Phase Ⅱ Study of Combined Sintilimab and Anlotinib with Gemcitabine plus Cisplatin in Advanced Biliary Tract Cancer: Efficacy, Safety and Optimize Dose

Phase Ⅱ Study of Combined Sintilimab and Anlotinib with Gemcitabine plus Cisplatin in Advanced Biliary Tract Cancer: Efficacy, Safety and Optimize Dose | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Phase Ⅱ Study of Combined Sintilimab and Anlotinib with Gemcitabine plus Cisplatin in Advanced Biliary Tract Cancer: Efficacy, Safety and Optimize Dose Jingjing Li, Shurui Zhou, Xiaoqing Xu, Qinhong Zheng, Fabiao Zhang, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4557891/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Background: The prognosis of biliary tract cancer (BTC) is poor, with limited efficacy of first-line chemotherapy. SAGC is a randomized, controlled, phase 2 trial evaluating the efficacy of sintilimab (an anti-PD-1 inhibitor) and anlotinib (an anti-angiogenic VEGF-targeted agent) combined with standard chemotherapy as a first-line treatment in advanced BTC. Methods: Eighty eligible patients with unresectable, locally advanced, or metastatic BTC were randomized 1:1 to SAGC group ( n = 40) to receive sintilimab (200 mg) and anlotinib (initial 10 mg, then adjusted for 8 mg on days 1-14) plus GC (gemcitabine 1,000 mg/m 2 and cisplatin 25 mg/m 2 on days 1 and 8) every 3 weeks for up to 8 cycles, followed by sintilimab and anlotinib until disease progression or unacceptable toxicity or to GC group ( n = 40), respectively. The primary endpoint was progression-free survival (PFS). The secondary endpoints included the objective response rate (ORR), overall survival (OS), and safety. The AKT/YAP-induced tumor-bearing mice model was established to study effect of anlotinib on the tumor immune microenvironment at varying doses (low-dose: 3 mg/kg, high-dose: 6 mg/kg). Results: The median follow-up was 13.4 months, and 77 of the 80 patients (96.3%) discontinued treatment. The median PFS was 8.5 months (SAGC group) and 6.2 months (GC group) (hazard ratio: 0.47 [95% CI, 0.22–0.64], P = 0.003). The ORR for the SAGC and GC groups were 51.4% and 29.4%, respectively. Overall, grade 3/4 treatment-related adverse events occurred in 75.0% (30/40) and 43.6% (17/39) of cases in the SAGC and GC groups, respectively. A post hoc analysis shown that patients in SAGC group who received 8mg (22 patients) of anlotinib daily had a higher ORR (54.5% vs. 38.8%) compared to those received 10mg (18 patients), and there was a trend towards an OS benefit (HR: 0.49 [95% CI, 0.14–1.18], P = 0.055). In vivo, the combination of low-dose anlotinib with anti-PD-1 resulted in heightened vascular pericyte coverage, improved vascular perfusion, enhanced cytotoxicity of activated T cells, and increased secretion of effector cytokines when compared to high-dose anlotinib. Conclusion: Sintilimab and anlotinib in addition to gemcitabine plus cisplatin treatment in patients with advanced BTC significantly improved PFS and had a manageable safety profile, and the survival benefit of anlotinib 8mg group is more superior. Low‐dose anlotinib plus anti–PD-1 immune therapy may synergistically improve the antitumor response with reducing adverse effects in vivo. Trial registration number ClinicalTrials.gov Identifier: NCT04300959. Health sciences/Diseases/Cancer/Cancer therapy/Cancer immunotherapy Health sciences/Diseases/Cancer/Gastrointestinal cancer/Biliary tract cancer/Bile duct cancer Biliary tract cancer Phase 2 trial Sintilimab Anlotinib Progression-free survival Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Biliary tract cancers (BTCs) are the second most common liver cancer type after hepatocellular carcinoma, and account for approximately 3% of all gastrointestinal malignancies worldwide [ 1 ]. They are mainly divided into gallbladder carcinoma (GBC), intrahepatic cholangiocarcinoma (ICC), and extrahepatic cholangiocarcinoma (ECC), based on their anatomical primary site. The incidence of BTC is high and has increased over the years, with 6.8 new cases per 100,000 in China in 2022[ 2 ]. Most patients with BTC are at an advanced stage at the time of diagnosis and are unsuitable for surgical resection; systemic chemotherapy is the only treatment option for most patients. The preferred first-line chemotherapy for advanced BTC is gemcitabine plus cisplatin, which provides limited survival benefits with a median overall survival (OS) of 11.7 months and a median progression-free survival (PFS) of 5.7–8.0 months [ 3 , 4 ]. Target-oriented therapy has progressed and changed the treatment landscape of BTC, with patients harboring FGFR2 fusions or rearrangements benefiting from pemigatinib, infigratinib, and futibatinib, and those with IDH1 -mutations benefiting from ivosidenib [ 5 – 8 ]. Additionally, some patients may be treated based on their molecular profiles of ERBB2 , BRAF , and NTRK [ 9 – 13 ]. However, the number of patients with these targetable characteristics is small (~ 7% with FGFR alterations and ~ 13% with IDH1 mutations in intrahepatic cholangiocarcinoma cases) [ 14 ]; thus, the overall benefits of target-oriented treatment remain limited for most patients with advanced BTC. The efficacy of immune checkpoint inhibitors (ICIs) in BTC is demonstrated in several studies, either as a single agent or in combination with chemotherapy, including pembrolizumab, nivolumab, and durvalumab, in patients who failed first-line chemotherapy [ 15 – 17 ] and chemotherapy-naïve patients [ 4 , 18 – 21 ]. However, the improvement in PFS and OS with ICIs plus chemotherapy as the first-line treatment remains moderate. Although the TOPAZ-1 trial met its primary endpoint OS in first-line advanced BTC, the incremental median OS and PFS with durvalumab plus gemcitabine and cisplatin versus chemotherapy alone were below 2 months (median OS: 12.8 vs. 11.6 months, median PFS: 7.2 vs. 5.7 months) [ 4 ]. A phase 3 Keynote 966 study also indicated that pembrolizumab plus gemcitabine and cisplatin could improve OS compared to chemotherapy as a first-line therapy for advanced BTC [ 21 ]. Vascular targeting is believed to enhance the antitumor efficacy of ICIs by modulating the tumor microenvironment (TME) and creating positive feedback loops that reinforce each other [ 22 ]. A retrospective real-world study indicated that treatment with an anti-angiogenic agent (lenvatinib) in combination with an ICI showed an active trend towards improved survival in patients with advanced BTCs after line treatment with manageable adverse events [ 23 ]. Another anti-angiogenic agent (anlotinib) in combination with ICI showed promising efficacy and a well-tolerated safety profile in advanced BTCs that had failed first-line chemotherapy [ 24 ]. The clinical benefit of chemotherapy in combination with an antiangiogenic agent and ICI was further observed in the first-line treatment of BTC in a prospective single-arm study in 2023 [ 25 ]. However, there is a lack of validated evidence from randomized controlled clinical studies. This multicenter, randomized, controlled, and phase 2 SAGC trial was conducted to evaluate the clinical activity and safety of the addition of sintilimab (an anti-PD-1 inhibitor) and anlotinib (an anti-angiogenic VEGF -targeted inhibitor) to chemotherapy as a first-line treatment in patients with advanced BTC. Furthermore, we conducted an animal experiment to investigate the potential optimization of dosage for the drug anlotinib. Methods Study design and participants The SAGC trial was a randomized, controlled, open label phase 2 study conducted at three centers in China, including Zhejiang Cancer Hospital, Quzhou People's Hospital and Taizhou Hospital. The main inclusion criteria were patients aged 18 years or older, with a baseline Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1, with biopsy/pathology-confirmed unresectable, locally advanced, or metastatic BTC (including GC, ICC, and ECC), who had not received previous systemic therapy for advanced or metastatic disease, had measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and had adequate hematological and organ function with a life expectancy exceeding 12 weeks. Patients who received neoadjuvant or adjuvant chemotherapy 6 months or more prior to randomization were eligible. Key exclusion criteria included active bleeding or clinically significant gastrointestinal (GI) abnormalities that might increase the risk for GI bleeding, immune-related conditions such as autoimmune disease and major inflammatory diseases, clinical evidence of metastatic disease to the brain and clinically significant cardiovascular disease, acute or chronic active hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, HBV DNA greater than 2,000 international units per milliliter (IU/mL) or 10⁴ copies per mL, and HCV RNA greater than 10³ copies per mL. Detailed inclusion and exclusion criteria are seen in the online supplemental material. The trial was registered at Clinicaltrials. gov (identifier: NCT04300959) and was conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. The protocol and amendments were approved by the Institutional Review Board and Ethics Committee of each site. All patients provided written informed consent prior to enrolment. Randomization and masking Eligible patients were randomly assigned (1:1) to the sintilimab plus anlotinib plus gemcitabine and cisplatin (SAGC) or gemcitabine and cisplatin (GC) group using a simple randomization method. No masking was performed because this was an open-label study. Procedures Patients in the SAGC group received sintilimab (200 mg intravenously administered on day 1 every 3 weeks) and anlotinib (10 mg orally once daily from day 1 to day 14, every 3 weeks) along with chemotherapy (25 mg/m 2 cisplatin followed by 1,000 mg/m 2 gemcitabine, each administered on days 1 and 8 every 3 weeks) for up to 8 cycles, followed by maintenance sintilimab plus anlotinib. Patients in the GC group received chemotherapy alone (cisplatin 25 mg/m 2 followed by gemcitabine 1,000 mg/m 2 , each administered on days 1 and 8 every 3 weeks) for up to 8 cycles. After 18 patients were enrolled in the SAGC group, the starting dose of anlotinib was modified from 10 to 8 mg daily owing to the observed bone marrow suppression events. Treatment was continued until disease progression, intolerable toxicity, withdrawal of consent, study completion, or study termination occurred, whichever occurred first. Dose adjustments were permitted for chemotherapy and anlotinib but not for sintilimab, according to the protocol. Considering that gemcitabine, cisplatin, and anlotinib are marketed drugs, dose adjustments were made by the investigators according to the treatment-related adverse events determined and medication instructions (details in Supplementary Protocol ). Patients who temporarily or permanently discontinued chemotherapy or sintilimab and anlotinib owing to adverse events were allowed to continue taking other agents as long as the investigator determined that there was a clinical benefit. Assessments Tumors were assessed by investigators using computed tomography or magnetic resonance imaging (MRI) at baseline and every 6 weeks as per RECIST 1.1. Safety was assessed for the patients who received at least one dose of the assigned treatment. Vital signs, clinical laboratory tests, and other adverse events were continuously monitored throughout the study and assessed according to the National Cancer Institute’s (NCI) Common Terminology Criteria for Adverse Events, version 4.0. Endpoints The primary endpoint was PFS, defined as the time from randomization to disease progression according to RECIST version 1.1, or death from any cause, whichever occurred first. The secondary endpoints were objective response rate (ORR), defined as the percentage of patients with radiologically confirmed complete or partial response according to RECIST version 1.1; OS, defined as the time from randomization to death from any cause; and safety, described as the occurrence of treatment-related adverse events (TRAEs) of any grade. Animal models Female C57BL/6 mice aged 6–8 weeks were procured from the Shanghai Model Organisms Center. The orthotopic cholangiocarcinoma (CCA) tumor-bearing models were established through hydrodynamic tail vein injection (HD inj) of 30 µg YapS127A, 20 µg AKT, and 2 µg SB100 transposase plasmids diluted in 2 ml of 0.9% NaCl. Anlotinib (kindly provided by Chia Tai Tianqing Pharmaceutical Group Co. Ltd) treatment at varying doses[ 26 – 28 ](low-dose: 3 mg/kg, high-dose: 6 mg/kg) was initiated 7 days post tumor cell inoculation and administered orally via gavage daily. Anti-mouse PD-1 antibody (CD279, BE0146, BioXcell) was administered at a dose of 200 mg per mouse through intraperitoneal injection every 3 days. The control group received intragastric administration of 0.9% saline and intraperitoneal injection of IgG, while tumor-bearing mice were euthanized at the conclusion of the experiment. Additional independent experiments were conducted to compare the survival times of mice in each group. Approval for all animal experiments was obtained from the Animal Experimental Ethics Committee of Zhejiang Cancer Hospital, and the experiments were conducted in accordance with guidelines for the care and use of laboratory animals. Statistical analysis The sample size was determined based on the primary endpoint of PFS under the assumption that the GC group had a median PFS of 6.5 months, which was based on the ABC-02 study [ 3 ]. This estimate also accounted for the potential impact on the median PFS estimate caused by a longer tumor assessment interval (12 weeks) used in ABC-02, and that SAGC would improve PFS with a hazard ratio (HR) of 50%; approximately 51 PFS events would be needed to provide 80% power to detect this difference under a type I error controlled at 10% (two-sided). Eighty subjects were randomized in a 1:1 ratio to achieve 51 PFS events to complete the study in a reasonable time, and considering the possible loss to follow-up. Patients who were lost to follow-up were censored at the randomization date for PFS analysis and at the last known survival date for OS analysis. The efficacy analysis set included patients randomly assigned to each treatment group, regardless of whether they had received treatment. The safety analysis set included patients who received at least one dose of the study treatment. Quantitative data were displayed as the median (range) or number of patients (percentage). Proportional comparisons between groups were performed using Fisher's exact test. Survival analyses (PFS and OS) were performed using Kaplan-Meier curves, P values were determined using the log-rank test, and HRs were calculated using the Cox proportional hazards model. A two-sided P value lower than 0.05 was considered statistically significant for all tests except for the primary endpoint, unless indicated otherwise. A two-sided P value ≤ 0.1 was used to determine statistical significance for the primary endpoint PFS. Univariate analysis was used to explore the associations between different variables and PFS, and the results are presented as HRs with 95% confidence intervals (CIs). All analyses were performed using R 3.4.0 [Vienna, Austria]. Results Patients and Treatment Eighty patients were enrolled between March 26, 2020, and May 25, 2022, with 40 patients in each group (Fig. 1 ). Overall, most patients were males (61.3%) and had ECOG PS 1 (97.5%), ICC (62.5%), and metastatic disease (100%); 17.5% patients were HBV positive. Baseline characteristics were generally well balanced between the treatment groups (Table 1 ). Eighteen patients in the SAGC group received a starting dose of 10 mg anlotinib daily under the initial protocol, while 22 patients received 8 mg daily under the amended protocol. Table 1 Clinical characteristics of patients at baseline Patient chracteristics a SAGC group ( n = 40) GC group ( n = 40) P Age (years), median 62.5 (54.8, 68.3) 59.5 (50.0, 66.3) 0.244 CA199 (U/ml), median 67.53 (21.8, 961.1) 172.84 (20.1, 1428.0) 0.796 Sex 1.000 Female 16 (40.0%) 15 (37.5%) Male 24 (60.0%) 25 (62.5%) ECOG b performance status score 0.494 0 2 (5.0%) 0 (0%) 1 38 (95.0%) 40(100.0%) Primary tumor type 0.643 Extrahepatic cholangiocarcinoma 6 (15.0%) 5 (12.5%) Gallbladder carcinoma. 