells using a Cell Trace Violet cell proliferation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cells were incubated with Cell Trace Violet for 20 min at room temperature and then washed. T cells were stimulated using anti-CD3 antibody-coated plates. After 72 h, the cells were harvested and the dilution of Cell Trace Violet was evaluated by flow cytometry.

      RNA isolation and gene expression analysis

      A total of 20 tumor samples with formalin-fixed paraffin-embedded (FFPE) slides were retrieved. RNA was extracted using Trizol (Thermo Fisher Scientific) and isolated using the AllPrep FFPE tissue kit (Qiagen, Germany), following the manufacturer’s instructions. The quality of FFPE-derived RNA was measured by the proportion of fragments > 200b (DV200). FFPE-derived RNA from all 20 samples was processed on FFPE-compatible RNAseq library preparation using QuantSeq 3’ mRNA-Seq Library Prep Kit FWD (Lexogen, USA). RNA sequencing was performed using the QuantSeq 3’ mRNA-sequencing kit (Lexogen, Vienna, Austria) to obtain the gene expression profiles of samples from the IL-6-high and -low groups.

      Estimation of immune cell infiltration into the TME

      The absolute score and relative fraction of the infiltrated immune subsets in the samples were estimated using the CIBERSORT algorithm. The leukocyte signature matrix LM22 was used as a signature gene set to infer the abundance of immune subsets in the gene expression data. The permutations were performed 100 times for statistical analysis. The Student’s t-test was used to compare the IL-6-high and -low groups, and to assess the statistical significance of IL-6-related enrichment of the immune subsets. Signatures of T cell dysfunction and exclusion were calculated based on algorithm of the previous report.
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      Immunohistochemical staining of tumor tissues was performed with anti-CD8 (Ventana) and anti-CD4 (Abcam) antibodies, as previously discribed.
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      Densities of CD8+ and CD4+ T cells were quantified using the ImageJ software (https://imagej.nih.gov/ij/download.html) as previously described.
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      Statistical analysis

      Independent sample t-tests, analysis of variance, and chi-square tests were used to compare variables. Survival analysis was performed using the Kaplan–Meier method, and the subgroups were compared using the log-rank test. Univariable and multivariable analyses of survival outcomes were performed using Cox proportional hazards regression analysis. The optimal cut-off value for IL-6 was determined using the maxstat (maximally selected rank statistics) package of the R software (https://www.r-project.org/).
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      • Schumacher M.
      Maximally selected rank statistics.
      All other statistical analyses were performed using the IBM SPSS version 18.0 software. Statistical significance was set at P < 0.05.

