Stereotactic body radiotherapy with sintilimab and bevacizumab biosimilar in anti-PD-1 refractory hepatocellular carcinoma: the ReUNION-1 phase 2 trial

Domain: neuroscience
Published: 2025-12-18
Editorial outcome: major_revision

Abstract
Immune checkpoint inhibitor (ICI) resistance in hepatocellular carcinoma (HCC) poses a major therapeutic challenge. Here we present a Phase 2 trial evaluating stereotactic body radiotherapy (SBRT) combined with sintilimab and bevacizumab biosimilar (PD-1/VEGF blockade) to overcome resistance in ICI-refractory HCC. Twenty-one patients with progressive HCC after ICI therapy receive SBRT followed by sintilimab 200 mg and bevacizumab biosimilar 15 mg/kg every 3 weeks. The primary outcome, objective response rate in non-irradiated lesions is 33.3%, with a disease control rate of 66.7%. Median progression-free survival is 6.2 months, and estimated median overall survival is 24.4 months. SBRT achieves 100% local control, with 33.3% experiencing grade 3 or higher adverse events. Proteomic profiling reveals that responders exhibit lower baseline IFN-γ and elevated IL-6, while post-SBRT increases in IFN-γ, IL-2, and IL-6 correlate with improved outcomes. These results indicate that combination of SBRT in ICI-refractory HCC is effective, well-tolerated, and may be guided by cytokine assessment.

Introduction
Hepatocellular carcinoma (HCC) accounts for 90% of all primary liver cancers; it ranks as the third leading cause of cancer-related death globally1, with an estimated 5-year survival rate of less than 4% for advanced-stage patients2. Immune checkpoint inhibitors (ICIs), particularly anti-PD-1/PD-L1 agents, have revolutionised systemic therapy, enabling durable responses in a subset of patients3. However, monotherapy yields limited objective response rates (ORRs) of 13–20% (e.g., durvalumab4 and tislelizumab5). Consequently, combination strategies integrating ICIs with antiangiogenic agents (anti-VEGF agents, e.g., bevacizumab), tyrosine kinase inhibitors (TKIs), or anti-CTLA-4 antibodies have emerged as first-line standards. Landmark phase 3 trials, including IMbrave150 (atezolizumab plus bevacizumab; ORR 27.3%6), ORIENT-32 (sintilimab plus bevacizumab biosimilar; ORR 21.0%7), CARES-310 (camrelizumab plus rivoceranib; ORR 26.8%8), HIMALAYA (tremelimumab plus durvalumab; ORR 20.1%4), and CheckMate-9DW (nivolumab plus ipilimumab; ORR 36%9), have demonstrated significantly improved outcomes over sorafenib or lenvatinib.

Despite these advances, at least 60% of patients receive no benefit from combination immunotherapy (IO), and the inevitable immunoresistance remains a formidable constraint to implementing ICIs4,6,8. These patients then become candidates for next-line treatment. Treatment options after progression on front-line ICI regimens remain poorly defined, as most approved second-line agents (e.g., regorafenib) have been validated in sorafenib-pretreated populations10. Currently, the choice for late-line therapy after front- or second-line ICI treatment is primarily empirical, with the shift between ICIs and anti-angiogenic agents largely dependent on drug safety, patient comorbidities, physician and patient preferences, and local reimbursement policies.

Currently, data are available from several retrospective studies exploring the feasibility and efficacy of rechallenging with ICIs in the context of prior exposure11–13. However, the protocols have mainly focused on the use of different ICI combinations and the replacement of previously unused ICI-based regimens. The main rationale is that inhibiting non-cross-resistance might promote antitumour responses in patients who did not respond to previous ICI therapy14–16. These types of regimens are reported to have tolerable toxicity, with ORRs ranging from 16–30% and disease control rates (DCRs) ranging from 40%–55%11–13. The therapeutic effectiveness of a simply substituted immunotherapy category remains unsatisfactory, yet prospective data are scarce.

In addition to systemic therapy, locoregional therapies, such as radioembolization and radiotherapy, have been explored and are promising, with favourable results17,18. Radiotherapy has rapidly become a crucial component of multidisciplinary treatment for liver cancer due to advances in technology, resulting in the transition to stereotactic body radiotherapy (SBRT)19. SBRT allows high doses of radiation to be applied to the targeted tumour area in a limited number of fractions with less damage to the surrounding healthy tissues. Recent prospective and retrospective studies have reported that SBRT can achieve high rates of local tumour control at 2 years, ranging from 68–95%20,21, and is an effective local treatment for inoperable early-, intermediate-, and advanced-stage HCCs19. However, many patients experience out-of-field progression, highlighting the need for concurrent systemic disease control22.

Accumulating evidence has demonstrated that radiotherapy rewires the tumour microenvironment (TME) to enhance immunotherapeutic sensitivity through dual mechanisms: stimulating tumour cells to upregulate immune-recognition molecules that potentiate cancer cell detection and antitumour immune activation23–27 while concurrently inducing pro-inflammatory cytokines and chemokines that recruit effector immune cells into the TME24,28. Consequently, SBRT is being actively investigated in combination with systemic agents, particularly ICIs, for the treatment of metastatic lesions across multiple malignancies, including HCC29. Antiangiogenic therapies such as VEGF inhibitors further augment this approach by demonstrating radiosensitizing properties in tumours such as glioma30 and potentiating radiation-induced abscopal effects when combined with ICIs in HCC31. Nevertheless, whether SBRT-mediated tumoral or circulating immune microenvironment reprogramming can resensitize patients with immunotherapy-refractory HCC to subsequent ICI regimens remains an unanswered clinical question.

Herein, we report the results of the ReUNION-1 study, a phase 2 trial of sintilimab (IBI308) and a bevacizumab biosimilar (IBI305) combined with SBRT after failure of anti-PD-1 treatment in patients with unresectable HCC. In light of the need to identify potential biomarkers and clarify the basis of the tumour response, we analysed serial blood samples obtained before treatment and at time points during treatment and identified many SBRT-induced and efficacy-correlated cytokines. This study will provide evidence for the efficacy of combining SBRT with ICI in patients with ICI-refractory HCC.

Results
Patient characteristics
The trial schema is summarised in Fig. 1A and Supplementary Fig. S1A. Between March 2022 and April 2024, a total of 25 patients were screened for eligibility. Among them, one patient had no target lesion other than the proposed radiotherapy lesion; two patients underwent gastroscopy, suggesting untreated or incompletely treated oesophageal or gastric varices with bleeding or a high risk of bleeding; and one patient declined enrolment for personal reasons. Ultimately, 21 patients were enroled (Fig. 1B). The baseline demographics of the patients are summarised in Supplementary Dataset 1 and Supplementary Dataset 2. All 21 patients were included in the intention-to-treat (ITT) analysis, with a cut-off date of 31 August 2024 (Fig. 1B). The median duration of the latest line immunotherapy before enrolment was 7.4 months (interquartile range [IQR], 5.9–18.4 months). The median age was 55 years, and 81.0% of the patients were men. Nine patients had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0, and 12 had a PS of 1. All 21 patients had a Child-Pugh grade of A at baseline. According to the Chinese Society of Clinical Oncology (CSCO) guidelines, the front-line TKI/PD-1 combination was selected based on availability and data from contemporary trials in China, despite differences in international guidelines. Nineteen patients were treated with at least one line of TKI plus PD-1. Specifically, 73.7% (14/19) of these patients received a combination of lenvatinib and camrelizumab or sintilimab, 21.1% (4/19) received apatinib and camrelizumab, and 5.3% (1/19) received both regimens. Additionally, one patient received anti-VEGF (bevacizumab) plus anti-PD-L1 (atezolizumab) treatment, and another received anti-CTLA-4 plus anti-PD-1 treatment. Details of the fractionation regimen for each patient receiving SBRT treatment (median dose 8 Gy/fraction, range 5–8 Gy in 5 fractions) and the target lesion dose-volume metrics used to assess efficacy, including the median volume receiving ≥5 Gy (V5, which was 0%), can be found in the Source data. Chronic HBV infection was the leading cause of liver disease (n=18, 85.7%). Most patients were characterised by the following: prior one-line systemic therapy (n=13, 61.9%), high baseline serum AFP levels (n=12, 57.1%), multiple intrahepatic lesions (n=18, 85.7%), and extrahepatic metastasis (n=15, 71.5%).

Treatment efficacy
With a median follow-up of 9.1 months (IQR, 7.6–16.1), 21 (100%) of the 21 patients were evaluable for the best overall response (BOR). A confirmed partial response (PR) was observed in 7 patients without complete response (CR), contributing to an ORR of 33.3%. Stable disease (SD) was observed in 7 patients, with a DCR of 66.7% (Fig. 2A and Table 1). Notably, reductions in the sizes of irradiated lesions after treatment compared with those at baseline were observed in 20 (95.2%) of the 21 enrolled patients (Fig. 2B). The local-regional control (LRC) rate was 100% (21/21, Fig. 2B), indicating excellent local control of SBRT treatment. Excitingly, lesions outside the irradiation field can also achieve very effective results (Supplementary Fig. S2A, S2B). Durable disease control, defined as continuous tumour control lasting ≥6 months, was observed in patients with both a PR and SD. One patient with a PR according to RECIST v1.1 maintained disease control for more than two years after IBI cessation. Three of the seven patients who experienced a PR remained alive, with no alternative systemic therapy required. Seven patients had progressive disease as their best response.

By the data cut-off date for the final analysis, 18 patients experienced disease progression, the median progression-free survival (PFS) was 6.2 months (Fig. 2C, D, 95% CI: 3.7–15.1 months), and 2 patients were still under treatment (Fig. 2C). The 6-month and 12-month PFS rates were 57.1% (95% CI, 36.5%–75.5%) and 14.3% (95% CI, 5.0%–34.6%), respectively (Fig. 2D). Additionally, eight patients died, and the estimated median overall survival (OS) was 24.4 months (Fig. 2C, D, 95% CI: 9.2 months-NA). Kaplan-Meier estimates of the 6-month and 12-month OS rates were 95.2% (95% CI 77.3%–99.8%) and 33.3% (95% CI 17.2%–54.6%), respectively. The median duration of response (mDOR) was 5.70 months (95% CI, 2.0–12.4). Overall, local responses were strong and durable (Fig. 2E, F, Supplementary Fig. S2B). Moreover, neither the location of the metastases nor the response to prior ICI therapy nor PFS to prior ICI therapy was significantly associated with treatment response or clinical benefit (Supplementary Fig. S2C, S2D, Supplementary Table S1), indicating the generalisability of this regimen among HCC patients who were refractory to immunotherapy.

Safety
All patients included in the ITT analysis completed radiotherapy and at least one cycle of sequential treatment with sintilimab plus a bevacizumab biosimilar. Treatment-related adverse events (TRAEs) and treatment-emergent AEs (TEAEs) are listed in Table 2 and Supplementary Table S2, respectively. During the therapy period, TRAEs of any grade occurred in 17 of 21 patients (81.0%; see details in Table 2). No unexpected adverse events or treatment-related deaths occurred. Commonly observed TRAEs included decreased lymphocyte count (n=8, 38.1%), decreased platelet count (n=8, 38.1%), and increased alanine aminotransferase (n=8, 38.1%) and aspartate aminotransferase (n=8, 38.1%). Grade 3 or higher TRAEs (serious TRAEs) occurred in 7 (33.3%) patients, commonly including decreased platelet count (n=2, 9.5%), decreased white blood cell count, neutropenia (n=2, 9.5%), decreased platelet count (n=2, 9.5%), and gastrointestinal haemorrhage (n=2, 9.5%). Discontinuation of therapy occurred in 3 patients (14.3%) due to serious TRAEs. Two cases (9.5%) of grade 4 gastrointestinal haemorrhage were observed (Patient 4 after 12 cycles; Patient 5 after 10 cycles of sintilimab and bevacizumab biosimilar). Both patients stabilised after endoscopic haemostasis. One participant (4.8%) with grade 3 anaphylaxis was documented (Patient 11 during cycle 2 of sintilimab and bevacizumab biosimilar). The patient gradually recovered after treatment with corticosteroids, and sintilimab was permanently discontinued. One patient (4.8%) with withdrew from the trial due to personal preference.