11 (27.5%) 8 (20.0%) Intrahepatic cholangiocarcinoma 23 (57.5%) 27 (67.5%) Extent of disease 0.453 Initially unresectable 31 (77.5%) 27 (67.5%) Recurrent 9 (22.5%) 13 (32.5%) Metastases Liver 26 (65.0%) 26 (65.0%) 1.000 Lung 9 (22.5%) 10 (25.0%) 1.000 Bone 3 (7.5%) 8 (20.0%) 0.194 Peritoneum 9 (22.5%) 8 (20.0%) 1.000 Distant lymph node 27 (67.5%) 22 (55.0%) 0.359 Hepatitis B Virus 0.769 No 34 (85.0%) 32 (80.0%) Yes 6 (15.0%) 8 (20.0%) Previous history of surgery 1.000 No 26 (65.0%) 25 (62.5%) Yes 14 (35.0%) 15 (37.5%) Previous adjuvant therapy 1.000 No 37 (92.5%) 36 (90.0%) Yes 3 (7.5%) 4 (10.0%) a Data are n (%) or median (IQR). b ECOG, Eastern Cooperative Oncology Group. Seventy-seven patients discontinued the study treatment at the data cutoff (December 30, 2023), mainly owing to disease progression (33/40 in the SAGC group vs. 32/40 in the GC group); 2 patients continued treatment (1 in the SAGC group and 1 in the GC group) (Fig. 1 ). Patients in the SAGC group received sintilimab plus anlotinib for a median of seven cycles (range: 1–22) combined with chemotherapy for a median of six cycles (range: 1–8) with a dose intensity of 784.2 mg/m 2 for gemcitabine and 18.9 mg/m 2 for cisplatin. The median chemotherapy exposure in the GC group was six cycles (range: 1–8) with a dose intensity of 806.1 mg/m 2 for gemcitabine and 20 mg/m 2 for cisplatin ( Supplementary Table S1 ). Overall, 14 patients in the SAGC group and 23 in the GC group received at least one anti-tumor therapy post study treatment discontinuation, including chemotherapy (11/40 [27.5%] vs. 13/40 [32.5%]), ICI (6/40 [15.0%] vs. 15/40 [37.5%]), and tyrosine kinase inhibitors (0/40 [0%] vs. 8/40 [20.0%]) ( Supplementary Table S2 ). Efficacy The median follow-up at the data cutoff was 13.4 months. Thirty-five (87.5%) of 40 patients in the SAGC group and 35 (87.5%) of 40 patients in the GC group had progressive disease or died. The median PFS was 8.5 months (95% CI, 5.6–11.0) in the SAGC group versus 6.2 months (95% CI, 4.4–7.8) in the GC group (HR: 0.47 [95% CI, 0.22–0.64], P = 0.003; Fig. 2 A). The 12-month PFS rates were 26.4% and 0% in the SAGC and GC groups, respectively. Consistent improvements in PFS were observed across the most clinically relevant subgroups (Fig. 3 ). The median PFS was 9.1 months with SAGC for patients with ICC versus 6.2 months with GC (HR: 0.34, 95% CI, 0.15–0.75; Supplementary Figure S1 ). Twenty-nine (72.5%) patients died in the SAGC group and 23 (57.5%) in the GC group; the median OS was 13.2 months (95% CI, 8.7–19.0) versus 13.7 months (95% CI, 10.2–15.3) (HR: 1.04 [95% CI, 0.40–1.49], P = 0.895 Fig. 2 B). Thirty-seven patients in the SAGC group and 34 in the GC group received at least one post-baseline imaging assessment. Nineteen (51.4%) of the 37 patients randomized to SAGC group achieved a partial response (PR), and 16/37 (43.2%) patients had stable disease (SD), contributing to an ORR of 51.4% and a DCR of 94.6%. In addition, 10/34 (29.4%) patients achieved PR, and 18/34 (52.9%) patients achieved SD in the GC group according to their randomized assignments, with an ORR of 29.4% and a DCR of 82.3% (Table 2 , Supplementary Figure S2 ). The ORR was significantly higher in the SAGC group than in the GC group ( P = 0.033). The median time to response was 2.5 (1.7–3.6) months in the SAGC group and 3.0 (2.7–4.0) months in the GC group. A longer median duration of response with SAGC versus GC (9.1 [4.9, NA] versus 3.4 [2.0, NA] months) was observed (Table 2 ). Table 2 Summary of best overall response in SAGC and GC groups Efficacy a SAGC group ( n = 37) GC group ( n = 34) P Best overall response c 0.033 Partial response 19 (51.4%) 10 (29.4%) Stable disease 16 (43.2%) 18 (52.9%) Progressive disease 2 (5.4%) 6 (17.6%) Objective response rate 51.4% 29.4% Disease control rate 94.6% 82.3% Median time to onset of response, months 2.5 (1.7, 3.6) 3.0 (2.7, 4.0) Median duration of response, months 9.1 (4.9, NA) 3.4 (2, NA) a Data are shown as n (%) and median (IQR). b Patients without any post-baseline imaging assessment. c Per RECIST version 1.1. A post hoc sensitivity analysis was performed to compare the efficacy between patients who received a starting dose of anlotinib of 10 mg daily and those who received an anlotinib dose of 8 mg daily in the SAGC group. The median PFS did not significantly differ between the 8mg and 10mg groups (8.5 vs. 7.6 months, HR: 0.87 [95% CI, 0.30–1.55], P = 0.691) with a trend longer ( Fig. 4 A ) . A trend towards longer median OS was observed in the 8mg group compared to the 10mg group (14.9 vs. 9.3 months, HR: 0.49 [95% CI, 0.14–1.18], P = 0.055) ( Fig. 4 B ) , with this difference remaining consistent during long-term follow-up. The ORRs were 38.8% at 10 mg daily and 54.5% at 8 mg daily. ( Supplementary Table S3 ). Safety Seventy-nine of the 80 patients received at least one dose of the assigned treatment and were included in the safety analyses. All patients experienced at least one treatment-related adverse event (TRAE);75% had at least one grade 3/4 TRAE in the SAGC group and 43.6% in the GC group. No adverse events in grade 5 were observed in either group. Adverse events led to dose reductions in 28 (70%) and 19 (48.7%) patients in the SAGC and GC groups, respectively. Furthermore, AEs led to treatment discontinuation in 1 (2.5%) and 0 patients in the SAGC and GC groups, respectively (Data not shown). The most common treatment-related AEs are shown in Table 3 . The most frequent grade 3–4 treatment-emergent AEs (occurring in ≥ 5% of patients) were leukopenia (9 [22.5%] of 40 patients in SAGC group vs. 11 [28.3%] of 39 patients in GC group), neutropenia (18 [45%] vs. 9 [23.1%]), thrombocytopenia (11 [27.5%] vs. 4 [10.3%]), anemia (6 [15%] vs. 6 [15.4%]), increased γ-glutamyltransferase (5 [12.5%] vs. 1 [2.6%]). Serious hematological toxicities (including neutropenia and thrombocytopenia) were more common in the SAGC group than in the GC group. The incidence of grade 4 AE in the SAGC group decreased from 23.5–13.0% after the starting dose of anlotinib was reduced to 8 mg daily (Supplementary Table S4) . Potentially immune-mediated AEs occurred in 9 (22.5%) of the 40 participants in the SAGC group, of all were grade 1–2 (Table 3 ). The GC group was not evaluated for immune-related AEs since this was an open-label trial. Common immune-mediated adverse events included abnormal thyroid function in 4 patients (10%) and rashes in4 patients 10%). The only potentially immune-mediated AE that led to death was grade 2 pneumonitis, which occurred in one participant in the SAGC group and was treated with corticosteroids. Table 3 Summary of treatment-related adverse events Safety a SAGC ( n = 40) GC ( n = 39) b* Any grade >= Grade 3 Grade 1 Grade 2 Grade 3 Grade 4 Any grade >= Grade 3 Grade 1 Grade 2 Grade 3 Grade 4 Any treatment-emergent adverse event 40 (100%) 30 (75%) 4 (10%) 6 (15%) 23 (57.5%) 7 (17.5%) 39 (100%) 17 (43.6%) 6 (15.4%) 16 (41%) 14 (35.9%) 3 (7.7%) Leukopenia 35 (87.5%) 9 (22.5%) 7 (17.5%) 19 (47.5%) 9 (22.5%) 0 (0%) 24 (61.5%) 11 (28.2%) 6 (15.4%) 7 (17.9%) 11 (28.2%) 0 (0%) Anaemia 34 (85%) 6 (15%) 12 (30%) 16 (40%) 6 (15%) 0 (0%) 33 (84.6%) 6 (15.4%) 14 (35.9%) 13 (33.3%) 6 (15.4%) 0 (0%) Neutropenia 33 (82.5%) 18 (45%) 5 (12.5%) 10 (25%) 16 (40%) 2 (5%) 24 (61.5%) 9 (23.1%) 4 (10.3%) 11 (28.2%) 6 (15.4%) 3 (7.7%) Thrombocytopenia 30 (75%) 11(27.5%) 11 (27.5%) 8 (20%) 6 (15%) 5 (10%) 25 (64.1%) 4 (10.3%) 13 (33.3%) 8 (20.5%) 4 (10.3%) 0 (0%) Increased γ-glutamyltransferase 29 (72.5%) 5 (12.5%) 18 (45%) 6 (15%) 5 (12.5%) 0 (0%) 27 (69.2%) 1 (2.6%) 22 (56.4%) 4 (10.3%) 0 (0%) 1 (2.6%) Hypoalbuminemia 27 (67.5%) 0 (0%) 22 (55%) 5 (12.5%) 0 (0%) 0 (0%) 17 (43.6%) 0 (0%) 17 (43.6%) 0 (0%) 0 (0%) 0 (0%) Hyponatremia 18 (45%) 0 (0%) 16 (40·0%) 2 (5·0%) 0 (0%) 0 (0%) 21 (53·8%) 0 (0%) 21 (53·8%) 0 (0%) 0 (0%) 0 (0%) Increased aspartate aminotransferase 17 (42.5%) 1 (2.5%) 14 (35%) 2 (5%) 1 (2.5%) 0 (0%) 12 (30.8%) 1 (2.6%) 10 (25.6%) 1 (2.6%) 1 (2.6%) 0 (0%) Fatigue 17 (42.5%) 0 (0%) 10 (25%) 7 (17.5%) 0 (0%) 0 (0%) 11 (28.2%) 0 (0%) 7 (17.9%) 4 (10.3%) 0 (0%) 0 (0%) Increased alanine aminotransferase 13 (32.5%) 2 (5%) 9 (22.5%) 2 (5%) 1 (2.5%) 1 (2.5%) 12 (30.8%) 2 (5.1%) 9 (23.1%) 1 (2.6%) 2 (5.1%) 0 (0%) Proteinuria 13 (32.5%) 0 (0%) 8 (20%) 5 (12.5%) 0 (0%) 0 (0%) 8 (20.5%) 0 (0%) 4 (10.3%) 4 (10.3%) 0 (0%) 0 (0%) Increase creatinine 10 (25%) 0 (0%) 8 (20%) 2 (5%) 0 (0%) 0 (0%) 8 (20.5%) 0 (0%) 7 (17.9%) 1 (2.6%) 0 (0%) 0 (0%) Hypokalemia 9 (22.5%) 2 (5·0%) 6 (15·0%) 1 (2·5%) 2 (5·0%) 0 (0%) 8 (20.5%) 0 7 (17·9%) 1 (2·6%) 0 (0%) 0 (0%) Nausea 9(22.5%) 0 (0%) 8 (20%) 1 (2.5%) 0 (0%) 0 (0%) 3 (7.7%) 1 (2.6%) 2 (5.1%) 0 (0%) 1 (2.6%) 0 (0%) Vomiting 9(22.5%) 0 (0%) 6(15%) 3 (7.5%) 0 (0%) 0 (0%) 10 (25.6%) 1 (2.6%) 9 (23.1%) 0 (0%) 1 (2.6%) 0 (0%) Hyperuricemia 8 (20%) 0 (0%) 8 (20%) 0 (0%) 0 (0%) 0 (0%) 5 (12.8%) 0 (0%) 5 (12.8%) 0 (0%) 0 (0%) 0 (0%) Hypertension 7 (17.5%) 2 (5%) 4 (10%) 1 (2.5%) 2 (5%) 0 (0%) 5 (12.8%) 0 (0%) 3 (7.7%) 2 (5.1%) 0 (0%) 0 (0%) Constipation 7 (17.5%) 0 5 (12.5%) 2 (5.0%) 0 (0%) 0 (0%) 8 (20.5%) 0 (0%) 6 (15.4%) 2 (5.1%) 0 (0%) 0 (0%) Fever 7 (17.5%) 0 (0%) 6 (15%) 1 (2.5%) 0 (0%) 0 (0%) 1 (2.6%) 0 (0%) 1 (2.6%) 0 (0%) 0 (0%) 0 (0%) Anorexia 7 (17.5%) 0 (0%) 6 (15%) 1 (2.5%) 0 (0%) 0 (0%) 5 (12.8%) 0 (0%) 3 (7.7%) 2 (5.1%) 0 (0%) 0 (0%) Increased conjugated bilirubin 6 (15%) 3 (7·5%) 2 (5·0%) 1 (2·5%) 3 (7·5%) 0 (0%) 5 (12.8%) 1 (2·6%) 2 (5·1%) 2 (5·1%) 1 (2·6%) 0 (0%) Increase serum amylase 4 (10%) 0 (0%) 3 (7.5%) 1 (2.5%) 0 (0%) 0 (0%) 4 (10.3%) 0 (0%) 4 (10.3%) 0 (0%) 0 (0%) 0 (0%) Rash 4 (10%) 0 (0%) 3 (7.5%) 1 (2.5%) 0 (0%) 0 (0%) 4 (10.3%) 0 (0%) 2 (5.1%) 2 (5.1%) 0 (0%) 0 (0%) Diarrhea 3 (7.5%) 1 (2.5%) 0 (0%) 2 (5%) 1 (2.5%) 0 (0%) 1 (2.6%) 0 (0%) 0 (0%) 1 (2.6%) 0 (0%) 0 (0%) Abdominal pain 2 (5%) 0 (0%) 2 (5%) 0 (0%) 0 (0%) 0 (0%) 5 (12.8%) 0 (0%) 4 (10.3%) 1 (2.6%) 0 (0%) 0 (0%) Hand-foot syndrome 2 (5%) 0 (0%) 2 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Hyperthyroidism 2 (5%) 0 (0%) 2 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Hypothyroidism 2 (5%) 0 (0%) 0 (0%) 2 (5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Interstitial pneumonia 1 (2.5%) 1 (2.5%) 0 (0%) 1 (2.5%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) a Data are shown as n (%). b One patient withdrew consent after randomization. In vivo validation Previous observations suggest that dose reduction of anlotinib may lead to improved outcomes and decreased adverse events. To explore the potential mechanisms of these dose-dependent responses, an orthotopic AKT/YAP-induced CCA tumor-bearing model was established ( Supplementary Figure S3 A ). Consistent with expectations, the combination of anlotinib and anti-PD-1 treatment demonstrated greater efficacy in inhibiting tumor growth compared to monotherapy or control groups. Our study revealed that there were no statistically significant differences in efficacy between high- and low-dose anlotinib treatment ( Supplementary Figure S4 ). However, the combination of low-dose anlotinib with anti-PD-1 demonstrated greater efficacy in inhibiting tumor growth compared to high-dose anlotinib combination therapy (Fig. 5 A-D). Additionally, survival outcomes were similar across treatment groups, with the most favorable prognosis observed in the low-dose anlotinib plus anti-PD-1 subgroup (Fig. 5 E). Furthermore, there were no significant differences in body weight among the treatment groups, suggesting that high-dose anlotinib monotherapy and combination therapy did not result in noticeable toxicity ( Supplementary Figure S3 B ). Moreover, the mice in the high-dose group exhibited a notable reduction in small intestinal villus and significant irregular morphologies ( Supplementary Figure S3 C ). These results collectively suggest that low-dose anlotinib treatments can improve the efficacy of anti-PD-1 therapy while minimizing adverse effects in the orthotopic CCA tumor-bearing model. Existing research suggests that antiangiogenic agents have the potential to restructure the tumor immunosuppressive microenvironment through the normalization of vascular structure and mitigation of hypoxia in a dose-dependent fashion. Therefore, our initial assessment focused on the alterations in tumor blood vessels. The findings indicated that high-dose anlotinib led to a statistically significant reduction in intratumoral microvascular density (MVD, defined as CD31 + vessel numbers per field) ( Supplementary Figure S5A-B ), whereas the low-dose group demonstrated a more pronounced effect in enhancing perivascular cell coverage (the ratio of a-SMA/CD31 double-positive area over CD31 + area) ( Supplementary Figure S5A&C ). In the context of combined treatment, the combination of low-dose anlotinib with anti-PD-1 therapy notably increased perivascular cell coverage and improved vascular perfusion compared to the high-dose group, demonstrating enhancements in vascular functionality and the normalization of vascular structure (Fig. 5 F-J). The findings depicted in Fig. 5 K-L indicate that the combination of low-dose anlotinib and anti-PD-1 may have a beneficial effect on hypoxia compared to other treatment groups. Furthermore, our analysis focused on the infiltration of immune effector cells, specifically examining the proliferation of