      Results

      Patient characteristics and treatment outcome

      From June 2020 to October 2021, 165 patients with unresectable HCC treated with Ate/Bev were prospectively registered in the Discovery (N=84) and Validation (N=81) cohorts at three tertiary cancer centers in Korea. A CONSORT diagram is shown in Fig. S1. The baseline clinical and laboratory characteristics are shown in Table 1 and Fig. 1, respectively. The median patient age was 61 years, and 84.2% of the patients were male. Most patients were Child-Pugh class A (87.3%) and BCLC stage C (79.4%). Hepatitis B (67.3 %) was the most common cause of HCC. Most patients (66.1%) had received at least one prior local therapy for HCC. Patients in the Validation cohort had worse ECOG performance status and more Child-Pugh B7 class than those in the Discovery cohort. The Discovery cohort showed an objective response rate (ORR) of 33.3% and a 1-year OS rate of 90.0%, with a median follow-up duration of 11.4 months. The Validation cohort showed an ORR of 24.7% and a 1-year OS rate of 82.4%, with a median follow-up of 6.6 months.
      Table 1Baseline demographics of patients with HCC.
      Total HCC cohort (N=165)Discovery cohort (N=84)Validation cohort (n=81)
      Median age (interquartile range, IQR)61 (55-68)61 (55-69)61 (56-68)
      Male sex139 (84.2%)70 (83.3%)69 (85.2%)
      ECOG performance status
       068 (41.2%)48 (57.1%)20 (24.7%)
       197 (58.8%)36 (42.9%)61 (75.3%)
      Child-Pugh classification
       A599 (60.0%)59 (70.2%)40 (49.4%)
       A645 (27.3%)20 (23.8%)25 (30.9%)
       B721 (12.7%)5 (6.0%)16 (19.7%)
      Barcelona Clinical liver cancer stage
       B34 (20.6%)21 (25.0%)13 (16.0%)
       C131 (79.4%)63 (75.0%)68 (84.0%)
      Alpha-fetoprotein ≥ 400 ng/ml, n (%)56 (33.9%)26 (31.0%)30 (37.0%)
      Presence of macrovascular invasion61 (37.0%)24 (28.6%)37 (45.7%)
      Presence of extrahepatic spread96 (58.2%)46 (54.8%)50 (61.7%)
      Etiology of HCC
       Hepatitis B117 (67.3%)60 (71.4%)57 (70.4%)
       Hepatitis C12 (6.7%)5 (6.0%)7 (8.6%)
       Alcohol19 (11.5%)12 (14.3%)7 (8.6%)
       Other or unknown17 (14.5%)7 (8.3%)10 (12.3%)
      Prior local therapy for HCC109 (66.1%)58 (69.0%)51 (63.0%)
      Median IL6 (IQR) at baseline2.89 (0.8-8.9)3.29 (1.3-8.3)2.32 (0.0-9.4)
      IL6 at baseline
       Negative or low (<18.49 pg/ml)140 (84.8%)74 (88.1%)66 (81.5%)
       High (≥18.49 pg/ml)25 (15.2%)10 (11.9%)15 (18.5%)
      Figure thumbnail gr1
      Fig. 1Baseline serum interleukin (IL)-6 levels are higher in patients without clinical benefit (CB) than in those with CB. (A) Comparisons of baseline serum cytokine levels, laboratory markers, and clinical characteristics. (B) Comparisons of baseline serum IL-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α levels according to clinical benefit 6 month (CB6m) (CB6m, n=47; non-CB6m, n=37). (C) Comparisons of serum IL-6 levels according to the best response. Values were compared using unpaired t-tests (B) or ANOVA with Tukey’s post hoc test (C).