Biomarkers for treatment response and outcomes
Serial Olink proteomics assays for Immuno-Oncology in plasma (see details in Supplementary Dataset 3 and Supplementary Dataset 4), in parallel with flow cytometry analysis for immune lineages in PBMCs, were performed to compare responders and non-responders to identify possible biomarkers that could predict efficacy. A total of 52 samples were obtained and tested at the time points indicated in Supplementary Fig. S1. All samples passed quality control and were included in the analysis (Supplementary Fig. S3A).

At the T1 time point, responders (Rs, defined as patients with PR) exhibited significantly lower enrichment of IFN-γ (p=0.025) and serum globulin (GLB, p=0.047) and higher levels of CXCL1 (p=0.036), TNF-α (p=0.048), and IL-6 (p=0.002) than non-responders (NRs, defined as patients with SD and PD; Fig. 3A, D, Supplementary Fig. S3B-C and S3G). Additionally, responders had a significantly higher percentage of CD4+ T cells (p=0.019), white blood cell (WBC) count (p=0.038), albumin-to-globulin ratios (A/G, p=0.025), and neutrophil count (p=0.046) than non-responders (Supplementary Fig. S3D and S3G). When the duration of response was considered, IFN-γ expression (p=0.014) and AFP levels (p=0.007) were lower in patients with clinical benefit response (CBR, patients with SD longer than 6 months and patients with PR) than in non clinical benefit (NCB) patients (patients with SD less than 6 months and patients with progression disease [PD]), whereas VEGFA (p=0.033), TWEAK (p=0.026), CD4 (p=0.049), IL-6 (p=0.043), and TNF-α (p=0.0031) levels were higher (Fig. 3B, D, Supplementary Fig. S3B and S3H). Additional biomarkers or characteristics, including age, sex, ECOG score, China liver cancer (CNLC) staging, enrichment of immune lineages, and serum AFP levels, were analysed for correlation with clinical efficacy (Supplementary Fig. S3I). Unsurprisingly, the levels of the classical tumour biomarker AFP were consistently lower in patients with CBR and long PFS at baseline.

Intriguingly, levels of plasma IFN-γ were also decreased in patients with longer PFS than in those with shorter PFS (p=0.0058, Fig. 3C, D). Survival analysis also showed that patients with a higher level of IFN-γ had poorer PFS and estimated OS (Fig. 3F). The median estimated OS was 24.4 months in the low-IFN-γ group and 9.3 months in the high-IFN-γ group (hazard ratio [HR], 4.12; 95% CI, 1.36–12.46; p=0.009). The median PFS was 7.8 months in the low-IFN-γ group and 3.7 months in the high-IFN-γ group (HR, 3.19; 95% CI, 1.5–6.78; p=0.025). Moreover, the existence of extrahepatic metastases (EHS) was correlated with shorter PFS (HR, 5.62; 95% CI: 1.23–25.62) but was not significantly correlated with a lower ORR (EHS vs. non-EHS: 26.7% vs. 50%, p=0.354), and AFP<400 μg/L was correlated with better PFS (HR, 0.09; 95% CI: 0.02–0.41) (Fig. 3E, F, Supplementary Fig. S3J and S3L).

We further investigated the dynamics of plasma proteins in patients at the time of BOR and disease progression. The results demonstrated a downward trend in the expression of PDCD1 (p=0.02) and VEGFA (p=0.0003), as well as in the ratio of CD4+ T cells to CD8+ T cells (Th/Ts, p=0.002). In contrast, an increase in the abundance of CD8+ T cells (p=0.006) and the ratio of terminal effector T cells (defined as CD45RA+CCR7-CD8+ T cells) to CD8+ T cells (p=0.031) was observed when the patients achieved BOR (Fig. 3I), irrespective of response. Impressively, when the disease progressed, the expression levels of arginase 1 (ARG1) and placental growth factor (PGF) and the neutrophil-lymphocyte ratio (NLR) significantly increased compared with those at baseline, whereas the levels of the T-cell activation marker OX-40 and the T-cell chemotactic factor CX3CL1 significantly decreased (Fig. 3J).

Dynamics of cytokines and immune cells in the circulation during SBRT
Subsequently, we enumerated the differences in the levels of circulating immune cells and immuno-oncology-related proteins before and after SBRT (Fig. 4). A significant SBRT-induced increase in IL15, IL6, and CD70 and a decline of VEGFA, B cells, and total lymphocytes were detected. After SBRT, the median frequency of B cell and lymphocyte counts in unirradiated PBMCs decreased from 12.01% (IQR 8.92–15.72%) to 7.46% (IQR 4.35–11.11%) (p=0.0017) and from 1.26 (IQR 0.96–1.75) G/L to 0.73 (IQR 0.46–1.14) G/L (p=0.016), respectively, and the circulating abundance of VEGFA decreased from 9.68 (IQR 9.26–9.94) to 9.28 (IQR 9.04–9.59) (p=0.0057). In contrast, the concentrations of circulating IL6 (3.756 versus 3.278, p=0.0442), CD70 (4.090 versus 3.872, p=0.0088), and IL15 (4.413 versus 4.030, p=0.0091) and two other specific inflammatory markers, the NLR (3.644 versus 2.090, p=0.0043) and the PLR (202.7 versus 111.2, p=0.0058), were significantly elevated after SBRT (Fig. 4A, C, Supplementary Fig. S4A).

While our findings indicated that radiotherapy-induced perturbations in plasma cytokine profiles and immune cell subsets were associated with therapeutic response and PFS, only variations displaying consistent statistical significance across subgroup analyses were deemed to hold substantive relevance to clinical efficacy (Fig. 4A, C). Specifically, SBRT triggered a more prominent augmentation in plasma concentrations of IFN-γ, IL-6, and IL-17A in the CBR group and long-term survival patients (Fig. 4D, E, G) than in the NCB group and patients with short PFS. Additionally, a higher expression of IL-2 in post-SBRT plasma was associated with a better response to the ICI-combined treatment (Fig. 4D, F, G). Moreover, a more pronounced decrease in circulating MMP12 in pre-SBRT and post-SBRT blood samples was associated with improved PFS and clinical benefit (Fig. 4D, E, G). Taken together, inflammation signalling, such as via IFN-γ, IL-6, and IL-2, might be particularly essential in mediating SBRT-triggered immune responses.

Other biomarkers and subgroup analysis
Additional biomarkers or characteristics, including age, sex, ECOG score, prior lines of treatment, expression of PD-L1, infiltration by CD8+ T and CD4+ T cells at diagnosis, existence of liver metastasis, and serum PIVKA-II levels, were analysed for correlation with clinical efficacy (Figure S4B, S4C, Supplementary Table S1, and Supplementary Dataset 1), and no significant differences were observed.

Methods
Study design and participants
The open-label, single-arm, phase 2 study was conducted in accordance with the Declaration of Helsinki and all patients provided written informed consent before enrolment. The institutional review boards of Wuhan Union Hospital approved the study (Ethics number: [2022] 0136-01). The trial was registered with chictr.org.cn (ChiCTR2200056068) on 31 January 2022, and the published study protocol59 was provided in the Supplementary Information. Analyses were conducted as planned in the preregistration. Patients did not receive financial compensation for participation in the study.

Patients aged 18 years or over with an ECOG performance status of ≤1 and clinically or pathologically diagnosed hepatocellular carcinoma (HCC) that had failed to respond to previous anti-PD-1 immunotherapy were eligible for enrolment. According to the Response Evaluation Criteria in Solid Tumour version 1.1 (RECIST v1.1), measurable disease was required if at least one tumour was amenable to SBRT treatment at a dose of 25–50 Gy in 5 fractions, and another measurable tumour lesion was present that did not undergo radiotherapy for efficacy assessment.

Other inclusion criteria included having an A Child-Pugh grade, a life expectancy of ≥3 months, and sufficient bone marrow and organ function. The exclusion criteria included a history of any other malignancy, metastases to the central nervous system, previous radiotherapy, autoimmune or interstitial lung disease, or serious comorbidities, such as congestive heart failure or uncontrolled diabetes.

Procedures
Patients underwent SBRT once daily for five consecutive days, targeting a single tumour lesion. Sintilimab (200 mg, Innovent Biologics, Suzhou, China) combined with bevacizumab biosimilar (15 mg/kg, Innovent Biologics, Suzhou, China) was administered intravenously every 3 weeks, starting 1–2 weeks after completion of SBRT. Treatment continued until disease progression, the occurrence of intolerable toxicity, patient withdrawal, death, or for a maximum duration of 24 months. All patients included in the ITT analysis had finished radiotherapy and at least one cycle of sequential treatment with sintilimab plus a bevacizumab biosimilar. Complete follow-up, including physical examination, information on adverse events, and radiographic evaluation, was performed every 9 weeks. Key laboratory tests and flow cytometry analysis of immune lineages in PBMCs were performed in the clinical laboratory of Wuhan Union Hospital.

Outcomes
The primary endpoint was the ORR, defined as the percentage of patients who had a RECIST v1.1-defined CR or PR; only non-irradiated (abscopal) lesions were RECIST eligible60. The secondary endpoints included DCR, PFS (time from the date of enrolment to the date of disease progression or death), OS (time from the date of enrolment to the date of death), and LRC rates. The PFS or OS times of patients who were still progression-free or alive at the time of the last follow-up were censored. Responders (Rs) were defined as patients with BOR of PR. Non-responders (NRs) were defined as patients with BOR of SD or PD. Clinical benefit responders (CBR) were defined as patients who achieved a BOR of PR or SD lasting longer than 6 months. Non-clinical benefit (NCB) was defined as patients with a BOR of PD or SD lasting less than 6 months. Long progression-free survival (LPFS) and short progression-free survival (SPFS) were defined as patients with PFS longer or shorter than the median PFS, respectively. LRC rates were defined as the proportion of patients without local progression of all irradiated target lesions from the baseline value according to the RECIST 1.1 assessment. Outfield lesions were defined as lesions outside the 500 cGy dosimetric line. The safety assessment spanned from informed consent through follow-up. Investigators evaluated adverse event (AE) incidence and severity per the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. AEs, including TEAEs, TRAEs and serious TRAEs, were monitored during treatment and up to 30 days after the last dose according to the study protocol. TRAEs were meticulously evaluated by investigators through a safety assessment protocol aligned with China NMPA’s Technical Guideline for Causality Assessment of Adverse Events in Drug Clinical Trials (2024 edition, https://www.cde.org.cn/main/news/viewInfoCommon/0a5ae4924881321c07cce100e99f2a5c). Three core criteria had to be fulfilled: temporal plausibility (onset/duration patterns matching known toxicity kinetics), biological gradient (dose/exposure-dependent changes confirmed via pharmacokinetic monitoring), and the exclusion of confounders (active ruling out disease progression, portal hypertension, and concurrent infection through serial imaging and laboratory profiling). The specific criteria are presented in Supplementary Dataset 5.

Plasma immune proteomics
Plasma samples were collected at the indicated time points according to the timeline described in Supplementary Fig. 1A, 1B. The main time points include baseline (T1) after SBRT and before IBIstreatment (T2), at best overall response (T3), and at disease progression (T4). All samples were tested in a single run in the same batch. Proteins in the plasma samples were measured using the Olink® Target Immuno-Oncology Panel (Olink Proteomics, Sweden) according to the manufacturer’s instructions, which includes the detection of 92 proteins by the proximity expansion assay (PEA) with 1 μL of each sample. In brief, pairs of oligonucleotide-labelled antibody probes bind to their target proteins. If the two probes come into proximity, their oligonucleotides hybridise in a pairwise manner.

The introduction of DNA polymerase initiates a proximity-dependent DNA polymerisation event, generating a unique PCR target sequence. This sequence is subsequently detected and quantified using a microfluidic real-time PCR instrument (Biomark HD, Fluidigm). The resulting Ct data undergo quality control and normalisation against a set of internal and external controls. The final assay output is reported as normalised protein expression (NPX) values, which are arbitrary units on a log2 scale, where higher values indicate greater protein expression. The four internal controls are designed to emulate and monitor the distinct stages of the PEA process. These include two incubation/immuno controls, an extension control, and a detection control. The internal controls are added to all samples as well as to the external controls, serving for quality assurance and data normalisation. The external controls comprise a negative control, which is utilised for determining the limit of detection (LOD), and triplicate interplate controls (IPCs), which are used for data normalisation.