immune cells through dual immunofluorescent staining of CD8 and Ki67. Quantitative analysis revealed that the proportion of Ki67+/CD8 + double-positive cells within CD8 + T cells was significantly higher in the treatment group following low-dose anlotinib treatment compared to the other groups (Fig. 5 M-N, Supplementary Figure S6 ). Additionally, the co-administration of low-dose anlotinib with anti-PD-1 immune therapy has been shown to synergistically enhance activated T cell cytotoxicity. Flow cytometry analysis revealed a significant increase in the secretion of effector cytokines, including GZMB (granzyme B), perforin, and TNF-α (tumor necrosis factor-α), in the group receiving the low-dose combination therapy (Fig. 6 ). The upregulation of PD-1 in CD8 + T cells exhibited a similar pattern as previously described. In line with existing experimental results, our findings established a mechanistic association between vasculature and the infiltration of immune cells within tumors, leading us to propose the utilization of a combination therapy involving low-dose anlotinib and anti-PD-1 to mitigate the adverse effects of anticancer drugs while preserving their therapeutic benefits. Discussion This trial is a randomized controlled trial to evaluate the clinical efficacy and safety of combining anti-PD-1 inhibitor and anti-VEGF therapy with chemotherapy as a first-line treatment in patients with advanced BTC. The addition of sintilimab plus anlotinib to the standard chemotherapy regimen of gemcitabine and cisplatin showed promising clinical benefit and manageable toxicity for patients with advanced BTC, with significant improvement in median PFS (8.5 months). However, this was a phase 2 study with a small sample size and the results should be interpreted with caution. The median PFS (6.2 months), median OS (13.7 months), and ORR (29.4%) with chemotherapy alone in this study were consistent with previous reports [ 4 , 29 , 30 ]. This study met its primary endpoint, with significantly improved PFS with SAGC versus GC (8.5 months vs. 6.2 months), which was generally consistent with other similar treatment regimens in first-line advanced BTC. The median PFS with gemcitabine and cisplatin plus atezolizumab (an anti-PD-L1 inhibitor) and bevacizumab was 8.4 months in patients with advanced BTC in a phase 2 IMbrave-151 study [ 31 ]. The lower median PFS in the SAGC group in this study than that of the currently reported PFS with gemcitabine plus oxaliplatin combined with toripalimab (an anti-PD-1 inhibitor) plus lenvatinib (10.2 months) in patients with advanced ICC [ 25 ] may be attributable to patients with older age (median: 62.5 vs. 56.5 years) and relatively poor performance status (ECOG PS 1: 97.5% vs. 0%). The present study enrolled more patients with TNM stage IV (100% vs. 40%), and less patients with ICC (57.5% vs. 100%). Patients with ICC have a better OS than those with biliary tract cancers of different primary origins [ 32 ]. Consistently, patients with ICC in the SAGC group had a higher PFS (9.1 months) than compared with overall patients (8.5 months). In addition, higher ORR and DCR were observed in this study. The ORR of the SAGC group in this study was 51.4%, which was almost twice that of the GC group (29.4%). Previous studies show that the ORR of ICIs combined with chemotherapy is 26.7–29% [ 4 , 21 ]. Taken together, the present results add evidence for the efficacy and feasibility of combining anti-PD-1 antibody, vascular-targeting tyrosine kinase inhibitor (TKI), and chemotherapy as a first-line treatment in patients with advanced BTC. This study did not show a statistically significant improvement in OS with SAGC, which might be explained that, more patients in the GC group than those in the SAGC group received subsequent antitumor treatment (57.5% vs. 35.0%), particularly ICI in the GC group after disease progression (37.5%). Another important reason is the relatively high starting dose of anlotinib (10 mg/day) at the beginning of this study. Although the higher dose of anlotinib (10 mg/day) had the same efficacy (measured by PFS) as the lower dose (8 mg/day), the PFS benefit may not be fully translated into OS owing to increased toxicity at a higher dose. Treatment options after disease progression and patient performance status may also be affected. In the SAGC group, patients receiving a lower dose of anlotinib tended to have better OS compared with patients receiving a higher dose (14.9 vs. 9.3 months). In vivo study, high doses of anlotinib may have a negative effect on efficacy when combined with ICIs compared with low doses. This study did not observe any new safety signals in the GC or SAGC groups. Hematological toxicity (such as neutropenia) was the main grade 3 or higher AE in the SAGC group, as well as the most common TRAE for toripalimab combined with lenvatinib and chemotherapy in a phase 2 study on advanced intrahepatic cholangiocarcinoma [ 25 ]. The most significant AE of SAGC with 10 mg anlotinib daily on treatment continuity was the high incidence of grade 4 thrombocytopenia, which is consistent with previous reports [ 33 – 35 ]. SAGC treated with anlotinib 8 mg once daily is generally well tolerated in patients with advanced BTC. Thus, the starting dose of anlotinib was adjusted from 10 mg to 8 mg after 18 patients were enrolled in the SAGC group, after which grade 3/4 thrombocytopenia decreased from 38.9–13.6%, and overall safety became more manageable. Notably, the anlotinib dose reduction did not affect the PFS of patients with SAGC and tended to provide better OS. Common immune-mediated AEs included abnormal thyroid function, fever, and rash, which were mainly grade 1 or 2. No new safety concerns were identified. These events were managed with appropriate supportive therapy. The use of systemic corticosteroids to manage immune-mediated AEs was generally low (< 10% of the participants). To validate the effectiveness of low-dose anlotinib in conjunction with anti-PD-1 immunotherapy in mitigating adverse effects and enhancing efficacy in this phase 2 trial, as well as to investigate potential mechanisms, the AKT/YAP-induced CCA tumor-bearing mice model was established for in vivo validation experiments. The findings indicated that the low-dose group exhibited a stronger impact on enhancing vascular normalization in comparison with the high-dose anlotinib and control groups in CCA. By implementing this enhancement, we may significantly facilitate the TME transition from abnormal structural and functional states to normal states through the increase of vascular perfusion and the decrease of hypoxia area. Moreover, the combination of low-dose anlotinib with anti-PD-1 therapy has been proven to markedly improve the infiltration and activation of CD8 + cytotoxic T cells and result in a reprogramming of TME, which may effortlessly enhance immunotherapy effectiveness as well as reduce the occurrence of adverse reactions in CCA. Our findings mentioned above are consistent with prior research, suggesting that there is a correlation between the dosage of TKI drugs and their impact on vascular normalization[ 36 ]. More specifically, antiangiogenic drugs with the lower doses may potentially ameliorate hypoxia, acidosis, and hypoperfusion in solid tumors by facilitating vascular normalization and enhancing immune cell infiltration, ultimately facilitating the transition of the immunosuppressive microenvironment to an immune-supportive state[ 37 , 38 ]. Conversely, there exist risks of excessive pruning of blood vessels and rebound angiogenesis when the doses of TKI drugs are too high in clinical administration, which can exacerbate hypoxia and hypoperfusion, inhibit the killing capacity of immune cells, and reduce the efficacy of immunotherapy[ 39 ]. Consequently, it is very important to select an appropriate dose of TKIs when combined with ICIs to achieve better clinical efficacy, lower side effects and smaller financial burden for BTC patients. This study has several limitations. The phase II design and safety-based sample size determination indicated that patient numbers were relatively low for efficacy analyses. Although assignment to the treatment arms was randomized, no stratification was applied; thus, the results might have been biased owing to potential confounding factors. Additionally, the open-label design may have influenced the evaluation of PFS, and no blinded review of the imaging was performed. The early design of the study meant that it had only two arms and lacked the ICI plus chemotherapy group, which was recently approved as standard treatment; therefore, it cannot directly explain whether the addition of targeted therapy to ICI plus chemotherapy can improve the prognosis of patients with advanced BTC. In conclusion, this phase Ⅱ study demonstrated that sintilimab plus anlotinib in combination with gemcitabine plus cisplatin was superior to gemcitabine plus cisplatin alone, with a significant improvement in PFS and manageable toxicity. Notably, reducing the dosage of anlotinib to 8mg may lead to a prolonged overall survival, potentially attributed to the improvement of tumor vascular and immune microenvironment at a lower anlotinib dose. Abbreviations BTC, biliary tract cancer; CCA, cholangiocarcinoma; PD-1, programmed death 1; VEGF , vascular endothelial growth factor; PFS, progression-free survival; ORR, objective response rate; OS, objective response rate; NGS, next generation sequencing; GBC, gallbladder carcinoma; ICC, intrahepatic cholangiocarcinoma; ECC, extrahepatic cholangiocarcinoma; ICI, immune checkpoint inhibitor; TME, tumor microenvironment; ECOG PS, Eastern Cooperative Oncology Group performance status; RECIST, Response Evaluation Criteria in Solid Tumors; GI, gastrointestinal; HBV, hepatitis B virus; HCV, hepatitis C virus; NCI, National Cancer Institute; TMB, tumor mutation burden; IQR, interquartile range; HR, hazard ratio; PR, partial response; SD, stable disease; DCR, disease control rate; TRAE, treatment related adverse event; AE, adverse event; TNM, tumor node metastasis; DoR, date of duration; NSCLC, non-small cell lung cancer; MMR, DNA mismatch repair; HRR, homologous recombination repair; GZMB, granzyme B; TNF‐α, tumor necrosis factor‐α; TKI, tyrosine kinase inhibitor. Declarations Contributors Jingjing Li, Weijing Sun, and Jieer Ying designed the study and wrote the manuscript. Jingjing Li, Qinhong Zheng, Xiaoqing Xu, Fabiao Zhang, Zhe Han, Wei Wu, Xinbao Wang, Yuhua Zhang, Bingchen Wu, Qing Wei, Da Li, X. Xu, and Jieer Ying recruited patients and collected data. Jingjing Li and Qi Xu were responsible for the patient randomization. Li, Xing Sun, Weijing Sun, Xinbao Wang, and Jieer Ying assessed and verified the data. Xing Sun, Jieer Ying, Junrong Yan, and Xiaonan Wang performed the statistical analyses. Shurui Zhou and Yiwen Zhou conducted the in vivo experiments. Jingjing Li, Shurui Zhou and Jieer Ying drafted the manuscript. All authors reviewed and revised the manuscript and approved the final version. The corresponding author had full access to all data and had the final responsibility for the decision to submit for publication. Ethics approval and consent to participate This trial was approved by all the independent institutional review boards and ethics committees at each participating site. All study-related procedures were performed with written informed consent from all patients. This trial was registered at ClinicalTrials.gov.gov (identifier: NCT04300959) and was performed in accordance with Good Clinical Practice and the Declaration of Helsinki. Consent for publication Not applicable. Conflict of interest statement We declare no competing interests. Data availability statement The study protocol is presented in Appendix A. Data supporting the findings of this study are available upon request from the corresponding authors. Funding information This trial was funded by Innovent Biologics (Suzhou) Co., Ltd. and Chia Tai Tianqing Pharmaceutical Group, which provided sintilimab and anlotinib hydrochloride, respectively. The study was supported by grants from Natural Science Fund of Zhejiang Province (LTGY23H160010, LQ20H160005), Medical and Health Science and Technology Program of Zhejiang Province (2021KY087), Zhejiang Provincial Clinical Medical Research Center for Hepatobiliary and Pancreatic Diseases (2018E50001), Quzhou Phoenix Medical Scholar Talent Training Fund (258 Key Discipline Talent Training Project)(2021ZYC-B31), Key Laboratory of Prevention, Diagnosis and Therapy of Upper Gastrointestinal Cancer of Zhejiang Province (2022E10021) and National Natural Science Foundation of China (82303080, 82303415). The funders played no role in the study design, data collection, data analysis, data interpretation, or manuscript writing. Acknowledgments We thank the patients, their families, and all participating clinical teams for making this study possible. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Clinical and biomarker analyses of sintilimab plus gemcitabine and cisplatin as first-line treatment for patients with advanced biliary tract cancer. Nat Commun. 2023;14(1):1340. Mpekris F, Voutouri C, Baish JW, Duda DG, Munn LL, Stylianopoulos T, et al. Combining microenvironment normalization strategies to improve cancer immunotherapy. Proc Natl Acad Sci U S A. 2020;117(7):3728-37. Huang Y, Yuan J, Righi E, Kamoun WS, Ancukiewicz M, Nezivar J, et al. Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Proc Natl Acad Sci U S A. 2012;109(43):17561-6. Li Q, Wang Y, Jia W, Deng H, Li G, Deng W, et al. Low-Dose Anti-Angiogenic Therapy Sensitizes Breast Cancer to PD-1 Blockade. Clin Cancer Res. 2020;26(7):1712-24. Huang Y, Stylianopoulos T, Duda DG, Fukumura D, Jain RK. Benefits of vascular normalization are dose and time dependent--letter. Cancer Res. 2013;73(23):7144-6. Additional Declarations There is NO Competing Interest. Supplementary Files Supp.Materials.docx Protocolversion1.0.pdf Trial Protocal(Version 1.0) protocol.