      Serum IL-6 levels were elevated in patients without clinical benefit

      Next, we compared the baseline serum cytokine levels, laboratory markers, and clinical characteristics between patients with and without clinical benefit 6 month (CB6m) (Fig. 1A). CB6m was defined as CR, PR, or stable disease with ≥ 6 months PFS after Ate/Bev treatment. Among the various markers, serum IL-6 levels were significantly higher in patients without CB6m than in those with CB6m (mean 11.56 vs. 5.05, P=0.02) (Fig. 1A and 1B). Moreover, patients who had PD at the first response evaluation had remarkably high baseline serum IL-6 levels compared to those of patients with objective responses (CR + PR) (mean 14.15 vs. 3.62, P=0.026).
      Then, using the maximally selected rank statistics, we determined the cut-off value for high IL-6 that could optimally predict PFS as 18.49 pg/mL (Fig. S2). Using this cut-off value, 15.2% of the patients in this study (11.9% in the Discovery cohort and 18.5% in the Validation cohort) were defined as IL-6-high (Table 1).
      Previous studies have shown that IL-6 levels are strongly associated with HCC development and serum IL-6 levels progressively increased from healthy liver status to hepatitis, liver cirrhosis, and HCC, according to disease stage.
      • Shakiba E.
      • Ramezani M.
      • Sadeghi M.
      Evaluation of serum interleukin-6 levels in hepatocellular carcinoma patients: a systematic review and meta-analysis.
      In this study, we confirmed that IL-6 levels increased proportionally with decreased liver function, and Child-Pugh B7 patients had higher IL-6 levels than Child-Pugh A patients. However, in terms of disease stage, BCLC stages B and C did not show a significant difference in IL-6 levels (Table 2). Recently, the C-reactive protein and AFP in immunotherapy (CRAFITY) score has been associated with clinical outcomes in HCC patients treated with PD-(L)1 immunotherapy.
      • Scheiner B.
      • Pomej K.
      • Kirstein M.M.
      • Hucke F.
      • Finkelmeier F.
      • Waidmann O.
      • et al.
      Prognosis of patients with hepatocellular carcinoma treated with immunotherapy - development and validation of the CRAFITY score.
      Intriguingly, there is a significant correlation between high IL-6 levels and high CRAFITY scores (P < 0.001) (Table 2).
      Table 2Clinical characteristics according to IL-6 status.
      IL-6-low (n=140)IL-6-high (n=25)P-value
      Median Age (IQR)61 (55-68)64 (58-69)0.120
      Male sex119 (85.0%)20 (80.0%)0.527
      ECOG performance status0.058
       062 (44.3%)6 (24.0%)
       178 (55.7%)19 (76.0%)
      Child-Pugh classification<0.001
       A592 (65.7%)7 (28.0%)
       A635 (25.0%)10 (40.0%)
       B713 (9.3%)8 (32.0%)
      Barcelona Clinical liver cancer stage0.287
       B31 (22.1%)3 (12.0%)
       C109 (77.9%)22 (88.0%)
      Alpha-fetoprotein ≥ 400 ng/ml, n (%)45 (32.1%)11 (44.0%)0.249
      Presence of macrovascular invasion48 (34.3%)13 (52.0%)0.091
      Presence of extrahepatic spread79 (56.4%)17 (68.0%)0.280
      Etiology of HCC0.720
       Hepatitis B100 (71.4%)17 (68.0%)
       Hepatitis C9 (6.4%)3 (12.0%)
       Alcohol17 (12.1%)2 (8.0%)
       Other or unknown14 (10.0%)3 (12.0%)
      CRAFITY score<0.001
       063 (45.0%)2 (8.0%)
       164 (45.7%)10 (40.0%)
       213 (9.3%)13 (52.0%)
      Prior local therapy for HCC98 (70.0%)11 (44.0%)0.011

      High IL-6 levels were associated with unfavorable clinical outcomes in response to Ate/Bev in HCC

      Next, we compared the clinical outcomes of Ate/Bev treatment according to baseline IL-6 levels in unresectable HCC. In the Discovery cohort, patients with high IL-6 levels at baseline had a lower ORR than those with low IL-6 levels (0% vs. 37.8%; Fig. 2A). Moreover, PFS and OS were significantly worse in patients with high IL-6 levels than in those with low IL-6 levels (hazard ratio [HR]=2.93, P=0.003 for PFS; HR=3.02, P=0.021 for OS) (Fig. 2B and 2C). These findings were consistent with those of the Validation cohort. Patients with high IL-6 levels showed a significantly lower ORR than those with low IL-6 levels (7% vs. 28.8%; Fig. 2D). Moreover, the IL-6-high group showed worse PFS and OS than the IL-6-low group (HR=2.15, P=0.018 for PFS; HR=4.64, P<0.001 for OS) (Fig. 2E and 2F). Of note, almost half of the patients with high IL-6 levels at baseline (60.0% in the Discovery cohort and 46.7% in the Validation cohort) showed disease progression at their first radiologic response evaluation, compared to only 17.1% of patients with low IL-6 levels (12.2% in the Discovery cohort and 22.7% in the Validation cohort).
      Figure thumbnail gr2
      Fig. 2High IL-6 levels are associated with reduced clinical outcomes following treatment with Ate/Bev in HCC. (A–C) Treatment outcomes of the Discovery cohort (N=84) and (D–F) Validation cohort (N=81). (A and D) Bar charts showing the best response to Ate/Bev as determined by IL-6 levels. (B, C, E, and F) Kaplan–Meier curves showing PFS and OS according to IL-6 levels. P values were calculated using the log-rank test. Hazard ratios (HRs) and 95% confidence intervals (CIs) are shown in the survival curves. Ate/Bev: atezolizumab and bevacizumab; PFS: progression-free survival; OS: overall survival.
      Because other variables such as Child-Pugh class may serve as confounding factors of survival outcome, we further validated the clinical impact of baseline IL-6 levels using a multivariable Cox proportional hazards model. On multivariable analysis, high baseline IL-6 levels were still the most significant factor associated with poor PFS and OS compared to other confounding variables, such as age, sex, ECOG performance status, Child-Pugh class, alpha-fetoprotein (AFP) levels, macrovascular invasion (MVI), and extrahepatic metastasis (HR 1.843 and P=0.029 for PFS; HR 2.462 and P=0.014 for OS) (Fig. 3A and 3B).
      Figure thumbnail gr3
      Fig. 3Multivariable survival analysis using the Discovery and Validation cohorts. (A and B) Forest plots showing multivariable analyses of PFS (A) and OS (B) by age, sex, Eastern Cooperative Oncology Group (ECOG) performance status, Child-Pugh score, alpha-fetoprotein (AFP), macrovascular invasion (MVI), extrahepatic spread, and serum IL-6 levels; N=165, pooled data from the Discovery and Validation cohorts. P values were calculated using Cox proportional hazards regression analysis.