Quality control of the data occurs in two stages. First, the run’s quality is assessed by calculating the standard deviation for both the detection control and the incubation/immuno controls. For a run to pass quality control, this standard deviation must be less than 0.2. Second, each sample undergoes quality control by comparing its detection control and one of the incubation control results against the run median. All assay validation data, including detection limits and intra- and inter-assay precision data, are available on the manufacturer’s website (www.olink.com). The complete dataset for the Olink analysis has been uploaded as Supplementary Dataset 4.

Immunohistochemical (IHC) analysis of the tumour immune microenvironment
We analysed 11 FFPE samples from pre-enrolled HCC primary or metastatic lesions: (a) 9 primary HCCs (8 surgical resections, 1 ultrasound-guided biopsy) and (b) 2 lung metastases (1 metastasectomy, 1 CT-guided biopsy). Tissues were collected at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (from 2019–2023; see details in Source data) with histopathological confirmation. IHC analysis was performed on 4-μm FFPE sections using antibodies against CD4 (Clone 4B12, Dako #M7310), CD8 (Clone C8/144B, Dako #M7103), and PD-L1 (Clone 22C3, Dako #SK006). Following EDTA-based antigen retrieval (pH 9.0, 95°C, 20 min) and overnight primary antibody incubation at 4°C, the sections were stained with EnVision FLEX/HRP (Dako) and DAB, and haematoxylin was used for counterstaining. The quantitative assessment included the following: 1) CD4+/CD8+ T-cell density (cells/mm²) counted in five random 400× HPFs spanning intratumoural and invasive margin regions and 2) PD-L1 combined positive score (CPS) = PD-L1+ cells (tumour/lymphocytes/macrophages)/viable tumour cells × 100 (CPS≥1=positive, per the FDA 22C3 pharmDx criteria). Experienced pathologists performed all assessments.

Statistical analysis
This trial adopted Simon’s two-stage optimal design to evaluate the primary endpoint of ORR. Historical benchmarks defined the null (π₀=15%)7 and alternative (π₁=40%) response hypotheses. Early termination criteria stipulated trial discontinuation if ≤1 response occurred among the initial 9 evaluable participants (stage I). Progression to stage II disease required ≥2 responses, with final efficacy declared if >5/19 patients achieved responses (α=0.05, power=80%). To account for 10% attrition, 21 patients were enrolled.

The efficacy-evaluable population comprised participants who received ≥1 treatment cycle with post-baseline imaging. The safety analysis included all patients who were administered ≥1 dose with follow-up toxicity assessments. Survival endpoints (PFS/OS) were analysed via Kaplan-Meier estimates with log-rank tests, whereas response rates (ORRs/DCRs) were calculated using Clopper-Pearson exact 95% CIs—Cox proportional hazard models generated HRs for survival comparisons.