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4557891","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":328317256,"identity":"e807f699-9bd0-4f30-97e6-678406a85834","order_by":0,"name":"Jingjing Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBACPmaGBIMPDGwkaGEDajGcQZoWIGbmIclhbOwMD4pt/vDJ8zcwH/v4hcEujyiHGefwsBnOOMCWPFuGIbmYSC0SbAkGDDzGzBIMBxIbiNJiYUCyFoYEiBbGD8RqMew5APTLYbZkZgaDZMJa+PnPpBn8+HNMnr+9+TDjjwo7wloYGHjSDBgYjgFjBxRBBoTVAwH74QcMDDVgJuMPonSMglEwCkbBSAMAXZYsuwSdC8wAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang Cancer Hospital, Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Li","suffix":""},{"id":328317257,"identity":"9d539480-fd1d-43a2-8ad4-d287d376c50c","order_by":1,"name":"Shurui Zhou","email":"","orcid":"","institution":"Zhejiang Cancer Hospital, Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shurui","middleName":"","lastName":"Zhou","suffix":""},{"id":328317258,"identity":"9d40cde1-f017-4406-8c97-6c0e2dbf88ce","order_by":2,"name":"Xiaoqing Xu","email":"","orcid":"","institution":"Zhejiang cancer hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqing","middleName":"","lastName":"Xu","suffix":""},{"id":328317259,"identity":"3355a97d-8578-40fd-a11b-19cd4197cdb3","order_by":3,"name":"Qinhong Zheng","email":"","orcid":"","institution":"Quzhou Affiliated Hospital of Wenzhou Medical University, Quzhou People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qinhong","middleName":"","lastName":"Zheng","suffix":""},{"id":328317260,"identity":"e0edba1f-80e8-4498-910a-bd0131b75e78","order_by":4,"name":"Fabiao Zhang","email":"","orcid":"","institution":"Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fabiao","middleName":"","lastName":"Zhang","suffix":""},{"id":328317261,"identity":"840659b5-3cc2-48f1-bdb1-291da57484a5","order_by":5,"name":"Cong Luo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Luo","suffix":""},{"id":328317262,"identity":"7f97cace-ed33-4d92-8a7c-4e39c93a97f5","order_by":6,"name":"Da Li","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Da","middleName":"","lastName":"Li","suffix":""},{"id":328317263,"identity":"a8d8fe2f-f6f3-4546-8a72-d00022ca3219","order_by":7,"name":"Xing Sun","email":"","orcid":"","institution":"Innovent Biologics, Inc","correspondingAuthor":false,"prefix":"","firstName":"Xing","middleName":"","lastName":"Sun","suffix":""},{"id":328317264,"identity":"11c06b08-995c-4590-9aa6-e21bb8c4b34f","order_by":8,"name":"Zhe Han","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Han","suffix":""},{"id":328317265,"identity":"eac67057-368a-454d-b0c8-da5a17190640","order_by":9,"name":"Wei Wu","email":"","orcid":"","institution":"The Cancer Hospital of the University of Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wu","suffix":""},{"id":328317266,"identity":"39f0e1c9-8532-41c9-b00d-bd2a54b87b29","order_by":10,"name":"Junrong Yan","email":"","orcid":"https://orcid.org/0000-0001-9750-7518","institution":"Nanjing Geneseeq Technology Inc.","correspondingAuthor":false,"prefix":"","firstName":"Junrong","middleName":"","lastName":"Yan","suffix":""},{"id":328317267,"identity":"be291d39-dda2-43bd-b299-f467754a9da6","order_by":11,"name":"Yang Shao","email":"","orcid":"https://orcid.org/0000-0003-4585-1792","institution":"Princess Margaret Cancer Centre","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Shao","suffix":""},{"id":328317268,"identity":"bf2091b5-e804-4786-abc8-fa4e3ffff7a1","order_by":12,"name":"Yuhua Zhang","email":"","orcid":"https://orcid.org/0000-0002-1308-1992","institution":"Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Yuhua","middleName":"","lastName":"Zhang","suffix":""},{"id":328317269,"identity":"0b895cf0-cff8-4fc1-be07-0252d1911938","order_by":13,"name":"Bingchen Wu","email":"","orcid":"","institution":"Hospital of Chinese Medicine of Changxing","correspondingAuthor":false,"prefix":"","firstName":"Bingchen","middleName":"","lastName":"Wu","suffix":""},{"id":328317270,"identity":"85822014-faf6-47b3-84dc-202f07bf619c","order_by":14,"name":"Qing Wei","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Wei","suffix":""},{"id":328317271,"identity":"2e9cfb22-8f2c-43db-b2ec-2d6cf708f037","order_by":15,"name":"xinbao wang","email":"","orcid":"","institution":"Zhejiang cancer hospital","correspondingAuthor":false,"prefix":"","firstName":"xinbao","middleName":"","lastName":"wang","suffix":""},{"id":328317272,"identity":"11ef5873-4d54-4146-9886-35896c51e99f","order_by":16,"name":"Yiwen Zhou","email":"","orcid":"","institution":"The Second School of Clinical Medicine, Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yiwen","middleName":"","lastName":"Zhou","suffix":""},{"id":328317273,"identity":"b656cb54-62f8-4494-aad3-bf759704c157","order_by":17,"name":"Weijing Sun","email":"","orcid":"https://orcid.org/0000-0001-6549-9605","institution":"University of Kansas Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Weijing","middleName":"","lastName":"Sun","suffix":""},{"id":328317274,"identity":"d9c55144-3bf6-4237-9189-ca4df1f98917","order_by":18,"name":"Qi Xu","email":"","orcid":"","institution":"Zhejiang Cancer Hospital, Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Xu","suffix":""},{"id":328317275,"identity":"0c1cc5b3-df9c-4209-9bab-d126027dfdce","order_by":19,"name":"Jieer Ying","email":"","orcid":"https://orcid.org/0000-0001-9452-1242","institution":"Zhejiang Cancer Hospital, Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jieer","middleName":"","lastName":"Ying","suffix":""}],"badges":[],"createdAt":"2024-06-10 12:05:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4557891/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4557891/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-60119-3","type":"published","date":"2025-07-01T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60703706,"identity":"47e8fc06-1940-4bbb-bcbc-fee52ab78ac2","added_by":"auto","created_at":"2024-07-19 18:53:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63881,"visible":true,"origin":"","legend":"\u003cp\u003eStudy flowchart illustrating patients' randomization and group allocation for treatments with SAGC and GC\u003c/p\u003e\n\u003cp\u003e*One patient did not receive assigned treatment after randomization and was subsequently excluded from safety analysis.\u003c/p\u003e\n\u003cp\u003eAbbreviations: SAGC, sintilimab plus anlotinib combined with gemcitabine and cisplatin; GC, gemcitabine plus cisplatin.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/4c85156ec8314ac11643085e.png"},{"id":60703705,"identity":"95e2934f-0247-43b5-b40d-61881c74e857","added_by":"auto","created_at":"2024-07-19 18:53:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62395,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan–Meier curve of progression-free survival and overall survival. (A) Progression-free survival. (B) Overall survival.\u003c/p\u003e\n\u003cp\u003eAbbreviations: SAGC, sintilimab plus anlotinib combined with gemcitabine and cisplatin; GC, gemcitabine plus cisplatin; CI, confidence interval; HR, hazard ratio.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/dde50c3590a7755732a8cb8b.png"},{"id":60703714,"identity":"1edea0eb-2975-4475-a317-ffe38af56496","added_by":"auto","created_at":"2024-07-19 18:53:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":224458,"visible":true,"origin":"","legend":"\u003cp\u003eForest plot of progression-free survival and overall survival in the subgroup-stratified full analysis set.\u003c/p\u003e\n\u003cp\u003e(A) Progression-free survival. (B) Overall survival.\u003c/p\u003e\n\u003cp\u003eAbbreviations: CI, confidence interval; ECOG, Eastern Cooperative Oncology Group; ECC, extrahepatic cholangiocarcinoma; GBC, gallbladder carcinoma;ICC, intrahepatic cholangiocarcinoma; SAGC, sintilimab plus anlotinib combined with gemcitabine and cisplatin; GC, gemcitabine plus cisplatin.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/281311b8a7fc01a9950241bb.png"},{"id":60703712,"identity":"9da55f3d-bb69-4515-82b9-381a8918b733","added_by":"auto","created_at":"2024-07-19 18:53:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62554,"visible":true,"origin":"","legend":"\u003cp\u003eKaplan–Meier curve of progression-free survival and overall survival in the SAGC group with different initial dose of anlotinib. (A) Progression-free survival. (B) Overall survival.\u003c/p\u003e\n\u003cp\u003eAbbreviations: CI, confidence interval; HR, hazard ratio; NA, not available.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/33e0d55f70ddf0bccb004a74.png"},{"id":60703710,"identity":"9644c05f-3f4f-4144-b499-d06e585fb72f","added_by":"auto","created_at":"2024-07-19 18:53:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":509265,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination of low‐dose Anlotinib with anti–PD-1 immune therapy may synergistically improve the antitumor response.\u003c/p\u003e\n\u003cp\u003e(A) Representative livers and (B) H\u0026amp;E staining of livers from AKT/YAP-induced CCA tumor-bearing C57BL/6 mice. Scale bar, 100μm. (C) Liver weight of tumor-bearing mice (n = 5 per group). (D) Quantification of tumor areas per liver section. (E) Kaplan–Meier survival curves of tumor-bearing mice treated as indicated since the day of tumor injection (n = 10 per group). (F) Representative immunofluorescent staining and (H) quantification of perivascular cell coverage. Red, CD31 staining; green, α-SMA staining; blue, DAPI staining. Scale bar, 20μm. (G) Quantification of MVD. (I) Representative immunofluorescent staining and (J) quantification of Ho33342 perfused tumor areas. Red, CD31 staining; green, sytogreen staining; blue, Hoechest 33342 staining. Scale bar, 100μm. (K) Representative immunofluorescent staining and (L) quantification of hypoxyprobe+ areas. Red, CD31 staining; green, hypoxyprobe staining; blue, DAPI staining. Scale bar, 100μm. (M) Representative immunofluorescent staining and (N) quantitation of CD8+ T cells infiltration. Red, CD8 staining; green, Ki67 staining; blue, DAPI staining. Scale bar, 20μm. Significance was determined by one-way ANOVA. Data are presented as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eAbbreviations：CCA, cholangiocarcinoma; P, anti-PD-1 therapy; A6, high-dose anlotinib (6mg/kg); A3, low-dose anlotinib (3mg/kg).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/fe0b3081b70b1b887bb5c1d4.png"},{"id":60703713,"identity":"1ed359df-03b8-4d3e-a515-0efe2b1a06fd","added_by":"auto","created_at":"2024-07-19 18:53:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":144497,"visible":true,"origin":"","legend":"\u003cp\u003eThe combination of low‐dose Anlotinib with anti–PD-1 immune therapy may synergistically enhanced CD8+ T cell cytotoxicity.\u003c/p\u003e\n\u003cp\u003e(Top) flow-cytometry analysis and (bottom) quantitation of GZMB, perforin, TNF-α, and PD-1 on CD8+ T cells in AKT/YAP-induced CCA tumors treated as indicated (n = 3 per group). All of the data are from one experiment representative of two independent experiments. Significance was determined by one-way ANOVA. Data are presented as mean ± SEM. *\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eAbbreviations：GZMB, granzyme B; TNF‐α, tumor necrosis factor‐α; PD-1, programmed cell death 1.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/de44d5e5c78ad97d0ff74d14.png"},{"id":85830980,"identity":"fb9b1042-ed49-4bfb-ac19-b089d56d68b2","added_by":"auto","created_at":"2025-07-02 07:43:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2353675,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/90ef5995-985c-4225-b444-02404f7f8466.pdf"},{"id":60703709,"identity":"59105cf9-d542-4edd-b055-ff2a41799788","added_by":"auto","created_at":"2024-07-19 18:53:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2303201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supp.Materials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/0026c7b097a9bdd3ab6b38d2.docx"},{"id":60704156,"identity":"c1363ef8-913f-44b6-8655-3faf95e9e67b","added_by":"auto","created_at":"2024-07-19 19:01:33","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":719892,"visible":true,"origin":"","legend":"\u003cp\u003eTrial Protocal(Version 1.0)\u003c/p\u003e","description":"","filename":"Protocolversion1.0.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/972f52ea2d30be6ad5db6af3.pdf"},{"id":60703707,"identity":"40792805-38d2-44c6-ae52-14f206aeb452","added_by":"auto","created_at":"2024-07-19 18:53:32","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":760106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"protocol.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4557891/v1/fea3e806fcddd6cf540a1eb9.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Phase Ⅱ Study of Combined Sintilimab and Anlotinib with Gemcitabine plus Cisplatin in Advanced Biliary Tract Cancer: Efficacy, Safety and Optimize Dose","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiliary tract cancers (BTCs) are the second most common liver cancer type after hepatocellular carcinoma, and account for approximately 3% of all gastrointestinal malignancies worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are mainly divided into gallbladder carcinoma (GBC), intrahepatic cholangiocarcinoma (ICC), and extrahepatic cholangiocarcinoma (ECC), based on their anatomical primary site. The incidence of BTC is high and has increased over the years, with 6.8 new cases per 100,000 in China in 2022[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost patients with BTC are at an advanced stage at the time of diagnosis and are unsuitable for surgical resection; systemic chemotherapy is the only treatment option for most patients. The preferred first-line chemotherapy for advanced BTC is gemcitabine plus cisplatin, which provides limited survival benefits with a median overall survival (OS) of 11.7 months and a median progression-free survival (PFS) of 5.7\u0026ndash;8.0 months [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Target-oriented therapy has progressed and changed the treatment landscape of BTC, with patients harboring \u003cem\u003eFGFR2\u003c/em\u003e fusions or rearrangements benefiting from pemigatinib, infigratinib, and futibatinib, and those with \u003cem\u003eIDH1\u003c/em\u003e-mutations benefiting from ivosidenib [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, some patients may be treated based on their molecular profiles of \u003cem\u003eERBB2\u003c/em\u003e, \u003cem\u003eBRAF\u003c/em\u003e, and \u003cem\u003eNTRK\u003c/em\u003e [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the number of patients with these targetable characteristics is small (~\u0026thinsp;7% with \u003cem\u003eFGFR\u003c/em\u003e alterations and ~\u0026thinsp;13% with \u003cem\u003eIDH1\u003c/em\u003e mutations in intrahepatic cholangiocarcinoma cases) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; thus, the overall benefits of target-oriented treatment remain limited for most patients with advanced BTC.