      Highly elevated serum IL-6 levels correlated with reduced cytokine production and CD8+ T cell proliferation

      To further unveil how high levels of serum IL-6 influence T cell immunity, we compared effector cytokine production by peripheral blood CD8+ T cells from IL-6-high and IL-6-low patients. CD8+ T cells from patients with high IL-6 levels produced less IFN-γ and TNF-α than those from patients with low IL-6 levels (Fig. 4A and 4B). To confirm that excess IL-6 affects CD8+ T cell function, we examined whether the function of CD8+ T cells from patients with low IL-6 levels can be impaired by exogenous IL-6 treatment. Indeed, IL-6-treated CD8+ T cells produced less IFN-γ and TNF-α than the control CD8+ T cells (Fig. 4C and 4D). Moreover, excess IL-6 suppressed CD8+ T cell proliferation (Fig. 4E and 4F). Therefore, high IL-6 levels may be associated with decreased effector function and proliferative capacity of cytotoxic T cells in patients with unresectable HCC.
      Figure thumbnail gr4
      Fig. 4Highly elevated serum IL-6 levels correlate with reduced cytokine production and CD8+ T cell proliferation. (A) IFN-γ and TNF-α production in CD8+ T cells according to serum IL-6 levels (n=102 for IL-6-Low; n=14 for IL-6-High); pooled data from the Discovery and Validation cohorts. (B) IFN-γ and TNF-α production by CD8+ T cells in the absence/presence of excess IL-6 (100 pg/mL); n=20 for each group. (C) CD8+ T cell proliferation in the absence/presence of excess IL-6 (100 pg/mL); n=20 for each group. Values were compared using unpaired (A) or paired t-tests (B and C).

      Highly elevated serum IL-6 levels may be associated with a non-T cell-inflamed TME in unresectable HCC

      To compare tumor immune phenotypes between the IL-6-high and -low groups, we examined intratumoral immune cell subsets from RNA sequencing data using CIBERSORT algorithms. Absolute scores from CIBERSORT algorithms estimated that the immune environment of tumors from the IL-6 high group consisted of a relatively lower quantity of immune subsets (Fig. 5A). To evaluate the difference in immune subsets between IL-6-high and -low tumors, hierarchical clustering was performed (Fig. S3A). In tumors with high IL-6 levels, CD8+ and CD4+ T cell CIBERSORT scores were remarkably lower than in those with low IL-6 levels (Fig. 5B and Fig. S3B). Moreover, T cell dysfunction and exclusion scores were remarkably higher in IL-6-high tumors compared with IL-6-low tumors (Fig. 5C). Consistent with the findings of the CIBERSORT analysis, histological analysis showed that intratumoral CD8+ and CD4+ T cells were lower in IL-6-high tumors compared with IL-6-low tumors (Fig. 5D). Therefore, patients with high serum IL-6 levels had non-T cell-inflamed TME in their tumor tissues.
      Figure thumbnail gr5
      Fig. 5Highly elevated serum IL-6 levels are associated with a non-T cell-inflamed tumor microenvironment in unresectable HCC. (A) Proportion of intratumoral immune cell subsets by CIBERSORT analysis according to serum IL-6 levels. (B) Comparison of CD8+ and CD4+ T cell CIBERSORT scores according to serum IL-6 levels. (C) Comparison of T cell dysfunction and exclusion scores according to serum IL-6 levels. (D) Images and comparisons of CD8+ and CD4+ T cells in tumor tissues according to serum IL-6 levels. Values were compared using unpaired t-tests. (A-C) n=10 for each group. (D) n=9 for IL-6-low, and n=8 for IL-6-high.