For further analysis, subgroup comparisons between responders and non-responders were conducted using the exact Wilcoxon rank-sum test. The relationship between potential biomarkers and the radiographic response was assessed via Spearman’s rank correlation coefficient. Additionally, biomarker levels before and after treatment were compared using the paired Wilcoxon signed-rank test to evaluate treatment effects. The Wilcoxon rank-sum test was used for intergroup comparisons. Correlations between biomarkers were quantified via Spearman’s ρ, with longitudinal changes assessed using the paired Wilcoxon signed-rank test. All analyses were conducted bidirectionally (SPSS Statistics 26; R, version 4.3.1) at p<0.05, with visualisation implemented in R and GraphPad Prism 9. The following R packages were employed: stats package for statistical testing; ggplot2 package for generating box plots, volcano plots and violin plots; swimplot package for patient-time trajectory plots; linkET package for correlation analysis; forestploter package for univariable forest plots; and the survminer and survival packages for Kaplan-Meier survival analysis and visualization. Data analysis of proteomics was conducted using the Olink NPX Signature Software (version 1.16.0) for quality control (QC) of Olink data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors reported the result of a single-site single-arm Phase II trial, in which they combined radiotherapy (SBRT) with anti-PD-1 (sintilimab) and Bev biosimilar to treat HCC patients who had progressed from the previous ICI-based systemic treatment. The outcome of the treatment was very impressive, the ORR in non-irradiated lesions reached 33.3% (7/21), with a DCR of 66.7% (14/21); when comes to the survival, the mPFS was 6.2 months and the mOS was estimated to reach 24.4 months. Despite the limited sample size, this is still a very successful results, esp. for HCC patients in their 2nd or even later line treatment.\nAlso, from my personal point of view, I agree that the ontology in this pilot trial worth more attention- for a patient with unresectable HCC, the chance of cure is very slim, so the goal is to extend the course of our treatment as much as possible and strive for a longer overall survival. Once the patient progresses from the 1st line systemic treatment, esp. for the “oligo-progressive” tumors, instead of turning immediately to the later line systemic treatment, we can also consider the loco-regional therapy (SBRT, TACE, RF, et al.), together with the current or later line systemic regime.\nMajor limitation\nThe authors argued that this excellent outcome was due to the fact that, radiotherapy reshapes the ICI-refractory TME, then re-sensitizes the anti-PD-1 treatment. The authors also did meticulous study with the peripheral blood and PBMC before and after the treatment, and had some very interesting findings. But, if we want to dig deeper and explore the underline mechanisms, the biomarker study within the TME from the clinical HCC specimen is definitely pivotal. We understand that obtaining biopsy samples from advanced HCC patients can be very tough, and this was not mentioned in the protocol, but this is just so important, or what we find will only be a phenomenon.\nMinor suggestions\n1. I think the submitting author mis-spelt the names of the first author, and last corresponding author, as well as one of the co-authors. I classify this as a “minor” mistake, but this is really the easiest mistake to avoid, as long as you really double-check the text carefully before you finally submit it.\n2. The linguistic issue, as well as some mistakes in the format. Just for an example, Line 92, “tumor microenvironment”, It appears for the first time in the text, and no formal abbreviation is given; then in line 96, “TME” was given directly. Line 135, same problem with “LRC” etc.\n3. 25 pts were screened and 4 were excluded, what were their specific reasons of been excluded?\n4. Line 161: Table 2; grade 3 or 4 should be specified, instead of mixed up.\n5. Line 181: The responders have higher WBC and NEU counts; is that possible that, responders with higher WBC and NEU just because they have better liver function?\n6. Line 192: Supplementary Table S2: in lab test, some results were only shown as “normal”, while some results were obvious within the normal range, but were presented in numerical data, why? The lab tests of all participants were not unified?", "reviewer_2": "The manuscript entitled “Clinical efficacy and biomarker analysis of stereotactic body radiotherapy combined with sintilimab and a bevacizumab biosimilar in anti-PD-1 refractory hepatocellular carcinoma” presents the outcomes of a phase II clinical trial evaluating the efficacy and safety of combining stereotactic body radiotherapy with sintilimab and bevacizumab biosimilar in patients with anti-PD-1-refractory hepatocellular carcinoma. Additionally, biomarker analyses were conducted to further explore the radiation-induced effects. Overall, this manuscript is well-structured and thorough, with high-quality figures. I recommend accepting the paper after the authors successfully address the following minor comments.\n1. Include some statistical data on HCC and immune checkpoint inhibitor treatment outcomes in the introduction section.\n2. For Figure 1, it would be better if the authors could increase the font sizes of certain plots for better readability.\n3. Include some discussion on the possible mechanisms of immune-resistance in current therapeutic approaches.\n4. I noticed that the sample size of this phase II study is limited to 21 patients. Please discuss the possible limitations of this small sample size.\n5. Abbreviations should be defined only at first mention. Please check the manuscript carefully to ensure consistency and avoid redundant definitions.", "reviewer_3": "This paper describes an interesting trial in which 21 advanced HCC patients previously refractory to checkpoint inhibitor(s) were evaluable for having received SBRT plus anti-PD1 plus anti VEGF. In addition patients were sequentially followed up for the concentration of soluble mediators in serum and surface markers of PBMC. The strategy is of interest and results are relevant for radioimmunotherapy of cancer.\nQuestions:\n1. More details regarding the SBRT regimens should be given. Perhaps examples of radiation fields are needed. What level of radiation was received. What were the scattered doses on the lesions left untreated.\n2. Although the protocol is published in BMJ open, it would make sense to have the last version uploaded as a companion file for this paper.\n3. Intervals of confidence should be given for all the parameters in table 1. And accordingly some of the conclusions perhaps need to be toned down.\n4. What was the response rate in extrahepatic metastasis in those patients who had them?\n5. Figure 2C is brilliant for summarized data presentation. Distinction of colors for the different treatments should be optimized.\n6. The spider plots of lesions outside the irradiation field should be given.\n7. Olink units of concentration in serum should be clarified (normalized to what?)\n8. Are there diagnostic biopsies to determine PD-L1. If so this and the infiltration by T cells at diagnosis would be important.\n9. The seminal work regarding immunotherapies for HCC should be referred to.\n10. More background on RT for HCC should be given.\n11. A review on HCC immunotherapy should be referred to.\n12. Previous experiences of antiangiogenics with radiotherapy should be discussed across diseases and ispecifically for HCC.", "reviewer_4": "I have read with great interest the study by Tang et al.\nTheir manuscript presents a phase 2 trial investigating SBRT combined with sintilimab (anti-PD-1) and a bevacizumab biosimilar (IBI305) in patients with ICI-refractory HCC. The study demonstrates a 33.3% ORR, a median PFS of 6.2 months, and an estimated median OS of 24.4 months. Furthermore, the study provides longitudinal biomarker analysis, identifying IFN-γ, IL-6, and IL-2 as key cytokines associated with therapeutic response.\nThis work is clinically relevant and mechanistically insightful, with several limitations I have detailed below.\n1. Without a control arm (e.g., ICI + Bevacizumab without SBRT), it is unclear whether SBRT directly contributes to improved outcomes. I would recommend to explicitly acknowledge this limitation in the Discussion.\n2. Due to the limited sample size, it is difficult to assess the clinical reproducibility of the ORR 33%. In particular, in their sample size calculation, the authors mention that the alternative hypothesis is ORR=40%. The study achieved an ORR of 33.3% - does this make the study positive or negative? It is not clear from the discussion and it should be clarified.\n3. Previous retrospective experiences (e.g. Scheiner et al. JHEP Reports 2023. https://doi.org/10.1016/j.jhepr.2022.100620 ) show that ICI rechallenge in ICI-refractory patients achieves an ORR of 26%. This is similar to the ORR showed in this manuscript, so I am not convinced of the added value of SBRT. I would recommend modifying the discussion accordingly.\n4. The authors state that most of the patients received TKI plus anti-PD1 as first line. They should expand on this treatment decision and clarify which agents were used, since the international guidelines do not recognise this combination as a treatment of choice in first line for patients with advanced HCC.\n5. PFS was 6.2 months, while median duration of response was 5.7 months. PFS is the time between treatment start to progression or death, so I would expect that patients responding to the treatment have a duration of response longer than the median PFS. The authors should clarify this point.\n6. I have some concerns regarding the treatment-related AEs. In particular, the most common trAEs reported are decreased lymphocytes and platelets. However, these are not part of the expected spectrum of AEs from ICI-antiVEGF. It is unlikely that this ICI combination causes a decrease in platelets, which might be due to the underlying disease but it cannot be attributed to an ICI combination. The authors should clarify how these AEs were considered related to the treatment.\n7. The authors should clarify which trAE caused the treatment discontinuation in 3 patients.\n8. Biomarkers analysis: how did the authors define \"decline vs non decline\" (line 198)?\n9. The authors report a decrease in PDCD1 and VEGFA as biologically relevant (lines 204-205). Maybe this is due to the targets of the anti-PD1 and the sintilimab used in the study?\n10. Lines 209-211: the targets increasing at progression are aspecific markers of inflammation. It is expected they would increase in patients progressing on treatment, in particular NLR has been extensively studied in HCC (Wu, Cancers 2022). Same for markers in lines 221-223: the increase in IL6 is expected after SBRT.\n11. Discussion: I would recommend reprhasing the first part as it does not read particularly well (242-248)\n12. Tables: I would recommend reporting all the adverse events, not only those deemed related to the treatment."}, "author_response": "POINT-TO-POINT RESPONSE TO REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, the authors reported the result of a single-site single-arm Phase II trial,\nin which they combined radiotherapy (SBRT) with anti-PD-1 (sintilimab) and Bev biosimilar\nto treat HCC patients who had progressed from the previous ICI-based systemic treatment. The\noutcome of the treatment was very impressive, the ORR in non-irradiated lesions reached 33.3%\n(7/21), with a DCR of 66.7% (14/21); when comes to the survival, the mPFS was 6.2 months\nand the mOS was estimated to reach 24.4 months. Despite the limited sample size, this is still\na very successful results, esp. for HCC patients in their 2nd or even later line treatment.\nAlso, from my personal point of view, I agree that the ontology in this pilot trial worth more\nattention- for a patient with unresectable HCC, the chance of cure is very slim, so the goal is to\nextend the course of our treatment as much as possible and strive for a longer overall survival.\nOnce the patient progresses from the 1st line systemic treatment, esp. for the \"oligo-\nprogressive\" tumours, instead of turning immediately to the later line systemic treatment, we\ncan also consider the loco-regional therapy (SBRT, TACE, RF, et al.), together with the current\nor later line systemic regime.\nResponse: Thank you for your thoughtful evaluation of our work and for recognising the\nunderlying rationale that supports our study. We are particularly grateful for your insightful\nsynthesis regarding the clinical ontology of our pilot trial, which eloquently elevates the\nsignificance of our findings within the broader context of unresectable HCC management. As\npreviously hypothesised, researches and reviews (Llovet et al. Nat Rev Gastroenterol Hepatol.\n2021; Hu, Y. et al., J Natl Cancer Cent, 2022; Scorsetti, M. et al., J Cancer Res Clin Oncol,2015,\nall included the revised reference section) offered valuable perspectives on both of the revised\nintroduction and discussion sections, emphasising the paradigm of prioritising sequential loco-\nregional therapies (e.g., SBRT, TACE, RF, etc.) combined with systemic regimens for oligo-\nprogressive disease, as opposed to prematurely transitioning to later-line systemic therapies.\nInterestingly, there are on-going prospective trial to interrogate efficacy and safety of\nradiotherapy to delay second-line systemic therapy in patients with oligoprogressive\nhepatocellular carcinoma. Your clear articulation of this concept has significantly enhanced the\ntranslational relevance of our work. We are highly grateful for your perspicuous and noteworthy\nfeedback.\nMajor limitation\nThe authors argued that this excellent outcome was due to the fact that, radiotherapy\nreshapes the ICI-refractory TME, then re-sensitises the anti-PD-1 treatment. The authors also\ndid meticulous study with the peripheral blood and PBMC before and after the treatment, and\nhad some very interesting findings. But, if we want to dig deeper and explore the underline\nmechanisms, the biomarker study within the TME from the clinical HCC specimen is definitely\npivotal. We understand that obtaining biopsy samples from advanced HCC patients can be very\ntough, and this was not mentioned in the protocol, but this is just so important, or what we find\nwill only be a phenomenon.\nResponse: We sincerely appreciate your discerning critique regarding the mechanistic\nexploration of our findings. We fully agree that direct analysis of tumour microenvironment\n(TME) biomarkers in clinical HCC specimens would provide the most conclusive evidence to\nsubstantiate our hypothesis about radiotherapy-induced TME remodelling. While the peripheral\nblood/PBMC analyses offered intriguing immunological insights, we noted this methodological\nlimitation more specifically in the revised discussion section (\"Critically, the absence of serial\ntumour biopsies precludes direct assessment of therapy-induced TME remodelling, diminishing\ntranslational insights. \"). Moreover, we also clarified in the inclusion criteria that patients with\nclinically diagnosed HCC (i.e., those without a pathological diagnosis) could be recruited\n(Methods section, \"Patients aged 18 years or over with an Eastern Cooperative Oncology Group\n(ECOG) performance status of ≤1 and a clinically or pathologically diagnosed hepatocellular\ncarcinoma (HCC) that has failed from previous anti-PD-1 immunotherapy are eligible for\nenrolment.