\u003c/p\u003e \u003cp\u003eThe efficacy of immune checkpoint inhibitors (ICIs) in BTC is demonstrated in several studies, either as a single agent or in combination with chemotherapy, including pembrolizumab, nivolumab, and durvalumab, in patients who failed first-line chemotherapy [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and chemotherapy-na\u0026iuml;ve patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the improvement in PFS and OS with ICIs plus chemotherapy as the first-line treatment remains moderate. Although the TOPAZ-1 trial met its primary endpoint OS in first-line advanced BTC, the incremental median OS and PFS with durvalumab plus gemcitabine and cisplatin versus chemotherapy alone were below 2 months (median OS: 12.8 vs. 11.6 months, median PFS: 7.2 vs. 5.7 months) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. A phase 3 Keynote 966 study also indicated that pembrolizumab plus gemcitabine and cisplatin could improve OS compared to chemotherapy as a first-line therapy for advanced BTC [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVascular targeting is believed to enhance the antitumor efficacy of ICIs by modulating the tumor microenvironment (TME) and creating positive feedback loops that reinforce each other [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A retrospective real-world study indicated that treatment with an anti-angiogenic agent (lenvatinib) in combination with an ICI showed an active trend towards improved survival in patients with advanced BTCs after line treatment with manageable adverse events [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Another anti-angiogenic agent (anlotinib) in combination with ICI showed promising efficacy and a well-tolerated safety profile in advanced BTCs that had failed first-line chemotherapy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The clinical benefit of chemotherapy in combination with an antiangiogenic agent and ICI was further observed in the first-line treatment of BTC in a prospective single-arm study in 2023 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, there is a lack of validated evidence from randomized controlled clinical studies.\u003c/p\u003e \u003cp\u003eThis multicenter, randomized, controlled, and phase 2 SAGC trial was conducted to evaluate the clinical activity and safety of the addition of sintilimab (an anti-PD-1 inhibitor) and anlotinib (an anti-angiogenic \u003cem\u003eVEGF\u003c/em\u003e-targeted inhibitor) to chemotherapy as a first-line treatment in patients with advanced BTC. Furthermore, we conducted an animal experiment to investigate the potential optimization of dosage for the drug anlotinib.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and participants\u003c/h2\u003e \u003cp\u003eThe SAGC trial was a randomized, controlled, open label phase 2 study conducted at three centers in China, including Zhejiang Cancer Hospital, Quzhou People's Hospital and Taizhou Hospital. The main inclusion criteria were patients aged 18 years or older, with a baseline Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1, with biopsy/pathology-confirmed unresectable, locally advanced, or metastatic BTC (including GC, ICC, and ECC), who had not received previous systemic therapy for advanced or metastatic disease, had measurable disease according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and had adequate hematological and organ function with a life expectancy exceeding 12 weeks. Patients who received neoadjuvant or adjuvant chemotherapy 6 months or more prior to randomization were eligible.\u003c/p\u003e \u003cp\u003eKey exclusion criteria included active bleeding or clinically significant gastrointestinal (GI) abnormalities that might increase the risk for GI bleeding, immune-related conditions such as autoimmune disease and major inflammatory diseases, clinical evidence of metastatic disease to the brain and clinically significant cardiovascular disease, acute or chronic active hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, HBV DNA greater than 2,000 international units per milliliter (IU/mL) or 10⁴ copies per mL, and HCV RNA greater than 10\u0026sup3; copies per mL. Detailed inclusion and exclusion criteria are seen in the online supplemental material.\u003c/p\u003e \u003cp\u003eThe trial was registered at Clinicaltrials. gov (identifier: NCT04300959) and was conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. The protocol and amendments were approved by the Institutional Review Board and Ethics Committee of each site. All patients provided written informed consent prior to enrolment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRandomization and masking\u003c/h2\u003e \u003cp\u003eEligible patients were randomly assigned (1:1) to the sintilimab plus anlotinib plus gemcitabine and cisplatin (SAGC) or gemcitabine and cisplatin (GC) group using a simple randomization method. No masking was performed because this was an open-label study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProcedures\u003c/h2\u003e \u003cp\u003ePatients in the SAGC group received sintilimab (200 mg intravenously administered on day 1 every 3 weeks) and anlotinib (10 mg orally once daily from day 1 to day 14, every 3 weeks) along with chemotherapy (25 mg/m\u003csup\u003e2\u003c/sup\u003e cisplatin followed by 1,000 mg/m\u003csup\u003e2\u003c/sup\u003e gemcitabine, each administered on days 1 and 8 every 3 weeks) for up to 8 cycles, followed by maintenance sintilimab plus anlotinib. Patients in the GC group received chemotherapy alone (cisplatin 25 mg/m\u003csup\u003e2\u003c/sup\u003e followed by gemcitabine 1,000 mg/m\u003csup\u003e2\u003c/sup\u003e, each administered on days 1 and 8 every 3 weeks) for up to 8 cycles. After 18 patients were enrolled in the SAGC group, the starting dose of anlotinib was modified from 10 to 8 mg daily owing to the observed bone marrow suppression events. Treatment was continued until disease progression, intolerable toxicity, withdrawal of consent, study completion, or study termination occurred, whichever occurred first. Dose adjustments were permitted for chemotherapy and anlotinib but not for sintilimab, according to the protocol. Considering that gemcitabine, cisplatin, and anlotinib are marketed drugs, dose adjustments were made by the investigators according to the treatment-related adverse events determined and medication instructions (details in \u003cb\u003eSupplementary Protocol\u003c/b\u003e). Patients who temporarily or permanently discontinued chemotherapy or sintilimab and anlotinib owing to adverse events were allowed to continue taking other agents as long as the investigator determined that there was a clinical benefit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAssessments\u003c/h2\u003e \u003cp\u003eTumors were assessed by investigators using computed tomography or magnetic resonance imaging (MRI) at baseline and every 6 weeks as per RECIST 1.1. Safety was assessed for the patients who received at least one dose of the assigned treatment. Vital signs, clinical laboratory tests, and other adverse events were continuously monitored throughout the study and assessed according to the National Cancer Institute\u0026rsquo;s (NCI) Common Terminology Criteria for Adverse Events, version 4.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEndpoints\u003c/h2\u003e \u003cp\u003eThe primary endpoint was PFS, defined as the time from randomization to disease progression according to RECIST version 1.1, or death from any cause, whichever occurred first. The secondary endpoints were objective response rate (ORR), defined as the percentage of patients with radiologically confirmed complete or partial response according to RECIST version 1.1; OS, defined as the time from randomization to death from any cause; and safety, described as the occurrence of treatment-related adverse events (TRAEs) of any grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimal models\u003c/h2\u003e \u003cp\u003eFemale C57BL/6 mice aged 6\u0026ndash;8 weeks were procured from the Shanghai Model Organisms Center. The orthotopic cholangiocarcinoma (CCA) tumor-bearing models were established through hydrodynamic tail vein injection (HD inj) of 30 \u0026micro;g YapS127A, 20 \u0026micro;g AKT, and 2 \u0026micro;g SB100 transposase plasmids diluted in 2 ml of 0.9% NaCl. Anlotinib (kindly provided by Chia Tai Tianqing Pharmaceutical Group Co. Ltd) treatment at varying doses[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e](low-dose: 3 mg/kg, high-dose: 6 mg/kg) was initiated 7 days post tumor cell inoculation and administered orally via gavage daily. Anti-mouse PD-1 antibody (CD279, BE0146, BioXcell) was administered at a dose of 200 mg per mouse through intraperitoneal injection every 3 days. The control group received intragastric administration of 0.9% saline and intraperitoneal injection of IgG, while tumor-bearing mice were euthanized at the conclusion of the experiment. Additional independent experiments were conducted to compare the survival times of mice in each group.\u003c/p\u003e \u003cp\u003e Approval for all animal experiments was obtained from the Animal Experimental Ethics Committee of Zhejiang Cancer Hospital, and the experiments were conducted in accordance with guidelines for the care and use of laboratory animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe sample size was determined based on the primary endpoint of PFS under the assumption that the GC group had a median PFS of 6.5 months, which was based on the ABC-02 study [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This estimate also accounted for the potential impact on the median PFS estimate caused by a longer tumor assessment interval (12 weeks) used in ABC-02, and that SAGC would improve PFS with a hazard ratio (HR) of 50%; approximately 51 PFS events would be needed to provide 80% power to detect this difference under a type I error controlled at 10% (two-sided). Eighty subjects were randomized in a 1:1 ratio to achieve 51 PFS events to complete the study in a reasonable time, and considering the possible loss to follow-up.\u003c/p\u003e \u003cp\u003ePatients who were lost to follow-up were censored at the randomization date for PFS analysis and at the last known survival date for OS analysis. The efficacy analysis set included patients randomly assigned to each treatment group, regardless of whether they had received treatment. The safety analysis set included patients who received at least one dose of the study treatment.\u003c/p\u003e \u003cp\u003eQuantitative data were displayed as the median (range) or number of patients (percentage). Proportional comparisons between groups were performed using Fisher's exact test. Survival analyses (PFS and OS) were performed using Kaplan-Meier curves, \u003cem\u003eP\u003c/em\u003e values were determined using the log-rank test, and HRs were calculated using the Cox proportional hazards model. A two-sided \u003cem\u003eP\u003c/em\u003e value lower than 0.05 was considered statistically significant for all tests except for the primary endpoint, unless indicated otherwise. A two-sided \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.1 was used to determine statistical significance for the primary endpoint PFS. Univariate analysis was used to explore the associations between different variables and PFS, and the results are presented as HRs with 95% confidence intervals (CIs). All analyses were performed using R 3.4.0 [Vienna, Austria].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Treatment\u003c/h2\u003e \u003cp\u003eEighty patients were enrolled between March 26, 2020, and May 25, 2022, with 40 patients in each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, most patients were males (61.3%) and had ECOG PS 1 (97.5%), ICC (62.5%), and metastatic disease (100%); 17.5% patients were HBV positive. Baseline characteristics were generally well balanced between the treatment groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Eighteen patients in the SAGC group received a starting dose of 10 mg anlotinib daily under the initial protocol, while 22 patients received 8 mg daily under the amended protocol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics of patients at baseline\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient chracteristics\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSAGC group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGC group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years), median\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62.5 (54.8, 68.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59.5 (50.0, 66.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.244\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCA199 (U/ml), median\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e67.53 (21.8, 961.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e172.84 (20.1, 1428.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.796\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16 (40.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24 (60.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25 (62.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eECOG\u003csup\u003eb\u003c/sup\u003e performance status score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.494\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2 (5.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38 (95.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary tumor type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.643\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtrahepatic cholangiocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6 (15.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGallbladder carcinoma.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11 (27.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntrahepatic cholangiocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23 (57.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27 (67.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtent of disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.453\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitially unresectable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31 (77.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27 (67.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRecurrent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9 (22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (32.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetastases\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26 (65.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 (65.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9 (22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (25.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3 (7.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeritoneum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9 (22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistant lymph node\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27 (67.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22 (55.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.359\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHepatitis B Virus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.769\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34 (85.