      Discussion

      Ate/Bev treatment has become the standard of care for unresectable HCC; however, a fraction of patients still exhibit primary resistance to this combination therapy. Pretreatment identification of these non-responders has been a crucial topic for both patients and clinicians. Herein, we found that a fraction (15%) of patients with unresectable HCC had high baseline IL-6 levels, and this was associated with reduced clinical benefits from Ate/Bev treatment. These findings were consistent even after adjusting for various confounding factors, including age, sex, ECOG, Child-Pugh class, AFP, MVI, and extrahepatic spread.
      Although PD-L1 expression, microsatellite instability (MSI)-high status, and tumor mutational burden in tumor tissues are well-known predictive markers of response to immunotherapy in other solid tumors, their clinical use is less established in HCC.
      • Yavuz B.G.
      • Hasanov E.
      • Lee S.S.
      • Mohamed Y.I.
      • Curran M.A.
      • Koay E.J.
      • et al.
      Current Landscape and Future Directions of Biomarkers for Immunotherapy in Hepatocellular Carcinoma.
      • Hyung J.
      • Cho E.J.
      • Kim J.
      • Kim J.H.
      • Kim J.E.
      • Hong Y.S.
      • et al.
      Histopathologic and Molecular Biomarkers of PD-1/PD-L1 Inhibitor Treatment Response among Patients with Microsatellite InstabilityHigh Colon Cancer.
      • Kim C.W.
      • Chon H.J.
      • Kim C.
      Combination Immunotherapies to Overcome Intrinsic Resistance to Checkpoint Blockade in Microsatellite Stable Colorectal Cancer.
      Especially in HCC, given that tissue biopsy is not mandatory for diagnosis, the discovery of tumor tissue predictive biomarkers is very limited. In this regard, the discovery of circulating biomarkers that can predict immunotherapy efficacy is clinically meaningful in HCC. Thus, serum IL-6 is a promising candidate as an easily accessible circulating predictive biomarker for Ate/Bev treatment in HCC.
      In this multicenter study, we verified the clinical implications of IL-6 using a multitude of evidence. First, we found that patients with high baseline IL-6 levels had unfavorable survival outcomes following Ate/Bev treatment. These findings were verified using a Validation cohort and multivariable analysis. Second, we elucidated the immunological impact of high IL-6 levels on CD8+ T cell function using serological and flow cytometric analyses. We demonstrated that patients with high IL-6 levels had reduced activation of peripheral CD8+ T cells compared to patients with low IL-6 levels. Moreover, excess IL-6 impaired cytokine production and proliferation of CD8+ T cells. These findings suggest that high baseline IL-6 levels may attenuate T cell immunity in unresectable HCC. Third, we evaluated immune cell infiltrates within the HCC tissue by comparing RNA sequencing data between the high and low IL-6 groups. Notably, CD8+ and CD4+ T cells were markedly reduced in IL-6 high tumors compared to IL-6 low tumors. Therefore, high serum IL-6 levels be associated with a non-T cell-inflamed TME in unresectable HCC.
      In this study, we defined clinical benefit 6 month (CB6m) as CR, PR, or SD with ≥ 6 months PFS. The disease control rate (DC) is generally defined as the sum of CR, PR, and SD, regardless of the PFS.
      • Villaruz L.C.
      • Socinski M.A.
      The clinical viewpoint: definitions, limitations of RECIST, practical considerations of measurement.
      When we compared CB6m and DC according to various cytokine levels, the overall trends were comparable, but CB better correlated with the differences in cytokine levels (Fig. S4). This may be because, in patients with SD, those with shorter PFS (< 6 months) have higher levels of IL-6 than those with longer PFS (≥ 6 months).
      Although we defined the IL-6 cut-off value for determining the clinical outcomes of first-line Ate/Bev therapy in unresectable HCC as 18.49 pg/mL, another recent study examining a small number of patients treated with Ate/Bev as first- or second-line therapy suggested that a relatively low IL-6 level (3.2 pg/mL) could be employed as a prognostic factor.
      • Myojin Y.
      • Kodama T.
      • Sakamori R.
      • Maesaka K.
      • Matsumae T.
      • Sawai Y.
      • et al.
      Interleukin-6 Is a Circulating Prognostic Biomarker for Hepatocellular Carcinoma Patients Treated with Combined Immunotherapy.
      However, this lower IL-6 cut-off value did not demonstrate significant differences in survival outcomes in our Discovery and Validation cohorts and T cell functional differences, which suggests that sufficiently high IL-6 levels need to be applied to reproducibly predict the therapeutic efficacy of Ate/Bev.
      The IL-6 signaling pathway is abnormally regulated in various malignancies,
      • Hoejberg L.
      • Bastholt L.
      • Schmidt H.
      Interleukin-6 and melanoma.
      • Ma Y.
      • Ren Y.