\").\nSpecifically, we emphasised the ethical and practical challenges of serial tumour biopsies\nin this heavily treated advanced HCC cohort; nevertheless, approved by the informed consent,\nwe collected surgical or biopsy tissues from accessible patients at the time of initial diagnosis\nand performed immunohistochemical staining for PD-L1 as well as T-cell infiltration to clarify\nwhether these two metrics are indirectly indicative of efficacy and prognosis. Unfortunately,\nthere was no correlation between these indicators and the efficacy of our treatment (See in\nrevised Supplementary Figure S4 and supplementary Table S6). We hypothesised that this could\nbe due to two reasons: 1) the number of tissue samples we obtained was too small to draw\nstatistically positive conclusions; 2) the tumour immune microenvironment of treated second-\nor later-line hepatocellular carcinoma patients had been substantially altered from the initial\nstate, and it is possible that the TME of the initial state could not be established as a definite\ncorrelation between the initial TME and the efficacy of the second-line treatments. That's why\nwe didn't examine these data in the first edition. In the revised version, we noted these results\nin the manuscript, described as \"infiltration by CD8+T and CD4+T cells at diagnosis,…, were\nanalysed for correlation with clinical efficacy\".\nAlternatively, we are now conducting a phase II trial (ChiCTR2300068781) to explore the\nefficacy of radiotherapy combined with Lenvatinib and immunotherapy in potentially\nresectable hepatocellular carcinoma. The protocol of this study requires appropriate pathology\nand subsequent surgical pathology before and after treatment, and we have employed the\ncorresponding single-cell and spatial analysis, hoping that the results of the following study\nwill be more straightforward in uncovering the mechanisms by which the TME of\nhepatocellular carcinoma is regulated by radiotherapy combined with Tyrosine kinase inhibitor\n(TKI) and immunotherapy.\nAlthough these attempts cannot fully compensate for the lack of human TME data in the\npresent study, they align with your vision for a deeper mechanistic interrogation. Thank you for\nhighlighting this critical gap, which significantly strengthens the translational implications of\nour work and guides future research priorities.\nMinor suggestions\n1. I think the submitting author mis-spelt the names of the first author, and last corresponding\nauthor, as well as one of the co-authors. I classify this as a \"minor\" mistake, but this is really\nthe easiest mistake to avoid, as long as you really double-check the text carefully before you\nfinally submit it.\nResponse: We would like to apologise to the editors and all reviewers for these most\nundeserved errors, which were the result of automated text grammar checking and our own\ncarelessness, leading to the misspelling of some authors' given names—many thanks for your\ntolerance and careful correction of our mistakes. We have thoroughly checked the names,\naffiliations, E-mail addresses and titles of all authors entered in Nature Communications'\nsubmission system and ensured their accuracy in the revised manuscripts, as well as in all\nattachments we submitted. There was no de facto change of author in the revision.\n2. The linguistic issue, as well as some mistakes in the format. Just for an example, Line 92,\n\"tumour microenvironment\", It appears for the first time in the text, and no formal abbreviation\nis given; then in line 96, \"TME\" was given directly. Line 135, same problem with \"LRC\" etc.\nResponse: We are sorry for the linguistic oversights in the manuscript and thank you for\npointing out errors and omissions in the terminology and formatting. All abbreviations (e.g.,\n\"tumour microenvironment (TME)\", \"local recurrence-free survival (LRC)\") have been\nrigorously standardised: full terms with abbreviations are now defined at first mention,\nfollowed by exclusive abbreviated usage. A comprehensive audit of the text has resolved\nanalogous issues across other clinical endpoints and biomarkers. To further ensure linguistic\nprecision, the manuscript underwent thorough editing for grammar, syntax, and adherence to\njournal style guidelines. These corrections are highlighted in the track-changes version. We\nacknowledge the importance of meticulous presentation and appreciate your vigilance in\nhelping us elevate the manuscript's technical quality.\n3. 25 pts were screened and 4 were excluded, what were their specific reasons of been\nexcluded?\nResponse: Thank you for your valuable suggestions and reminders. One patient had no target\nlesion other than the proposed radiotherapy lesion; two patients underwent gastroscopy\nsuggesting untreated or incompletely treated oesophageal or gastric varices with bleeding or\nhigh risk of bleeding; and one patient declined enrolment for personal reasons. We have\nannotated these patients in Figure 1 and detailed the situations in the revised manuscript in the\nsection of patient characteristics.\n4. Line 161: Table 2; grade 3 or 4 should be specified, instead of mixed up.\nResponse: We thank the reviewer for this valuable input. As recommended by you and other\nreviewers, we have restructured Tables for AEs to differentiate: 1) All-grade treatment-\nemergent adverse events (TEAEs) were replenished in the revised Supplementary Table S4; 2)\nGrade 3 and 4 treatment-related adverse events (TRAEs), were separated listed in Table 2 (see\nthe Criteria for Adverse Events in Drug Clinical Trials in Table S7 and the website:\nhttps://www.cde.org.cn/main/news/viewInfoCommon/0a5ae4924881321c07cce100e99f2a5c);\n3) All TRAEs, as well as TEAEs, are rearranged in the tables in order of decreasing incidence\nrate. This stratification would distinguish event severity and attribution more precisely in the\nrevised manuscript.\n5. Line 181: The responders have higher WBC and NEU counts; is that possible that,\nresponders with higher WBC and NEU just because they have better liver function?\nResponse: It is a particularly instructive query why white blood cell and neutrophil counts are\nassociated with treatment response. To answer this question, we re-analysed the blood test\nresults from the hospital's central laboratory and found no significant differences in Child-Pugh\n(CP) scores, serum bilirubin, or transaminases between responders and non-responders (Figure\nR1). However, more interestingly, we found that the sera globulin concentration at baseline was\nwithin the normal range for most treatment responders (normal reference range: 20-30 g/l),\nwhereas there was markedly abnormal elevation of globulin in the plasma of non-responders,\nwhich led to a reduction in the A/G ratio in non-responders. We have added these results into\nFigure 3H, Figure S3D, and the relevant results section. It has been reported that high pre-\ntreatment serum GLB and GLB/ALB ratio were independent factor which predicted worse OS\nin HCC patients receiving curative surgical resection1. Moreover, individuals with a high GLB\nhad markedly longer median of PFS and OS in non-small cell lung cancer (NSCLC) patients\ntreated with ICIs2, as well as in colorectal cancer CRC patients3. High globulin levels may be\nattributed to chronic inflammatory diseases such as chronic viral or bacterial infection and liver\ndisease1,4,5. In summary, we would argue there is insufficient direct evidence that decreased\nWBC and NEU may be directly associated with non-responders' liver function.\nFigure R1. Pre-treatment parameters correlated with liver function are plotted and compared\nbetween Rs and NRs. Wilcoxon rank-sum test was used to identify significant differences.\n6. Line 192: Supplementary Table S2: in the lab test, some results were only shown as\n\"normal\", while some results were obvious within the normal range, but were presented in\nnumerical data, why? The lab tests of all participants were not unified?\nResponse: During the raw data collection process at our centre, the Clinical Research\nCoordinator (CRCs) routinely annotate laboratory blood test results within the range of\ninstitutional normal reference values as \"normal\" in the source documents. For specific\nindicators of particular interest (e.g., tumour markers, cytokines or counts of immune lineages),\nwe intentionally extracted and recorded numerical values from medical records to facilitate\npotential efficacy correlation analyses. To eliminate ambiguity, we have now systematically\nreaudited and supplemented all previously labelled \"normal\" laboratory parameters in the\nrevised Supplementary Table S2 by cross-referencing raw data in the electronic medical record\nsystem. Correspondingly, the volcano points in Figures 3H, 4B, 4D-4F, and S3D-S3F have been\nsubstituted with revised data. The parameters that are statistically distinct between groups have\nbeen updated and illustrated in the box plots (Figures S3G-S3H). Should additional questions\narise regarding laboratory data, please do not hesitate to request clarification, and we will\nprovide corresponding data within the bounds of patient privacy protections. We sincerely\nappreciate this critical inquiry, which prompted an essential refinement of our dataset integrity.\nReviewer #2 (Remarks to the Author):\nThe manuscript entitled \"Clinical efficacy and biomarker analysis of stereotactic body\nradiotherapy combined with sintilimab and a bevacizumab biosimilar in anti-PD-1 refractory\nhepatocellular carcinoma\" presents the outcomes of a phase II clinical trial evaluating the\nefficacy and safety of combining stereotactic body radiotherapy with sintilimab and\nbevacizumab biosimilar in patients with anti-PD-1-refractory hepatocellular carcinoma.\nAdditionally, biomarker analyses were conducted to further explore the radiation-induced\neffects. Overall, this manuscript is well-structured and thorough, with high-quality figures. I\nrecommend accepting the paper after the authors successfully address the following minor\ncomments.\nResponse: We are deeply grateful for your thoughtful evaluation and generous recognition\nof our manuscript's scientific rigour, structural clarity, and graphical presentation. It is\nparticularly encouraging to receive your endorsement of the trial's clinical and translational\nvalue. We have meticulously addressed all raised comments in the revised manuscript and\nsupplemented additional methodological details to enhance reproducibility further. Moreover,\nwe proactively conducted a self-audit to refine ambiguous statements in the biomarker analysis\nsection and ensure consistency in data annotation across figures. Your constructive feedback,\nalongside our self-identified refinements, collectively elevate the manuscript's scholarly impact.\nThank you for your time and expertise in guiding this work toward publication readiness.\n1. Include some statistical data on HCC and immune checkpoint inhibitor treatment outcomes\nin the introduction section.\nResponse: We appreciate your valuable suggestions for strengthening the epidemiologic\ncontext and statistical descriptions. In the revised Introduction, we have integrated the\nfollowing updates: (1) 2024 GLOBOCAN data highlighting HCC's rising mortality-to-\nincidence ratio (Siegel, R. L. et al., 2024. CA Cancer J Clin); (2) Statistics from several Phase\nIII trials that included IMbrave-150, ORIENT-32, SHR-1210-III-310, HIMALAYA, and\nCheckMate-9DW that enrolled patients with hepatocellular carcinoma receiving front-line\nimmunotherapy (Ref.4,6-9 in the revised manuscript). These contemporary benchmarks now\nexplicitly frame the unmet need in ICI-refractory populations that motivated our SBRT-based\nsalvage strategy.\n2. For Figure 1, it would be better if the authors could increase the font sizes of certain plots\nfor better readability.\nResponse: We are grateful for your perceptive feedback regarding figure clarity. In the\nrevised manuscript, we have increased font sizes across all subplots of Figure 1 and Figure S1\n(particularly axis labels, tick marks, and survival curve annotations) to enhance visual\naccessibility. These adjustments were implemented using vector-based scaling to preserve\ngraphical resolution and are now fully compliant with the journal's formatting guidelines.\nAdditionally, we conducted a comprehensive audit of all figures to ensure consistency in\ntypography and spacing of elements. The updated high-resolution versions are embedded in\nboth the main text and supplemental files.\n3. Include some discussion on the possible mechanisms of immune-resistance in current\ntherapeutic approaches.\nResponse: We appreciate your emphasis on elucidating mechanisms of immune resistance. In\nthe revised discussion section, we have added a paragraph of \"Although the precise mechanisms\nunderlying innate or acquired resistance to ICIs are not yet fully elucidated, … inadequate\ncancer cell recognition by T cells, and the presence of T-cell inhibitory molecules or\nimmunosuppressive cells within the TME.\", incorporating recent advances, a modification that\nintensifies the analysis of mechanisms of resistance to current HCC therapies.\n4. I noticed that the sample size of this phase II study is limited to 21 patients. Please discuss\nthe possible limitations of this small sample size.\nResponse: Thanks for raising this important question. We acknowledge that the sample size of\nthis Phase 2 study (n = 21) was determined a priori by statistical power calculations; however,\nwe fully recognise the inherent limitations associated with this modest cohort. These constraints\ninclude reduced statistical power for subgroup analyses and potential impacts on\ngeneralizability. To address these limitations, we explicitly recommend future randomised\ncontrolled trials with larger sample sizes to validate our findings. As emphasised in the\ndiscussion section, we detailed the limitation of our study, as “Our study has inherent limitations.\nThe single-arm design and modest cohort size (n = 21) — though statistically predetermined\nby Simon's two-stage phase II framework — constrain statistical power for subgroup analyses\nand may impact generalizability…they underscore the imperative for randomized phase III\ntrials to further validate our observations”.\n5. Abbreviations should be defined only at first mention. Please check the manuscript carefully\nto ensure consistency and avoid redundant definitions.\nResponse: We are sorry for the linguistic oversights in the manuscript and thank you for\npointing out errors and omissions in the terminology format. All abbreviations (including but\nnot limited to TME, LCR, and ICI et al.) have been rigorously standardised: full terms with\nabbreviations are now defined at first mention, followed by exclusive abbreviated usage. We\nacknowledge the importance of meticulous presentation and appreciate your vigilance in\nhelping us elevate the manuscript's technical quality.\nReviewer #3 (Remarks to the Author):\nThis paper describes an interesting trial in which 21 advanced HCC patients previously\nrefractory to checkpoint inhibitor(s) were evaluable for having received SBRT plus anti-PD1\nplus anti VEGF. In addition, patients were sequentially followed up for the concentration of\nsoluble mediators in serum and surface markers of PBMC. The strategy is of interest and results\nare relevant for radioimmunotherapy of cancer.