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32 (80.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6 (15.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (20.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious history of surgery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26 (65.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25 (62.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14 (35.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (37.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrevious adjuvant therapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37 (92.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (90.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3 (7.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4 (10.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003eData are \u003cem\u003en\u003c/em\u003e (%) or median (IQR). \u003csup\u003eb\u003c/sup\u003eECOG, Eastern Cooperative Oncology Group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSeventy-seven patients discontinued the study treatment at the data cutoff (December 30, 2023), mainly owing to disease progression (33/40 in the SAGC group vs. 32/40 in the GC group); 2 patients continued treatment (1 in the SAGC group and 1 in the GC group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Patients in the SAGC group received sintilimab plus anlotinib for a median of seven cycles (range: 1\u0026ndash;22) combined with chemotherapy for a median of six cycles (range: 1\u0026ndash;8) with a dose intensity of 784.2 mg/m\u003csup\u003e2\u003c/sup\u003e for gemcitabine and 18.9 mg/m\u003csup\u003e2\u003c/sup\u003e for cisplatin. The median chemotherapy exposure in the GC group was six cycles (range: 1\u0026ndash;8) with a dose intensity of 806.1 mg/m\u003csup\u003e2\u003c/sup\u003e for gemcitabine and 20 mg/m\u003csup\u003e2\u003c/sup\u003e for cisplatin (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Overall, 14 patients in the SAGC group and 23 in the GC group received at least one anti-tumor therapy post study treatment discontinuation, including chemotherapy (11/40 [27.5%] vs. 13/40 [32.5%]), ICI (6/40 [15.0%] vs. 15/40 [37.5%]), and tyrosine kinase inhibitors (0/40 [0%] vs. 8/40 [20.0%]) (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy\u003c/h2\u003e \u003cp\u003eThe median follow-up at the data cutoff was 13.4 months. Thirty-five (87.5%) of 40 patients in the SAGC group and 35 (87.5%) of 40 patients in the GC group had progressive disease or died. The median PFS was 8.5 months (95% CI, 5.6\u0026ndash;11.0) in the SAGC group versus 6.2 months (95% CI, 4.4\u0026ndash;7.8) in the GC group (HR: 0.47 [95% CI, 0.22\u0026ndash;0.64], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The 12-month PFS rates were 26.4% and 0% in the SAGC and GC groups, respectively. Consistent improvements in PFS were observed across the most clinically relevant subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The median PFS was 9.1 months with SAGC for patients with ICC versus 6.2 months with GC (HR: 0.34, 95% CI, 0.15\u0026ndash;0.75; \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Twenty-nine (72.5%) patients died in the SAGC group and 23 (57.5%) in the GC group; the median OS was 13.2 months (95% CI, 8.7\u0026ndash;19.0) versus 13.7 months (95% CI, 10.2\u0026ndash;15.3) (HR: 1.04 [95% CI, 0.40\u0026ndash;1.49], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.895 Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThirty-seven patients in the SAGC group and 34 in the GC group received at least one post-baseline imaging assessment. Nineteen (51.4%) of the 37 patients randomized to SAGC group achieved a partial response (PR), and 16/37 (43.2%) patients had stable disease (SD), contributing to an ORR of 51.4% and a DCR of 94.6%. In addition, 10/34 (29.4%) patients achieved PR, and 18/34 (52.9%) patients achieved SD in the GC group according to their randomized assignments, with an ORR of 29.4% and a DCR of 82.3% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). The ORR was significantly higher in the SAGC group than in the GC group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033). The median time to response was 2.5 (1.7\u0026ndash;3.6) months in the SAGC group and 3.0 (2.7\u0026ndash;4.0) months in the GC group. A longer median duration of response with SAGC versus GC (9.1 [4.9, NA] versus 3.4 [2.0, NA] months) was observed (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of best overall response in SAGC and GC groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEfficacy\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSAGC group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;37)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGC group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;34)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBest overall response\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.033\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePartial response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19 (51.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (29.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStable disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (43.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (52.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProgressive disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (5.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (17.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eObjective response rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDisease control rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e94.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian time to onset of response, months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5 (1.7, 3.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0 (2.7, 4.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMedian duration of response, months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.1 (4.9, NA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.4 (2, NA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea\u003c/sup\u003eData are shown as \u003cem\u003en\u003c/em\u003e (%) and median (IQR). \u003csup\u003eb\u003c/sup\u003ePatients without any post-baseline imaging assessment. \u003csup\u003ec\u003c/sup\u003ePer RECIST version 1.1.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA post hoc sensitivity analysis was performed to compare the efficacy between patients who received a starting dose of anlotinib of 10 mg daily and those who received an anlotinib dose of 8 mg daily in the SAGC group. The median PFS did not significantly differ between the 8mg and 10mg groups (8.5 vs. 7.6 months, HR: 0.87 [95% CI, 0.30\u0026ndash;1.55], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.691) with a trend longer \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. A trend towards longer median OS was observed in the 8mg group compared to the 10mg group (14.9 vs. 9.3 months, HR: 0.49 [95% CI, 0.14\u0026ndash;1.18], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.055) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e, with this difference remaining consistent during long-term follow-up. The ORRs were 38.8% at 10 mg daily and 54.5% at 8 mg daily. (\u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSafety\u003c/h2\u003e \u003cp\u003eSeventy-nine of the 80 patients received at least one dose of the assigned treatment and were included in the safety analyses. All patients experienced at least one treatment-related adverse event (TRAE);75% had at least one grade 3/4 TRAE in the SAGC group and 43.6% in the GC group. No adverse events in grade 5 were observed in either group. Adverse events led to dose reductions in 28 (70%) and 19 (48.7%) patients in the SAGC and GC groups, respectively. Furthermore, AEs led to treatment discontinuation in 1 (2.5%) and 0 patients in the SAGC and GC groups, respectively (Data not shown).\u003c/p\u003e \u003cp\u003eThe most common treatment-related AEs are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The most frequent grade 3\u0026ndash;4 treatment-emergent AEs (occurring in \u0026ge;\u0026thinsp;5% of patients) were leukopenia (9 [22.5%] of 40 patients in SAGC group vs. 11 [28.3%] of 39 patients in GC group), neutropenia (18 [45%] vs. 9 [23.1%]), thrombocytopenia (11 [27.5%] vs. 4 [10.3%]), anemia (6 [15%] vs. 6 [15.4%]), increased γ-glutamyltransferase (5 [12.5%] vs. 1 [2.6%]). Serious hematological toxicities (including neutropenia and thrombocytopenia) were more common in the SAGC group than in the GC group. The incidence of grade 4 AE in the SAGC group decreased from 23.5\u0026ndash;13.0% after the starting dose of anlotinib was reduced to 8 mg daily \u003cb\u003e(Supplementary Table S4)\u003c/b\u003e. Potentially immune-mediated AEs occurred in 9 (22.5%) of the 40 participants in the SAGC group, of all were grade 1\u0026ndash;2 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The GC group was not evaluated for immune-related AEs since this was an open-label trial. Common immune-mediated adverse events included abnormal thyroid function in 4 patients (10%) and rashes in4 patients 10%). The only potentially immune-mediated AE that led to death was grade 2 pneumonitis, which occurred in one participant in the SAGC group and was treated with corticosteroids.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of treatment-related adverse events\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSafety\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eSAGC (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c13\" namest=\"c8\"\u003e \u003cp\u003eGC (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;39)\u003csup\u003eb*\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAny grade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;= Grade 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGrade 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGrade 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGrade 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGrade 4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAny grade\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026gt;= Grade 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eGrade 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eGrade 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eGrade 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eGrade 4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAny treatment-emergent adverse event\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23 (57.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e39 (100%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e17 (43.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6 (15.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e16 (41%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e14 (35.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e3 (7.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeukopenia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35 (87.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7 (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19 (47.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 (22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24 (61.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11 (28.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6 (15.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e7 (17.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e11 (28.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnaemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34 (85%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12 (30%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e33 (84.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e6 (15.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e14 (35.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e13 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6 (15.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutropenia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33 (82.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10 (25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16 (40%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24 (61.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9 (23.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e11 (28.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e6 (15.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e3 (7.7%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThrombocytopenia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 (75%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11(27.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (27.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25 (64.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e13 (33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e8 (20.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIncreased γ-glutamyltransferase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29 (72.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18 (45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e27 (69.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e22 (56.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypoalbuminemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 (67.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22 (55%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e17 (43.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e17 (43.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHyponatremia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18 (45%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (40\u0026middot;0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (5\u0026middot;0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e21 (53\u0026middot;8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e21 (53\u0026middot;8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIncreased aspartate aminotransferase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (42.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14 (35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12 (30.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e10 (25.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatigue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (42.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 (25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11 (28.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e7 (17.