      • Dai Z.-J.
      • Wu C.-J.
      • Ji Y.-H.
      • Xu J.
      IL-6, IL-8 and TNF-α levels correlate with disease stage in breast cancer patients.
      • Feng L.
      • Qi Q.
      • Wang P.
      • Chen H.
      • Chen Z.
      • Meng Z.
      • et al.
      Serum levels of IL-6, IL-8, and IL-10 are indicators of prognosis in pancreatic cancer.
      and several recent studies have reported that IL-6 may be involved in mechanisms of resistance to immunotherapy.
      • Kalbasi A.
      • Ribas A.
      Tumour-intrinsic resistance to immune checkpoint blockade.
      • Flint T.R.
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      IL-6 activates STAT3 in dendritic cells (DC), leading to the downregulation of major histocompatibility complex class II expression on the surface of DC.
      • Kitamura H.
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      • Ohtake J.
      • Homma S.
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      Interleukin‐6/STAT 3 signaling as a promising target to improve the efficacy of cancer immunotherapy.
      IL-6 is also capable of upregulating Agr-1 expression and chemokine receptor CCR5 in myeloid-derived suppressor cells (MDSCs), leading to MDSCs recruitment and activation.
      • Weber R.
      • Groth C.
      • Lasser S.
      • Arkhypov I.
      • Petrova V.
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      • et al.
      IL-6 as a major regulator of MDSC activity and possible target for cancer immunotherapy.
      Other recent studies have also revealed an association between high baseline IL-6 levels and reduced benefits from immunotherapy in patients with melanoma, renal cell carcinoma, and lung cancer.
      • Sang Y.B.
      • Yang H.
      • Lee W.S.
      • Lee S.J.
      • Kim S.G.
      • Cheon J.
      • et al.
      High Serum Levels of IL-6 Predict Poor Responses in Patients Treated with Pembrolizumab plus Axitinib for Advanced Renal Cell Carcinoma.
      ,
      • Hardy-Werbin M.
      • Rocha P.
      • Arpi O.
      • Taus Á.
      • Nonell L.
      • Durán X.
      • et al.
      Serum cytokine levels as predictive biomarkers of benefit from ipilimumab in small cell lung cancer.
      ,
      • Laino A.S.
      • Woods D.
      • Vassallo M.
      • Qian X.
      • Tang H.
      • Wind-Rotolo M.
      • et al.
      Serum interleukin-6 and C-reactive protein are associated with survival in melanoma patients receiving immune checkpoint inhibition.
      These findings suggest that high IL-6 levels may attenuate the efficacy of immunotherapy, and that IL-6 blockade could augment antitumor immunity. A recent preclinical study revealed that IL-6 blockade could suppress tumor growth by enhancing CD4+/CD8+ effector T cells while suppressing Th17 and macrophages.
      • Hailemichael Y.
      • Johnson D.H.
      • Abdel-Wahab N.
      • Foo W.C.
      • Bentebibel S.-E.
      • Daher M.
      • et al.
      Interleukin-6 blockade abrogates immunotherapy toxicity and promotes tumor immunity.
      Based on these previous findings, an ongoing phase Ib/II clinical trial (NCT04524871) is currently evaluating the efficacy and safety of anti-IL-6 add-on therapy in patients with unresectable HCC treated with Ate/Bev.
      Our study was limited in that it enrolled only East Asian patients from a hepatitis B virus-endemic region, therefore necessitating external validation in other ethnic groups. Furthermore, we did not evaluate the source of peripheral blood IL-6 levels. In HCC, IL-6 levels may be affected by various conditions, such as deterioration of liver function, progression of the HCC stage, and the immune phenotype of HCC, as shown by the RNA sequencing results of our study. Thus, further investigation is needed to clarify the source of elevated IL-6 levels. Moreover, in this study, we activated T cells in vitro for a short time; thus, some T cells may have remained in a naïve state, which could have affected the degree of cytokine production. Therefore, further mechanistic studies on the effect of IL-6 on T cell function are needed to validate the findings of this study.
      In conclusion, high baseline serum IL-6 level is associated with worse clinical outcomes in patients with unresectable HCC treated with Ate/Bev. Clinicians need to carefully monitor and perform early response evaluations when treating patients with high IL-6 levels; however, these patients should not be excluded from receiving a potentially effective standard of care therapy. Further validation and standardization of IL-6 assays are warranted to optimize the monitoring and management of the Ate/Bev combination therapy. In addition, future investigations are needed to explore whether the association between reduced clinical benefit in response to Ate/Bev treatment and high IL-6 levels observed in the present study can also be applied to other combination immunotherapies, such as anti-CTLA-4 and anti-PD-1/PD-L1.