\nResponse: We are deeply appreciative of your recognition of the clinical relevance and\ntranslational implications of our radioimmunotherapy strategy for ICI-refractory HCC. Your\naffirmation of the study's design and biomarker-driven approach is particularly encouraging. In\nalignment with your emphasis on methodological rigor, we acknowledge the inherent\nlimitations of this phase II trial, including its single-arm design and modest sample size, which\nrestrict causal attribution of SBRT's immunomodulatory effects.\nQuestions:\n1. More details regarding the SBRT regimens should be given. Perhaps examples of radiation\nfields are needed. What level of radiation was received. What were the scattered doses on the\nlesions left untreated.\nResponse: We are grateful for your meticulous scrutiny of the radiotherapy protocols. As\nrequested, we have specified the SBRT dose per fraction (median 8 Gy, range 5–8 Gy × 5\nfractions) for the irradiated lesion and provided scatter dose metrics in Revised results section\nand Table S2. The representative radiation field diagrams for hepatic and pulmonary lesions\nwere shown as below (see isodose distributions in Figure R2). Of 21 patients, 4 had non-\nirradiated target lesions within PTV margins (mean scatter dose 2.5-5.1 Gy with V5≤20%),\nwhereas 17 had lesions fully outside radiation fields with negligible exposure (<1 Gy). Target\nselection prioritised anatomical isolation to minimise off-target effects, consistent with ASTRO\nguidelines for oligoprogressive HCC. We hope these clarifications would address the requested\ndetails regarding SBRT administration, and if any further questions arise, additional\ninformation can be promptly provided.\nFigure R2. Dose-volume metrics of irradiated lesions and target lesions for RECIST v1.1\nevaluation. Representative radiation field diagrams for hepatic (A, patient #6 with BOR of\nPR, V5 = 2%) and pulmonary lesions (B, patient #10 with BOR of PD, V5 = 10%) were\nshown.\n2. Although the protocol is published in BMJ open, it would make sense to have the last version\nuploaded as a companion file for this paper.\nResponse: Actually, the study protocol file was uploaded along with the original manuscript\nand we apologise for not describing it in detail due to our oversight. We have emphasised about\nthe uploaded Study protocol V2 in the Methods section of the revised manuscript, and thank\nyou for your suggestion to make our data appear more intact.\n3. Intervals of confidence should be given for all the parameters in table 1. And accordingly\nsome of the conclusions perhaps need to be toned down.\nResponse: We apologize for not giving a complete presentation of the statistical data, and as\nyou suggested, confidence intervals for all parameters are presented in the revised Table 1.\nAccordingly, the conclusion section has been adjusted to be relevant.\n4. What was the response rate in extrahepatic metastasis in those patients who had them?\nResponse: This is really a very constructive question. Following the stratification of patients\naccording to the presence or absence of liver cancer metastases, it was found that patients with\nextrahepatic metastases (EHS) exhibited lower treatment efficacy rates of 26.7% (4/15)\ncompared to patients without EHS of 50% (3/3) for ORR (see in Figure R3 and revised\nsupplementary Figure S3J). The description in the relevant part was also changed to\n\"Meanwhile, the existence of extrahepatic metastases (EHS) was correlated with shorter PFS\n(HR, 5.62, 95% CI: 1.23–25.62), insignificantly but noteworthily lower ORR (EHS vs non-\nEHS: 26.7% vs 50%, p = 0.354) and AFP<400μg/L was correlated with better PFS (HR, 0.09,\n95% CI: 0.02–0.41) (Figure 3E-3F, Supplementary Figure S3J and S3L)\". While this\ndiscrepancy did not attain statistical significance, it warrants further consideration due to its\npotential implications. It is imperative that larger sample cohort studies are conducted in the\nfuture to provide robust evidence for this phenomenon.\nFigure R3. Clinical benefit of patients (left) and Response by RECIST v1.1 of patients (right)\nstratified by the existence of extrahepatic metastases.\n5. Figure 2C is brilliant for summarised data presentation. Distinction of colors for the different\ntreatments should be optimised.\nResponse: We sincerely appreciate your positive feedback on Figure 2C and the insightful\nsuggestion regarding colour optimisation. The colour contrast distinguishing different treatment\ngroups has been enhanced in the revised high-resolution figure (now Figure 2C), to improve\nvisual discrimination. Should any aspect of the revised visualisation remain suboptimal, we\nwelcome your specific guidance and will diligently implement further refinements.\n6. The spider plots of lesions outside the irradiation field should be given.\nResponse: We appreciate the reviewer's critical inquiry regarding lesion assessment. The\nmanuscript originally presented spider plots of unirradiated lesions per RECIST 1.1 criteria.\nHere we illustrated the spider plots and the waterfall plots for outfield lesions, which defined\nas the lesions out of the field of 500cGy .As shown in Figure R4, lesions outside the irradiation\nfield can also achieve very effective results, the relavant data were shown in the revised\nSupplementary Figure S2A-S2B.\nFigure R4. (A) Maximal change of tumor size from baseline in lesions outside the irradiation\nfield (outfield lesions). (B) Change of individual tumor burden regarding the outfield lesions.\nThank you again for your helpful feedback. The extra data you provided has made our data\non efficacy more complete.\n7. Olink units of concentration in serum should be clarified (normalised to what?)\nResponse: Thank you for broaching the subject. Olink's NPX (Normalised Protein eXpression)\nis a relative quantification unit, log2-transformed and normalised to internal controls,\nbackground levels to ensure comparability across runs, but it does not represent absolute\nconcentration. That is, the assay quantified protein expression levels using normalised log2-\ntransformed arbitrary units, with elevated values corresponding to increased protein abundance.\nSeveral other studies using Olink to detect plasma proteomes have also used NPX as a relative\nreference value with no actual concentration, such as Temba's study published in Nature\nMedicine6 and Xie's study published in STTT7. Specifically, the raw data were normalised and\nNPX values were calculated using Olink NPX Signature (version 1.16.0). Accordingly, the\ndescription of normalisation is presented in the Methods section, which we hope will satisfy\nyour requirement for rigor in the article.\n8. Are there diagnostic biopsies to determine PD-L1. If so this and the infiltration by T cells at\ndiagnosis would be important.\nResponse: This is a matter of great importance. We acknowledge the critical importance of\ntumour microenvironment dynamics. A key translational limitation of this trial is the inability\nto acquire paired tumour biopsies pre- and post-treatment, which precludes direct assessment\nof therapy-induced immune remodelling in late-line HCC. As hepatocellular carcinoma is often\nclinically diagnosed without biopsy, only 11/21 patients had archival tissue (diagnostic\nbiopsies/surgical specimens) available for analysis.\nImmunohistochemistry revealed no significant correlation between pre-treatment PD-L1\nCPS scores, CD8+/CD4+ T-cell infiltration densities, and clinical response to the therapeutic\nregimen (P > 0.05; Figure R5; revised Figure S4B-S4C and Table S6). Crucially, most samples\nwere obtained during initial diagnosis—prior to any systemic therapy—and may have been\naltered by subsequent front-line treatments, potentially obscuring true microenvironmental\nassociations.\nWe concur that serial tissue sampling is essential to decipher predictive biomarkers. Future\ntrials incorporating on-treatment biopsies (with rigorous ethical safeguards) are needed to\nidentify actionable immune signatures for precision management of anti-PD-1-refractory HCC.\nFigure R5. PD-L1, infiltration of CD8+T and CD4+T cells are plotted and compared between\nRs and NRs, as well as CBRs and NCBs. Wilcoxon rank-sum test was used to identify\nsignificant differences.\n9. The seminal work regarding immunotherapies for HCC should be referred to.\nResponse: We appreciate the insightful suggestion regarding seminal immunotherapy studies\nin HCC. Key foundational works have been incorporated into the Introduction and Discussion,\nincluding: 1) IMbrave1508 (atezolizumab/bevacizumab), ORIENT-329 and CARES-31010 for\nestablishing first-line standards; 2) Predictive biomarkers of PD-1 inhibitor efficacy, such as\ninflammatory gene signatures (Sangro et al., J Hepatol 2020)11, neutrophil-to-lymphocyte ratio\n(NLR)12, platelet-to-lymphocyte ratio (PLR)13, and the CRAFTY14 score—most\ncomprehensively reviewed by Llovet et al.15 A number of predictive biomarkers of PD-1\ninhibitor efficacy have been identified, including inflammatory gene signatures11, neutrophil-\nto-lymphocyte ratios (NLR) 12, platelet-to-lymphocyte ratios (PLR)13, and CRAFTY scores14.\nA comprehensive review of this subject can be found in the work of Llovet et al.15; 3)\nMechanistic insights into immunosuppression, including in neoantigen processing and\nrecognition16, suboptimal priming/activation of tumour-specific T cells17,18, insufficient T-cell\nexpansion or co-stimulatory signals, impaired trafficking of effector T cells to tumour sites19,\ninadequate cancer cell recognition by T cells, and the presence of T-cell inhibitory molecules\nor immunosuppressive cells within the TME20.\nThese additions contextualise our study within the evolving HCC immunotherapy\nlandscape. Should any critical omissions be noted, we welcome guidance to further strengthen\nthe scientific discourse.\n10. More background on RT for HCC should be given.\nResponse: We apologise for the omission of both the quotation and the background details from\nthe introduction. The revised References 19-22 and the relevant part of introduction delves into\nthe application of radiation therapy, with a particular emphasis on the use of SBRT in the\nmanagement of HCC. Due to an oversight in the original manuscript, the term \"hepatocellular\ncarcinoma\" was not qualified, which resulted in a misinterpretation. To rectify this, the\nstatement has been meticulously corrected in the revised manuscript, and a review on the\napplication of SBRT in HCC has been appended. The assistance rendered in the revision of the\nmanuscript is greatly appreciated.\n11. A review on HCC immunotherapy should be referred to.\nResponse: Thank you very much for your valuable suggestion. While we have detailed several\nphase III trials of immunotherapies, a comprehensive review of immunotherapies for HCC has\nnot been properly cited. We refer to Llovet's review (Immunotherapies for hepatocellular\ncarcinoma. Nat Rev Clin Oncol 19, 151–172 (2022). Ref. 3 of the revised manuscript) in the\nreference part, and we are confident that it will make our manuscript more convincing.\n12. Previous experiences of antiangiogenics with radiotherapy should be discussed across\ndiseases and specifically for HCC.\nResponse: This is a very bright suggestion. We reviewed the latest literature and found a recent\nstudy published in the Red Journal (Sheng et al., 2025, Int J Radiat Oncol Biol Phys)\ndemonstrating that anti-angiogenesis may potentiate the distant effects of the combination of\nradiotherapy combined with immunotherapy in hepatocellular carcinoma21. Furthermore,\nKashiwagi, S. et al. had previously reported that anti-angiogenesis promoted radiosensitivity in\nglioma cells22 (Nature Medicine, 2008). The addition of these citations in the revised manuscript\nreinforces the scientific validity and rationale of our study, and again, thank you for your advice\non refining the citations.\nReviewer #4 (Remarks to the Author):\nI have read with great interest the study by Tang et al.\nTheir manuscript presents a phase 2 trial investigating SBRT combined with sintilimab (anti-\nPD-1) and a bevacizumab biosimilar (IBI305) in patients with ICI-refractory HCC. The study\ndemonstrates a 33.3% ORR, a median PFS of 6.2 months, and an estimated median OS of 24.4\nmonths. Furthermore, the study provides longitudinal biomarker analysis, identifying IFN-γ,\nIL-6, and IL-2 as key cytokines associated with therapeutic response.\nThis work is clinically relevant and mechanistically insightful, with several limitations I have\ndetailed below.\n1. Without a control arm (e.g., ICI + Bevacizumab without SBRT), it is unclear whether SBRT\ndirectly contributes to improved outcomes. I would recommend to explicitly acknowledge this\nlimitation in the Discussion.\nResponse: We deeply appreciate your constructive feedback and acknowledge this limitation\nregarding the absence of a control arm. As explicitly stated in the revised discussion section: \"\nOur study has inherent limitations. The single-arm design and modest cohort size (n = 21) —\nthough statistically predetermined by Simon's two-stage phase II framework — constrain\nstatistical power for subgroup analyses and may impact generalizability.\" This limitation is\nfurther contextualised with other constraints (e.g., lack of serial biopsies) in the designated\ndiscussion subsection.\n2. Due to the limited sample size, it is difficult to assess the clinical reproducibility of the ORR\n33%. In particular, in their sample size calculation, the authors mention that the alternative\nhypothesis is ORR=40%. The study achieved an ORR of 33.3% - does this make the study\npositive or negative? It is not clear from the discussion and it should be clarified.\nResponse: We appreciate the reviewers' constructive comments. Although the observed\nORR of 33.3% is clinically meaningful in second- or third-line treatment, it failed to meet the\npredefined threshold of 40%. Our study design was established in 2022, referencing\ncontemporaneous data from immunotherapy, the ORIENT-32 study, and efficacy rates from\nprevious radiotherapy studies incorporating abscopal effects. We aimed to validate efficacy\nexceeding expectations with a limited sample size, hence setting the threshold at 40%. However,\nemerging data from 2024 indicated that the ORR for the ICI-resistant group receiving\nregorafenib plus pembrolizumab in a prospective trial range from 5.9% to 11.1%23, suggesting\nour initial 40% projection may have been too optimistic. While the expectations were not met,\nthis remains an encouraging approach, and we have revised the limitations significance of this\nstudy in the discussion section (\" Although the primary endpoint (ORR≥40%) was not achieved,\nthe 33.3% response rate substantially exceeds historical controls for anti-PD-1-refractory HCC,\nwhich signals potential clinical utility warranting validation in larger cohorts, especially given\nthe limited treatment options available in this situation. \").\n3. Previous retrospective experiences (e.g., Scheiner et al. JHEP Reports 2023.\nhttps://doi.org/10.1016/j.jhepr.2022.100620) show that ICI rechallenge in ICI-refractory\npatients achieves an ORR of 26%. This is similar to the ORR showed in this manuscript, so I\nam not convinced of the added value of SBRT. I would recommend modifying the discussion\naccordingly.