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIncreased alanine aminotransferase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (32.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12 (30.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e9 (23.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProteinuria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (32.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (20.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIncrease creatinine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (25%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (20.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e7 (17.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypokalemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 (22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5\u0026middot;0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (15\u0026middot;0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2\u0026middot;5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (5\u0026middot;0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (20.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e7 (17\u0026middot;9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1 (2\u0026middot;6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNausea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9(22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3 (7.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVomiting\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9(22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6(15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (7.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10 (25.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e9 (23.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHyperuricemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (20%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (12.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5 (12.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypertension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (12.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3 (7.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConstipation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (12.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (5.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (20.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e6 (15.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFever\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnorexia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (17.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (12.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3 (7.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIncreased conjugated bilirubin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (7\u0026middot;5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (5\u0026middot;0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2\u0026middot;5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3 (7\u0026middot;5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (12.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (2\u0026middot;6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2 (5\u0026middot;1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2 (5\u0026middot;1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1 (2\u0026middot;6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIncrease serum amylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (7.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRash\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (10%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3 (7.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2 (5.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiarrhea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (7.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbdominal pain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (12.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4 (10.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1 (2.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHand-foot syndrome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHyperthyroidism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypothyroidism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInterstitial pneumonia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0 (0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003csup\u003ea\u003c/sup\u003eData are shown as n (%). \u003csup\u003eb\u003c/sup\u003eOne patient withdrew consent after randomization.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo validation\u003c/h2\u003e \u003cp\u003ePrevious observations suggest that dose reduction of anlotinib may lead to improved outcomes and decreased adverse events. To explore the potential mechanisms of these dose-dependent responses, an orthotopic AKT/YAP-induced CCA tumor-bearing model was established (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA\u003c/b\u003e). Consistent with expectations, the combination of anlotinib and anti-PD-1 treatment demonstrated greater efficacy in inhibiting tumor growth compared to monotherapy or control groups. Our study revealed that there were no statistically significant differences in efficacy between high- and low-dose anlotinib treatment (\u003cb\u003eSupplementary Figure S4\u003c/b\u003e). However, the combination of low-dose anlotinib with anti-PD-1 demonstrated greater efficacy in inhibiting tumor growth compared to high-dose anlotinib combination therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Additionally, survival outcomes were similar across treatment groups, with the most favorable prognosis observed in the low-dose anlotinib plus anti-PD-1 subgroup (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, there were no significant differences in body weight among the treatment groups, suggesting that high-dose anlotinib monotherapy and combination therapy did not result in noticeable toxicity (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB\u003c/b\u003e). Moreover, the mice in the high-dose group exhibited a notable reduction in small intestinal villus and significant irregular morphologies (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC\u003c/b\u003e). These results collectively suggest that low-dose anlotinib treatments can improve the efficacy of anti-PD-1 therapy while minimizing adverse effects in the orthotopic CCA tumor-bearing model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExisting research suggests that antiangiogenic agents have the potential to restructure the tumor immunosuppressive microenvironment through the normalization of vascular structure and mitigation of hypoxia in a dose-dependent fashion. Therefore, our initial assessment focused on the alterations in tumor blood vessels. The findings indicated that high-dose anlotinib led to a statistically significant reduction in intratumoral microvascular density (MVD, defined as CD31\u0026thinsp;+\u0026thinsp;vessel numbers per field) (\u003cb\u003eSupplementary Figure S5A-B\u003c/b\u003e), whereas the low-dose group demonstrated a more pronounced effect in enhancing perivascular cell coverage (the ratio of a-SMA/CD31 double-positive area over CD31\u0026thinsp;+\u0026thinsp;area) (\u003cb\u003eSupplementary Figure S5A\u0026amp;C\u003c/b\u003e). In the context of combined treatment, the combination of low-dose anlotinib with anti-PD-1 therapy notably increased perivascular cell coverage and improved vascular perfusion compared to the high-dose group, demonstrating enhancements in vascular functionality and the normalization of vascular structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-J). The findings depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK-L indicate that the combination of low-dose anlotinib and anti-PD-1 may have a beneficial effect on hypoxia compared to other treatment groups.\u003c/p\u003e \u003cp\u003eFurthermore, our analysis focused on the infiltration of immune effector cells, specifically examining the proliferation of immune cells through dual immunofluorescent staining of CD8 and Ki67. Quantitative analysis revealed that the proportion of Ki67+/CD8\u0026thinsp;+\u0026thinsp;double-positive cells within CD8\u0026thinsp;+\u0026thinsp;T cells was significantly higher in the treatment group following low-dose anlotinib treatment compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM-N, \u003cb\u003eSupplementary Figure S6\u003c/b\u003e). Additionally, the co-administration of low-dose anlotinib with anti-PD-1 immune therapy has been shown to synergistically enhance activated T cell cytotoxicity. Flow cytometry analysis revealed a significant increase in the secretion of effector cytokines, including GZMB (granzyme B), perforin, and TNF-α (tumor necrosis factor-α), in the group receiving the low-dose combination therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The upregulation of PD-1 in CD8\u0026thinsp;+\u0026thinsp;T cells exhibited a similar pattern as previously described. In line with existing experimental results, our findings established a mechanistic association between vasculature and the infiltration of immune cells within tumors, leading us to propose the utilization of a combination therapy involving low-dose anlotinib and anti-PD-1 to mitigate the adverse effects of anticancer drugs while preserving their therapeutic benefits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis trial is a randomized controlled trial to evaluate the clinical efficacy and safety of combining anti-PD-1 inhibitor and anti-VEGF therapy with chemotherapy as a first-line treatment in patients with advanced BTC. The addition of sintilimab plus anlotinib to the standard chemotherapy regimen of gemcitabine and cisplatin showed promising clinical benefit and manageable toxicity for patients with advanced BTC, with significant improvement in median PFS (8.5 months). However, this was a phase 2 study with a small sample size and the results should be interpreted with caution. The median PFS (6.2 months), median OS (13.7 months), and ORR (29.4%) with chemotherapy alone in this study were consistent with previous reports [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study met its primary endpoint, with significantly improved PFS with SAGC versus GC (8.5 months vs. 6.2 months), which was generally consistent with other similar treatment regimens in first-line advanced BTC. The median PFS with gemcitabine and cisplatin plus atezolizumab (an anti-PD-L1 inhibitor) and bevacizumab was 8.4 months in patients with advanced BTC in a phase 2 IMbrave-151 study [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The lower median PFS in the SAGC group in this study than that of the currently reported PFS with gemcitabine plus oxaliplatin combined with toripalimab (an anti-PD-1 inhibitor) plus lenvatinib (10.2 months) in patients with advanced ICC [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] may be attributable to patients with older age (median: 62.5 vs. 56.5 years) and relatively poor performance status (ECOG PS 1: 97.5% vs. 0%). The present study enrolled more patients with TNM stage IV (100% vs. 40%), and less patients with ICC (57.5% vs. 100%). Patients with ICC have a better OS than those with biliary tract cancers of different primary origins [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consistently, patients with ICC in the SAGC group had a higher PFS (9.1 months) than compared with overall patients (8.5 months). In addition, higher ORR and DCR were observed in this study. The ORR of the SAGC group in this study was 51.4%, which was almost twice that of the GC group (29.4%). Previous studies show that the ORR of ICIs combined with chemotherapy is 26.7\u0026ndash;29% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Taken together, the present results add evidence for the efficacy and feasibility of combining anti-PD-1 antibody, vascular-targeting tyrosine kinase inhibitor (TKI), and chemotherapy as a first-line treatment in patients with advanced BTC.\u003c/p\u003e \u003cp\u003eThis study did not show a statistically significant improvement in OS with SAGC, which might be explained that, more patients in the GC group than those in the SAGC group received subsequent antitumor treatment (57.5% vs. 35.0%), particularly ICI in the GC group after disease progression (37.5%). Another important reason is the relatively high starting dose of anlotinib (10 mg/day) at the beginning of this study. Although the higher dose of anlotinib (10 mg/day) had the same efficacy (measured by PFS) as the lower dose (8 mg/day), the PFS benefit may not be fully translated into OS owing to increased toxicity at a higher dose. Treatment options after disease progression and patient performance status may also be affected. In the SAGC group, patients receiving a lower dose of anlotinib tended to have better OS compared with patients receiving a higher dose (14.9 vs. 9.3 months). In vivo study, high doses of anlotinib may have a negative effect on efficacy when combined with ICIs compared with low doses.\u003c/p\u003e \u003cp\u003eThis study did not observe any new safety signals in the GC or SAGC groups. Hematological toxicity (such as neutropenia) was the main grade 3 or higher AE in the SAGC group, as well as the most common TRAE for toripalimab combined with lenvatinib and chemotherapy in a phase 2 study on advanced intrahepatic cholangiocarcinoma [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The most significant AE of SAGC with 10 mg anlotinib daily on treatment continuity was the high incidence of grade 4 thrombocytopenia, which is consistent with previous reports [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. SAGC treated with anlotinib 8 mg once daily is generally well tolerated in patients with advanced BTC. Thus, the starting dose of anlotinib was adjusted from 10 mg to 8 mg after 18 patients were enrolled in the SAGC group, after which grade 3/4 thrombocytopenia decreased from 38.9\u0026ndash;13.6%, and overall safety became more manageable. Notably, the anlotinib dose reduction did not affect the PFS of patients with SAGC and tended to provide better OS. Common immune-mediated AEs included abnormal thyroid function, fever, and rash, which were mainly grade 1 or 2. No new safety concerns were identified. These events were managed with appropriate supportive therapy. The use of systemic corticosteroids to manage immune-mediated AEs was generally low (\u0026lt;\u0026thinsp;10% of the participants).