      Conflict of interest

      HJC has a consulting or advisory role at Eisai, Roche, Bayer, ONO, MSD, BMS, Celgene, Sanofi, Servier, AstraZeneca, Sillajen, Menarini, GreenCross Cell and has received research grants from Roche, Dong-A ST, Boryung Pharmaceuticals. CK has a consulting or advisory role at Roche, ONO, MSD, BMS, Oncocross, Virocure, Sillajen, and Panolos Biosciences, and has received research grants from Boryung Pharmaceuticals, Oncocross, Sillajen, and Virocure. JC has a consulting or advisory role at Roche, MSD China, Eisai, and Servier, and has received research grants from Bayer. VEG is an employee of F. Hoffmann-La Roche Ltd. and has stock options from F. Hoffmann-La Roche Ltd. The other authors have no potential conflicts of interest to disclose.

      Data availability statement

      Data are available upon request.

      Financial support statement

      This work was supported by the National Research Foundation of Korea [NRF] grant funded by the Korea government [MSIT] [NRF-2020R1C1C1010722 to HJC, NRF-2020R1A2C2004530 to CK, and NRF-2021R1F1A106196311 to BK].

      Authors contributions

      Supervision: CK and HJC; Conceptualization: HY, JC, BK, SYR, CK, and HJC; Funding acquisition: BK, CK, and HJC; Patient recruitment: JC, IK, HK, SJ, BK, CK, and HJC; Laboratory analysis: HY, YH, SHL, and WSL; Data Analysis and Visualization: HY, YH, GK, and SHL, CK, and HJC; Writing: HY, SHL, VEG, CK, and HJC

      Appendix A. Supplementary data

      The following is/are the supplementary data to this article:

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