\nResponse: We sincerely appreciate the reviewer's critical perspective regarding the added value\nof SBRT in the context of ICI rechallenge efficacy. While the Scheiner et al. retrospective\nanalysis24 reported a 26% ORR in ICI-refractory HCC—numerically comparable to our 33.3%\nORR—several methodological distinctions necessitate cautious interpretation. Crucially, as a\nprospective phase II trial, our study design inherently minimises confounding factors that\nfrequently challenge retrospective analyses. Upon detailed re-evaluation of the Scheiner's\ncohort, two key differences emerge that may overstate ICI rechallenge efficacy:\nFirst, 17 patients (29%) in their cohort received one or more lines of systemic therapy\nbetween initial ICI (ICI1) and rechallenge (ICI2). These intervening treatments—potentially\nincluding loco-regional therapies or novel agents—may have exerted immune-priming effects\nanalogous to radiotherapy's abscopal response, thus conflating the true rechallenge contribution.\nSecond, 45% of their ICI1 regimens were monotherapy (vs. only 7% in ICI2), suggesting that\nenhanced efficacy during rechallenge may partly reflect intensified combination strategies\nrather than the activity of rechallenge with ICI.\nIn contrast, our cohort exclusively enrolled patients who progressed after dual-ICI\ncombinations or ICI plus TKI/anti-VEGF therapy—representing a more treatment-refractory\npopulation where prior ICI exposure was maximally optimised. Within this context, the\nobserved 33.3% ORR achieved with SBRT combinatorial rechallenge signals meaningful\nclinical activity. We fully acknowledge that randomised trials remain essential to definitively\nestablish SBRT's incremental benefit, and have revised the introduction and the discussion to\nincorporate:1) Comment on the rationle of Scheiner's study (JHEP Reports 2023); 2) Emphasis\non the biological plausibility of radiotherapy-induced immune sensitisation in rechallenge\nsettings.\nWe are grateful for this insightful critique, which has strengthened our manuscript's\nscholarly rigor. The added discussion on objectively contextualises SBRT's potential role\nwithin evolving ICI rechallenge paradigms.\n4. The authors state that most of the patients received TKI plus anti-PD1 as first line. They\nshould expand on this treatment decision and clarify which agents were used, since the\ninternational guidelines do not recognise this combination as a treatment of choice in first line\nfor patients with advanced HCC.\nResponse: We appreciate this critical inquiry regarding first-line treatment selection. The\npredominant use of TKI/anti-PD-1 combinations in our cohort reflects real-world practice\npatterns in China, driven by two key considerations: 1) Accessibility Constraints: The\nIMbrave150 regimen (atezolizumab/bevacizumab) faces significant reimbursement barriers\nand limited accessibility in China's public healthcare system; 2) Emerging Evidence: Since\n2021, compelling phase II/III data from Chinese trials supported TKI/PD-1 combinations:\nRESCUE Study (Clin Cancer Res. 2021): Camrelizumab + apatinib (ORR 46%), APOLLO\nTrial (The LANCET Oncol 2025): Anlotinib + Penpulimab (mPFS 6.9 mo; PFS benefit vs\nsorafenib, HR 0.52), and CARES-310 (THE LANCET 2023): Camrelizumab + rivoceranib\n(25.4% (RECIST v1.1)/33.1% (mRECIST); PFS benefit vs sorafenib, HR 0.52). This type of\nregimen received Category 1 recommendations in the CSCO HCC Guidelines from 2022. They\nwere more affordable than drugs imported and most of them are reimbursed by the national\nhealth insurance. Nineteen patients were treated with at least one line of TKI plus PD-1 regimen.\nSpecifically, 73.7% (14/19) of these patients received a combination of lenvatinib and\ncamrelizumab or sintilimab, 21.1% (4/19) received apatinib and camrelizumab, and 5.3% (1/19)\nreceived both regimens.\nPer your suggestion, we have added this rationale to the results section, clarifying as \"\nAccording to the Chinese Society of Clinical Oncology (CSCO) guidelines, the front-line\nTKI/PD-1 combination was selected based on availability and data from contemporary trials in\nChina, despite differences in international guidelines. \"\nBesides, the following was added to the Discussion: \"These findings should be interpreted\nconsidering regional therapeutic preferences. The reliance on TKI/PD-1 combinations—while\njustified by local accessibility constraints and CSCO guidelines—may limit generalizability to\nhealthcare systems where atezolizumab/bevacizumab is first-line standard.\"\nThank you for highlighting this contextual nuance, we believe this clarification strengthens\nthe manuscript's transparency.\n5. PFS was 6.2 months, while median duration of response was 5.7 months. PFS is the time\nbetween treatment start to progression or death, so I would expect that patients responding to\nthe treatment have a duration of response longer than the median PFS. The authors should\nclarify this point.\nResponse: We thank the reviewer for raising this vital methodological point. PFS was\ndefined as the time from enrolment to the first imaging of disease progression or death,\nwhichever occurs first; DoR: Time from first confirmed response to progression (only in\nresponders, see Figure R6)\nFigure R6. Examples of participants' DOR\nThe apparent discrepancy—where median PFS (6.2 months from enrolment) exceeds\nmedian DoR (5.7 months from first response)—stems from endpoint definitions aligned with\nHCC trial standards and reflects that there is no inherent dependency requiring DoR to exceed\nPFS. Notably, two patients achieved best-practice PR at the second and third evaluations,\nrespectively. The delay in achieving PR also led to a shortening of DOR.\n6. I have some concerns regarding the treatment-related AEs. In particular, the most common\ntrAEs reported are decreased lymphocytes and platelets. However, these are not part of the\nexpected spectrum of AEs from ICI-antiVEGF. It is unlikely that this ICI combination causes a\ndecrease in platelets, which might be due to the underlying disease but it cannot be attributed\nto an ICI combination. The authors should clarify how these AEs were considered related to the\ntreatment.\nResponse: We would like to express our sincere gratitude to the reviewer for this perspicacious\nobservation regarding treatment-related adverse events (TRAEs), with particular reference to\nthe hematologic AEs. The attribution of decreased lymphocytes and platelets as TRAEs was\nmeticulously evaluated by investigators through a predefined safety assessment protocol\naligned with China NMPA's Technical Guideline for Causality Assessment of Adverse Events\nin Drug Clinical Trials (2024 edition, see in the website\nhttps://www.cde.org.cn/main/news/viewInfoCommon/0a5ae4924881321c07cce100e99f2a5c).\nIn order to achieve this, three core criteria had to be fulfilled: temporal plausibility\n(onset/duration patterns matching known toxicity kinetics), biological gradient (dose/exposure-\ndependent changes confirmed via pharmacokinetic monitoring), and exclusion of confounders\n(active ruling out of disease progression, portal hypertension, and concurrent infections through\nserial imaging and laboratory profiling). The specific criteria were presented in Supplementary\nTable S7.\nThe results indicate a high proportion of TRAEs in TEAE. One potential explanation for\nthis phenomenon is the determination of TRAE by the investigators when the occurrence is\ndeemed to be a potentially treatment-related AE, as per the stipulated criteria.\nIn cases of lymphocyte decrease, the investigators primarily attributed these TRAEs to\nsintilimab-induced T-cell exhaustion (in line with irAE patterns observed in ORIENT-329),\nwhich may be further exacerbated by bone marrow suppression induced by stereotactic body\nradiotherapy (SBRT). Regarding thrombocytopenia, the triple mechanisms of bevacizumab-\nmediated inhibitory action on platelets (by suppressing sP-selectin, interfering with platelet\nactivation, leading to downregulation of platelet activation markers)25,26, PD-1-driven\nthrombocytopenic (as documented in recent case series of immune thrombocytopenia)27 and\nimpaired liver function caused by treatment provided biological plausibility. Crucially, these\nevents manifested cyclically with treatment administration and improved upon cessation of\ntherapy, strengthening the causal link.\nWe have made the following amendments to enhance transparency in the revised\nmanuscript: 1) Table 2 and Supplementary Table S4 now stratifie all TRAEs and TEAEs\ndepending on the severity, with 3 and 4 grade separately; 2) The clarifications of TRAEs were\nstated in the methods section and Supplementary Appendix includes complete causality\nassessment worksheets per NMPA criteria.\n7. The authors should clarify which TRAE caused the treatment discontinuation in 3 patients.\nResponse: Patients #4 and #5 discontinued treatment due to grade 4 gastrointestinal\nhaemorrhage, having completed 12 and 10 cycles of sintilimab and bevacizumab biosimilar,\nrespectively. Patient #11 withdrew after experiencing grade 3 anaphylaxis during her second\ncycle of the same regimen. This clarification has been added to the results section as follows: \"\nDiscontinuation of therapy occurred in 3 patients (14.3%) due to treatment-related adverse\nevents (TRAEs). Two cases (9.5%) of grade 4 gastrointestinal haemorrhage were observed\n(Patient 4 after 12 cycles; Patient 5 after 10 cycles of sintilimab and bevacizumab biosimilar).\nBoth patients stabilized after endoscopic hemostasis. One participant (4.8%) of grade 3\nanaphylaxis was documented (Patient 11 during cycle 2 of sintilimab and bevacizumab\nbiosimilar). The patient gradually recovered after treatment with corticosteroids, and sintilimab\nwas permanently discontinued\".\n8. Biomarkers analysis: how did the authors define \"decline vs non decline\" (line 198)?\nResponse: We apologise very much for the clerical error. What we actually meant to state was\nthat there was a difference in the PFS and OS of the patients after stratification according to the\nlevel of plasma IFN-γ (cutoff at the median), which we have changed in the revised article to\n\"Survival analysis also showed that patients with a higher level of IFN-γ possessed poorer PFS\nand estimated OS (Figure 3F). Median estimated OS was 24.4 months in the low-IFN-γ group\nand 9.3 months in the high-IFN-γ group (Hazard Ratio [HR], 4.12; 95% CI, 1.36 - 12.46; p =\n0.009). Median PFS was 7.8 months in the low-IFN-γ group and 3.7 months in the high-IFN-γ\ngroup (HR, 3.19; 95% CI, 1.5–6.78; p = 0.025)\" in result section.\n9. The authors report a decrease in PDCD1 and VEGFA as biologically relevant (lines 204-205).\nMaybe this is due to the targets of the anti-PD1 and the sintilimab used in the study?\nResponse: The points you have enumerated are highly compelling. The administration of anti-\nPD1 and anti-VEGF agents has been identified as a contributing factor to this downward trend.\nIt is posited that a substantial decline in PDCD1 and VEGFA at the T3 time point (when the\npatient exhibited optimal responses) can be discerned precisely in the patient's plasma. However,\nas the disease progressed (T4 time point), although slightly, both absolute value of VEGFA and\nPDCD1 increased compared to T3 (Figure R7). This indicated that monitoring plasma VEGFA\nand PDCD1 abundance might facilitate disease progression prediction. However, this inference\nstill needs to be checked in expanded clinical samples, so we did not emphasize it in the\nmanuscript.\nFigure R7. Expressions of PDCD1 and VEGFA tested by olink from timepoint 1 to timepoint\n4. The plots and lines represent the mean and upper and lower quartiles, respectively. Paired\nWilcoxon signed rank test was used to identify significant differences. Only significant p\nvalues (p < 0.05) were illustrated.\n10. Lines 209-211: the targets increasing at progression are aspecific markers of inflammation.\nIt is expected they would increase in patients progressing on treatment, NLR has been\nextensively studied in HCC (Wu, Cancers 2022). Same for markers in lines 221-223: the\nincrease in IL6 is expected after SBRT.\nResponse: The theory you mentioned is indeed well-established. As a typical tumour involves\nan inflammation-mediated carcinogenesis process, patients with HCC, particularly in advanced\nstages, are frequently already in the pathophysiological process of chronic inflammation prior\nto treatment. A multitude of studies have demonstrated that specific markers of inflammation\nare associated with postoperative recurrence, prognosis, and ICI resistance11,28 in hepatocellular\ncarcinoma. However, existing research suggests that the role of inflammatory factors in tumours\nis highly heterogeneous. For instance, IL-6 can exhibit an antitumour effect in certain\ncircumstances, yet in others, it may stimulate tumour growth29. The time interval between T1\nand T2 is very short (from enrolment to the completion of 5 fractions of SBRT), while the time\ninterval between T1/T2 and T4 covers the period in which most patients undergo SBRT plus\ntwo or more cycles of systemic treatment. Previous evidence suggests that high-dose, low-\nfraction SBRT has a pro-inflammatory effect and promotes antigen presentation30,31. The results\nof multiple clinical studies also demonstrate that patients with Chinese hepatocellular\ncarcinoma caused by hepatitis B may benefit more from PD-1/PD-L1, either in combination\nwith anti-angiogenic or CTLA-4 antibodies32. In the context of our study, high-dose SBRT\nappeared to provoke acute inflammation yet in ICI-treated HCC, which may potentiate the\nantitumor efficacy of ICIs plus anti-VEGF therapy, making this rechallenge feasible. During\nsubsequent systemic treatment, the chronic elevation of the inflammatory factor IL-6 may\nportend disease progression or resistance. However, a surprising finding was that both NLR\nand PLR exhibited a gradual decline during subsequent systemic treatment (Figure R8). We\nhypothesise that this may be attributable to impaired haematopoiesis and liver function, leading\nto a reduction in neutrophil and platelet production in patients undergoing later-line therapy.\nTherefore, NLR and PLR were inadequate as inflammatory indicators for predicting efficacy\nor disease progression in our cohort. The underlying molecular mechanisms remain to be\nelucidated through relevant molecular biology experiments.\nFigure R8. NLR, PLR and the expressions of IL-6 tested from timepoint 1 to timepoint 4. The\nplots and lines represent the mean and upper and lower quartiles, respectively. Paired\nWilcoxon signed rank test was used to identify significant differences. Only significant p\nvalues (p < 0.05) were illustrated.*, p<0.05; **, p<0.01; ***, p<0.001.\n11. Discussion: I would recommend reprhasing the first part as it does not read particularly well\n(242-248)\nResponse: Our sincere thanks. We have revised the first paragraph of the discussion section in\nresponse to your comments, aiming for greater conciseness and accuracy. If you remain\ndissatisfied, please do not hesitate to share your specific concerns.\n12. Tables: I would recommend reporting all the adverse events, not only those deemed related\nto the treatment.\nResponse: Thanks for the advice. As recommended by you and other reviewers, we have\nrestructured Tables for AEs to differentiate: 1) All-grade treatment-emergent adverse events\n(TEAEs) were replenished in the revised Supplementary Table S4; 2) Grade 3 and 4 treatment-\nrelated adverse events (TRAEs), were separated listed in Table 2 (see the Criteria for Adverse\nEvents in Drug Clinical Trials in supplementary Table S7); 3) All TRAEs, as well as TEAEs,\nare rearranged in the tables in order of decreasing incidence rate. This stratification would\ndistinguish event severity and attribution more precisely in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have tried to answer all questions carefully, and I do believe that all questions about the minor limitations have been addressed. But when it comes to the major limitation, I have to say that it cannot be solved retrospectively, it has to be considered prospectively, when the trial was initially designed.