\u003c/p\u003e \u003cp\u003eTo validate the effectiveness of low-dose anlotinib in conjunction with anti-PD-1 immunotherapy in mitigating adverse effects and enhancing efficacy in this phase 2 trial, as well as to investigate potential mechanisms, the AKT/YAP-induced CCA tumor-bearing mice model was established for in vivo validation experiments. The findings indicated that the low-dose group exhibited a stronger impact on enhancing vascular normalization in comparison with the high-dose anlotinib and control groups in CCA. By implementing this enhancement, we may significantly facilitate the TME transition from abnormal structural and functional states to normal states through the increase of vascular perfusion and the decrease of hypoxia area. Moreover, the combination of low-dose anlotinib with anti-PD-1 therapy has been proven to markedly improve the infiltration and activation of CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T cells and result in a reprogramming of TME, which may effortlessly enhance immunotherapy effectiveness as well as reduce the occurrence of adverse reactions in CCA. Our findings mentioned above are consistent with prior research, suggesting that there is a correlation between the dosage of TKI drugs and their impact on vascular normalization[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. More specifically, antiangiogenic drugs with the lower doses may potentially ameliorate hypoxia, acidosis, and hypoperfusion in solid tumors by facilitating vascular normalization and enhancing immune cell infiltration, ultimately facilitating the transition of the immunosuppressive microenvironment to an immune-supportive state[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Conversely, there exist risks of excessive pruning of blood vessels and rebound angiogenesis when the doses of TKI drugs are too high in clinical administration, which can exacerbate hypoxia and hypoperfusion, inhibit the killing capacity of immune cells, and reduce the efficacy of immunotherapy[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Consequently, it is very important to select an appropriate dose of TKIs when combined with ICIs to achieve better clinical efficacy, lower side effects and smaller financial burden for BTC patients.\u003c/p\u003e \u003cp\u003eThis study has several limitations. The phase II design and safety-based sample size determination indicated that patient numbers were relatively low for efficacy analyses. Although assignment to the treatment arms was randomized, no stratification was applied; thus, the results might have been biased owing to potential confounding factors. Additionally, the open-label design may have influenced the evaluation of PFS, and no blinded review of the imaging was performed. The early design of the study meant that it had only two arms and lacked the ICI plus chemotherapy group, which was recently approved as standard treatment; therefore, it cannot directly explain whether the addition of targeted therapy to ICI plus chemotherapy can improve the prognosis of patients with advanced BTC.\u003c/p\u003e \u003cp\u003eIn conclusion, this phase Ⅱ study demonstrated that sintilimab plus anlotinib in combination with gemcitabine plus cisplatin was superior to gemcitabine plus cisplatin alone, with a significant improvement in PFS and manageable toxicity. Notably, reducing the dosage of anlotinib to 8mg may lead to a prolonged overall survival, potentially attributed to the improvement of tumor vascular and immune microenvironment at a lower anlotinib dose.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBTC, biliary tract cancer; CCA, cholangiocarcinoma; PD-1, programmed death 1; \u003cem\u003eVEGF\u003c/em\u003e, vascular endothelial growth factor; PFS, progression-free survival; ORR, objective response rate; OS, objective response rate; NGS, next generation sequencing; GBC, gallbladder carcinoma; ICC, intrahepatic cholangiocarcinoma; ECC, extrahepatic cholangiocarcinoma; ICI, immune checkpoint inhibitor; TME, tumor microenvironment; ECOG PS, Eastern Cooperative Oncology Group performance status; RECIST, Response Evaluation Criteria in Solid Tumors; GI, gastrointestinal; HBV, hepatitis B virus; HCV, hepatitis C virus; NCI, National Cancer Institute; TMB, tumor mutation burden; IQR, interquartile range; HR, hazard ratio; PR, partial response; SD, stable disease; DCR, disease control rate; TRAE, treatment related adverse event; AE, adverse event; TNM, tumor node metastasis; DoR, date of duration; NSCLC, non-small cell lung cancer; MMR, DNA mismatch repair; HRR, homologous recombination repair; GZMB, granzyme B; TNF‐α, tumor necrosis factor‐α; TKI, tyrosine kinase inhibitor.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributors\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJingjing Li, Weijing Sun, and Jieer Ying designed the study and wrote the manuscript. Jingjing Li, Qinhong Zheng, Xiaoqing Xu, Fabiao Zhang, Zhe Han, Wei Wu, Xinbao Wang, Yuhua Zhang, Bingchen Wu, Qing Wei, Da Li, X. Xu, and Jieer Ying recruited patients and collected data. Jingjing Li and Qi Xu were responsible for the patient randomization. Li, Xing Sun, Weijing Sun, Xinbao Wang, and Jieer Ying assessed and verified the data. Xing Sun, Jieer Ying, Junrong Yan, and Xiaonan Wang performed the statistical analyses. Shurui Zhou and Yiwen Zhou conducted the in vivo experiments. Jingjing Li, Shurui Zhou and Jieer Ying drafted the manuscript. All authors reviewed and revised the manuscript and approved the final version. The corresponding author had full access to all data and had the final responsibility for the decision to submit for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis trial was approved by all the independent institutional review boards and ethics committees at each participating site. All study-related procedures were performed with written informed consent from all patients. This trial was registered at ClinicalTrials.gov.gov (identifier: NCT04300959) and was performed in accordance with Good Clinical Practice and the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol is presented in Appendix\u0026nbsp;A.\u0026nbsp;Data supporting the findings of this study are available upon request from the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis trial was funded by Innovent Biologics (Suzhou) Co., Ltd.\u0026nbsp;and Chia Tai Tianqing Pharmaceutical Group, which provided sintilimab and anlotinib hydrochloride, respectively.\u0026nbsp;The study was supported by grants from Natural Science Fund of Zhejiang Province (LTGY23H160010, LQ20H160005), Medical and Health Science and Technology Program of Zhejiang Province (2021KY087), Zhejiang Provincial Clinical Medical Research Center for Hepatobiliary and Pancreatic Diseases (2018E50001), Quzhou Phoenix Medical Scholar Talent Training Fund (258 Key Discipline Talent Training Project)(2021ZYC-B31), Key Laboratory of Prevention, Diagnosis and Therapy of Upper Gastrointestinal Cancer of Zhejiang Province (2022E10021) and National Natural Science Foundation of China (82303080, 82303415).\u0026nbsp;The funders played no role in\u0026nbsp;the study design, data collection, data analysis, data interpretation, or\u0026nbsp;manuscript writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the patients, their families, and all participating clinical teams for making this study possible.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. 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Mol Cancer. 2024;23(1):59.\u003c/li\u003e\n \u003cli\u003eLiang L, Hui K, Hu C, Wen Y, Yang S, Zhu P, et al. Autophagy inhibition potentiates the anti-angiogenic property of multikinase inhibitor anlotinib through JAK2/STAT3/VEGFA signaling in non-small cell lung cancer cells. J Exp Clin Cancer Res. 2019;38(1):71.\u003c/li\u003e\n \u003cli\u003eMorizane C, Okusaka T, Mizusawa J, Katayama H, Ueno M, Ikeda M, et al. Combination gemcitabine plus S-1 versus gemcitabine plus cisplatin for advanced/recurrent biliary tract cancer: the FUGA-BT (JCOG1113) randomized phase III clinical trial. Ann Oncol. 2019;30(12):1950-8.\u003c/li\u003e\n \u003cli\u003eOkusaka T, Nakachi K, Fukutomi A, Mizuno N, Ohkawa S, Funakoshi A, et al. Gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer: a comparative multicentre study in Japan. Br J Cancer. 2010;103(4):469-74.\u003c/li\u003e\n \u003cli\u003eEl-Khoueiry AB, Ren Z, Chon H, Park JO, Kim JW, Pressiani T, et al. IMbrave151: A phase 2, randomized, double-blind, placebo-controlled study of atezolizumab with or without bevacizumab in combination with cisplatin plus gemcitabine in patients with untreated, advanced biliary tract cancer. Journal of Clinical Oncology. 2023;41(4_suppl):491-.\u003c/li\u003e\n \u003cli\u003eMcNamara MG, Lopes A, Wasan H, Malka D, Goldstein D, Shannon J, et al. Landmark survival analysis and impact of anatomic site of origin in prospective clinical trials of biliary tract cancer. J Hepatol. 2020;73(5):1109-17.\u003c/li\u003e\n \u003cli\u003eKuter DJ. Managing thrombocytopenia associated with cancer chemotherapy. Oncology (Williston Park). 2015;29(4):282-94.\u003c/li\u003e\n \u003cli\u003eLi Q, Su T, Zhang X, Pan Y, Ma S, Zhang L, et al. A Real-World Study of Optimal Treatment with Anlotinib First-Line Therapy in Advanced Hepatocellular Carcinoma. Cancer Manag Res. 2022;14:3037-46.\u003c/li\u003e\n \u003cli\u003eZeng TM, Yang G, Lou C, Wei W, Tao CJ, Chen XY, et al. Clinical and biomarker analyses of sintilimab plus gemcitabine and cisplatin as first-line treatment for patients with advanced biliary tract cancer. Nat Commun. 2023;14(1):1340.\u003c/li\u003e\n \u003cli\u003eMpekris F, Voutouri C, Baish JW, Duda DG, Munn LL, Stylianopoulos T, et al. Combining microenvironment normalization strategies to improve cancer immunotherapy. Proc Natl Acad Sci U S A. 2020;117(7):3728-37.\u003c/li\u003e\n \u003cli\u003eHuang Y, Yuan J, Righi E, Kamoun WS, Ancukiewicz M, Nezivar J, et al. Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Proc Natl Acad Sci U S A. 2012;109(43):17561-6.\u003c/li\u003e\n \u003cli\u003eLi Q, Wang Y, Jia W, Deng H, Li G, Deng W, et al. Low-Dose Anti-Angiogenic Therapy Sensitizes Breast Cancer to PD-1 Blockade. Clin Cancer Res. 2020;26(7):1712-24.\u003c/li\u003e\n \u003cli\u003eHuang Y, Stylianopoulos T, Duda DG, Fukumura D, Jain RK. Benefits of vascular normalization are dose and time dependent--letter. Cancer Res. 2013;73(23):7144-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biliary tract cancer, Phase 2 trial, Sintilimab, Anlotinib, Progression-free survival","lastPublishedDoi":"10.21203/rs.3.rs-4557891/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4557891/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThe prognosis of biliary tract cancer (BTC) is poor, with limited efficacy of first-line chemotherapy. SAGC is a randomized, controlled, phase 2 trial evaluating the efficacy of sintilimab (an anti-PD-1 inhibitor) and anlotinib (an anti-angiogenic VEGF-targeted agent) combined with standard chemotherapy as a first-line treatment in advanced BTC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eEighty eligible patients with unresectable, locally advanced, or metastatic BTC were randomized 1:1 to SAGC group (\u003cem\u003en\u003c/em\u003e = 40) to receive sintilimab (200 mg) and anlotinib (initial 10 mg, then adjusted for 8 mg on days 1-14) plus GC (gemcitabine 1,000 mg/m\u003csup\u003e2\u003c/sup\u003e and cisplatin 25 mg/m\u003csup\u003e2\u003c/sup\u003e on days 1 and 8) every 3 weeks for up to 8 cycles, followed by sintilimab and anlotinib until disease progression or unacceptable toxicity or to GC group (\u003cem\u003en\u003c/em\u003e = 40), respectively. The primary endpoint was progression-free survival (PFS). The secondary endpoints included the objective response rate (ORR), overall survival (OS), and safety. The AKT/YAP-induced tumor-bearing mice model was established to study effect of anlotinib on the tumor immune microenvironment at varying doses (low-dose: 3 mg/kg, high-dose: 6 mg/kg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe median follow-up was 13.4 months, and 77 of the 80 patients (96.3%) discontinued treatment. The median PFS was 8.5 months (SAGC group) and 6.2 months (GC group) (hazard ratio: 0.47 [95% CI, 0.22–0.64], \u003cem\u003eP\u003c/em\u003e = 0.003). The ORR for the SAGC and GC groups were 51.4% and 29.4%, respectively. Overall, grade 3/4 treatment-related adverse events occurred in 75.0% (30/40) and 43.6% (17/39) of cases in the SAGC and GC groups, respectively. A post hoc analysis shown that patients in SAGC group who received 8mg (22 patients) of anlotinib daily had a higher ORR (54.5% vs. 38.8%) compared to those received 10mg (18 patients), and there was a trend towards an OS benefit (HR: 0.49 [95% CI, 0.14–1.18], \u003cem\u003eP\u003c/em\u003e = 0.055). In vivo, the combination of low-dose anlotinib with anti-PD-1 resulted in heightened vascular pericyte coverage, improved vascular perfusion, enhanced cytotoxicity of activated T cells, and increased secretion of effector cytokines when compared to high-dose anlotinib.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Sintilimab and anlotinib in addition to gemcitabine plus cisplatin treatment in patients with advanced BTC significantly improved PFS and had a manageable safety profile, and the survival benefit of anlotinib 8mg group is more superior. Low‐dose anlotinib plus anti–PD-1 immune therapy may synergistically improve the antitumor response with reducing adverse effects in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration number ClinicalTrials.gov Identifier:\u003c/strong\u003e NCT04300959.\u003c/p\u003e","manuscriptTitle":"Phase Ⅱ Study of Combined Sintilimab and Anlotinib with Gemcitabine plus Cisplatin in Advanced Biliary Tract Cancer: Efficacy, Safety and Optimize Dose","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-19 18:53:28","doi":"10.21203/rs.3.rs-4557891/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"69eeaa97-57ec-4a70-b559-bd6c297f086e","owner":[],"postedDate":"July 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34750320,"name":"Health sciences/Diseases/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":34750321,"name":"Health sciences/Diseases/Cancer/Gastrointestinal cancer/Biliary tract cancer/Bile duct cancer"}],"tags":[],"updatedAt":"2025-07-02T07:43:23+00:00","versionOfRecord":{"articleIdentity":"rs-4557891","link":"https://doi.org/10.1038/s41467-025-60119-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-01 04:00:00","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-07-19 18:53:28","video":"","vorDoi":"10.1038/s41467-025-60119-3","vorDoiUrl":"https://doi.org/10.1038/s41467-025-60119-3","workflowStages":[]},"version":"v1","identity":"rs-4557891","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4557891","identity":"rs-4557891","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}