\nThe most important novelty of the manuscript lies in the point that, RT remodels the TME to “re-sensitise” the anti-PD-1 treatment, then the results from the paired clinical samples will be absolutely pivotal.\nI must say, this is still a very good work that took loads of hard work, and provide us (including myself) very interesting clinical data and info, but this piece of missing data limits it to reach a higher level.", "reviewer_2": "NCOMMS-25-10699A\nThe authors have successfully addressed all comments raised by the reviewers. The improvements made to the discussion section, as well as the enhanced clarity of the figures and tables, have significantly strengthened the overall quality of the manuscript. After careful evaluation of the revised version, I believe this paper meets the standards for publication in Nature Communications.", "reviewer_3": "My comments have been addressed.", "reviewer_4": "The authors have provided detailed and thoughtful responses to all of my comments, and I appreciate the clarifications and additional data included in the revised manuscript. However, while the study is clinically interesting and the authors acknowledge its limitations, I remain unconvinced about the added benefit of SBRT in this setting, particularly given the comparable response rates reported in retrospective ICI rechallenge studies. I believe this question will ultimately require validation in a randomised controlled trial.\nAlso, I still think that decreased lymphocytes and platelets should not be reported as treatment-related adverse events, because this is not in keeping with known spectrum of toxicity from sintilimab-bevacizumab. The fact that they are reported as the most frequent trAEs raises the question on how the toxicity data have been collected and reported in this study.\nI would also recommend a language revision, as some parts might sound not natural to the readers (e.g. \"Survival analysis also showed that patients [...] possessed poorer PFS\" )"}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have tried to answer all questions carefully, and I do believe that all questions\nabout the minor limitations have been addressed. But when it comes to the major limitation, I\nhave to say that it cannot be solved retrospectively, it has to be considered prospectively,\nwhen the trial was initially designed.\nThe most important novelty of the manuscript lies in the point that, RT remodels the TME to\n“re-sensitise” the anti-PD-1 treatment, then the results from the paired clinical samples will be\nabsolutely pivotal.\nI must say, this is still a very good work that took loads of hard work, and provide us (including\nmyself) very interesting clinical data and info, but this piece of missing data limits it to reach a\nhigher level.\nResponse:\nWe are deeply grateful for your thoughtful evaluation of our work and for recognizing both the\nclinical significance and inherent limitations of our study. We sincerely appreciate your\ninsightful synthesis regarding the clinical ontology of radiotherapy in oligoprogressive HCC—\nparticularly your articulation that \"instead of turning immediately to later-line systemic\ntreatment, loco-regional therapy combined with current regimens may extend therapeutic\ncourses\". This perspective profoundly elevates the translational relevance of our findings, and\nwe have integrated this concept into the revised Discussion.\nRegarding the critical limitation of lacking paired tumour samples:\n1. We fully concur that prospective TME biopsy integration is essential to validate radiation-\ninduced immune remodelling. As you rightly emphasized, \"this piece of missing data limits the\nmechanistic depth\".\n2. Immediate actions taken:\n1) Added pre-treatment IHC data from 11 archival specimens (Supplementary Figure S4,\nTable S6), revealing no correlation between baseline PD-L1/CD8+ T-cell density and clinical\nresponse (*p* > 0.05). This suggests that initial TME status may not predict salvage therapy\nefficacy in ICI-refractory HCC.\n2) Explicitly acknowledged this limitation in the Discussion: \" Critically, the absence of serial\ntumour biopsies precludes direct assessment of therapy-induced TME remodelling,\ndiminishing translational insights.\"\n3. Prospective validation:\n1) Our ongoing phase II trial (ChiCTR2300068781) in potentially resectable HCC mandates:\npre-/post-treatment biopsies with single-cell RNA-seq and spatial transcriptomics; Protocol\nspecifically designed to interrogate SBRT-induced changes in T-cell clonality, macrophage\npolarization, and cytokine networks.\nWhile we cannot retroactively address this limitation, your critique has fundamentally\nshaped our future research trajectory. We hold your rigorous scientific standards in the highest\nregard and hope that our continued work will advance this field toward precision radio-\nimmunotherapy.\nReviewer #2 (Remarks to the Author):\nNCOMMS-25-10699A\nThe authors have successfully addressed all comments raised by the reviewers. The\nimprovements made to the discussion section, as well as the enhanced clarity of the figures and\ntables, have significantly strengthened the overall quality of the manuscript. After careful\nevaluation of the revised version, I believe this paper meets the standards for publication in\nNature Communications.\nReviewer #3 (Remarks to the Author):\nMy comments have been addressed.\nResponse:\nWe thank Reviewers #2 and #3 for their endorsement. All prior requests (e.g., statistical clarity,\nfigure optimization, biomarker discussion) were incorporated in the previous revision.\nReviewer #4 (Remarks to the Author):\nThe authors have provided detailed and thoughtful responses to all of my comments, and I\nappreciate the clarifications and additional data included in the revised manuscript. However,\nwhile the study is clinically interesting and the authors acknowledge its limitations, I remain\nunconvinced about the added benefit of SBRT in this setting, particularly given the comparable\nresponse rates reported in retrospective ICI rechallenge studies. I believe this question will\nultimately require validation in a randomised controlled trial.\nResponse:\nWe acknowledge that randomized controlled trials (RCTs) are the gold standard to\ndefinitively establish SBRT’s contribution. Scheiner et al.'s findings on ICI rechallenge\nrepresent breakthrough research in this field, excitingly demonstrating that ICI rechallenge\ncan still achieve promising therapeutic outcomes even after resistance to immunotherapy in\nHCC. Nevertheless, our data suggest that SBRT may facilitate the resensitization of immune\ncheckpoint inhibitors (ICIs) in this refractory population, a conclusion supported by two lines\nof evidence:\n1. Our primary endpoint evaluation was based on target lesions that had NOT been\ntreated with SBRT, which could reflect the systemic effects induced by SBRT. Biological\nplausibility: 1) SBRT-induced cytokine surges (IFN-γ, IL-6, IL-2) were correlated with\nimproved PFS and response (Figure 4D–G), suggesting radiation primes antitumor immunity;\n2) 100% local control of irradiated lesions highlights SBRT’s ability to eliminate local\nimmunosuppressive niches, potentially enabling systemic efficacy.\n2. Although no head-to-head comparison was performed, our ORR, DCR, and survival\nwere numerically superior to those of the two ICI-resistant cohorts. We created Table R1 to\nmore directly compare the designs and efficacy results of different studies:\nTable R1: Comparison of the data of three ICI-rechallenge cohorts.\nRegorafenib +\nThis Study Scheiner et al.\nEndpoint Pembrolizumab\n(Prospective) (Retrospective)1\n(Prospective)2\nSample Size 21 58 95\nORR (non-irradiated) 33.3% (7/21) 26% (15/58) 5.9–11.1%\nDCR 66.7% (14/21) 55% (32/58) 40.7–51.9%\nMedian PFS 6.2 months 5.2 months (TTP) 4.0–4.2 months\nMedian OS 24.4 months 12.0 months Not reported\nIndeed, neither our data nor that of the above two studies currently provides a definitive\nanswer, the benefits of adding SBRT in this setting still need to be further confirmed through\nRCTs. Nevertheless, we can gain some insights from a series of studies on SBRT combined\nwith immunotherapy in non-small cell lung cancer (NSCLC). Paul Baas & Valsamo\nAnagnostou's team has reported that combining radiotherapy with immunotherapy in patients\nwith NSCLC significantly increases the ORR from 20% to 50%, compared to immunotherapy\nalone. It has a significant systemic immune regulatory effect, can induce a sustained immune\nresponse, thereby improving the treatment response rate and long-term survival rate of patients\n(JAMA Oncology, 2019, doi: 10.1001/jamaoncol.2019.1478; Nature Cancer, 2025, doi:\n10.1038/s43018-025-01018-w). We are currently designing a randomized controlled clinical\ntrial to investigate the optimal timing and benefits of SBRT in patients with HCC receiving ICI\ntherapies. Hopefully, these trials would provide us with more evidence in this field.\nWe agree with your concern about overemphasizing the contribution of SBRT in this\ntreatment regimen. Therefore, in the revised manuscript, the emphatic language in the\ndiscussion and conclusion sections has been replaced with more neutral, inferential descriptions\n(detailed in line 291, 299 and 318 of the unmarked manuscript). Furthermore, the description\nof the efficacy of SBRT in the text has been modified to refer to the combination of SBRT and\nimmunotherapy, as detailed in line 295, 297, and 299 of the unmarked manuscript. These\nmodifications will provide a more objective description of the benefits to patients in this study,\nwhich are provided by this triple therapy regimen. We look forward to your understanding and\nsupport for these revisions.\nAlso, I still think that decreased lymphocytes and platelets should not be reported as\ntreatment-related adverse events, because this is not in keeping with known spectrum of toxicity\nfrom sintilimab-bevacizumab. The fact that they are reported as the most frequent trAEs raises\nthe question on how the toxicity data have been collected and reported in this study.\nResponse:\nWe appreciate the reviewer’s scrutiny regarding the attribution of decreased lymphocyte\nand platelet counts as TRAEs, but we respectfully disagree with the suggestion that these events\nfall outside the expected spectrum for this combination regimen.\nTo further address your concerns, a timeline-based curve diagram of lymphocyte counts\nand platelet counts was plotted for each patient (Figure R1-B, and R2-B). Interestingly, we\nfound that lymphopenia is likely closely associated with SBRT, as we did observe transient\nlymphocyte decreases or upgraded AE ratings in most patients following SBRT.\nFigure R1-B. The counts of lymphocytes reported by blood routine test of all 8 patients who\nexperienced lymphocytopenia. Triangles: Self-reported cold (possible viral infection); LLN,\nlower limit of normal. The Blue squares represented the timing of the first report of TRAE.\nFigure R2-B. The counts of platelets reported by blood routine test and the symptomatic\ntreatment of all 8 patients who experienced thrombocytopenia. LLN, lower limit of normal.\nThe Blue squares represented the timing of the first report of TRAE.\nIn addition to the temporal association between treatment and adverse events (AEs), there\nis mechanistic evidence supporting the classification of these phenomena as TRAEs.\nSpecifically, SBRT is a well-documented cause of lymphopenia, as demonstrated in the\nliterature on combined modality therapy (SBRT is a well-established cause of lymphopenia,\nsupported by literature in combined modality therapy (e.g., Li, et al., 2024, Nature\nCommunications3; Paganetti, 2023, Frontiers in Oncology4; Chen et al., 2023, World J\nGastroenterol5). The significant post-SBRT drop in lymphocytes observed in our cohort\n(Figure R1-B and Figure R2-B) strongly supports this mechanism. Furthermore, the regimen\ncombining bevacizumab and sintilimab is known to impact platelet levels, with an incidence\nrate of approximately 26% reported in the ORIENT-326, which is comparable to the 38%\nobserved in our study. These findings suggest that the novel triplet combination therapy may\nexert synergistic or additive hematologic effects, contributing to the observed AEs.\nAlso, we would like to stress that reporting these events as TRAEs strictly followed the\nstandardized guidelines of NMPA and accurately reflects the safety profile of SBRT combined\nwith sintilimab and bevacizumab biosimilar in the population of anti-PD-1 resistant HCC,\nrather than just TRAEs specific to systemic immunotherapy. We have detailed the causality\nassessment process in the Methods section and provided Supplementary Table S7 for full\ntransparency.\nI would also recommend a language revision, as some parts might sound not natural to the\nreaders (e.g. \"Survival analysis also showed that patients [...] possessed poorer PFS\" )\nResponse:\nMany thanks for your criticism regarding the lack of linguistic accuracy in our paper. We\nhave thoroughly revised the manuscript for language and clarity. The awkward phrasing\nhighlighted by you have been corrected as “Survival analysis also showed that patients with a\nhigher level of IFN-γ had poorer PFS and estimated OS.” in the revised version. Furthermore,\nthe entire manuscript has undergone professional language editing by native speaker editing\nfrom Springer Nature, with the editing certificate number being: D75B-B57D-DC90-BF31-\nBE44. The verification was uploaded along with the revised manuscript.\nOnce again, thank you for your valuable feedback. We sincerely hope that these revisions\nwill address all of your concerns regarding this study.\nReference:\nwith hepatocellular carcinoma. JHEP Rep 5, 100620, doi:10.1016/j.jhepr.2022.100620 (2023).\npembrolizumab in patients with advanced hepatocellular carcinoma (HCC) previously treated\nwith immune checkpoint inhibitors (ICI). Journal of Clinical Oncology 42, 4007-4007,\ndoi:10.1200/JCO.2024.42.16_suppl.4007 (2024).\ntislelizumab in early-stage resectable hepatocellular carcinoma: the Notable-HCC phase 1b trial.\nNat Commun 15, 3260, doi:10.1038/s41467-024-47420-3 (2024).\nOncol 13, 1201500, doi:10.3389/fonc.2023.1201500 (2023).\nrecurrent or oligometastatic hepatocellular carcinoma: A phase II clinical trial. World J\nGastroenterol 29, 3871-3882, doi:10.3748/wjg.v29.i24.3871 (2023).\nunresectable hepatocellular carcinoma (ORIENT-32): a randomised, open-label, phase 2-3 study.\nLancet Oncol 22, 977-990, doi:10.1016/S1470-2045(21)00252-7 (2021)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I appreciate the works from the authors, as well as their contributions to the treatment of HCC. Unfortunately, from the viewpoint of a reviewer, I have to insist on my previous evaluation: the major limitation of this manuscript cannot be solved retrospectively, it has to be considered prospectively, when the trial was initially designed. Upon this design, the results from the study were just not strong enough to support the novelty that expected in the conclusions.", "reviewer_4": "I do not have any further comment to add."}, "author_response": ""}]

Decision letter