Study Design
This prospective, single-arm phase II investigator-initiated study evaluated the efficacy and safety of a PD-1 inhibitor (toripalimab) in combination with anlotinib. Patients who were pathologically diagnosed with HCC between January 1, 2019, and April 1, 2022, and who experienced disease progression or drug intolerance after first-line targeted therapy were included in this study. All patients provided written informed consent, and the protocol was approved by the Clinical Research Ethics Committee of the Eastern Hepatobiliary Surgical Hospital (Ethics No. EHBHKY2018-02-023) following the Good Clinical Practice (GCP) guidelines, the Declaration of Helsinki, and applicable local laws and regulations.
The primary endpoint of this study was the objective response rate (ORR), as assessed by the investigators based on RECIST v1.1. Secondary endpoints were progression-free survival (PFS), overall survival (OS), and disease control rates (DCR). The exploration endpoints were biomarkers and safety.
Patient Selection
The inclusion criteria were as follows: 1) histological or cytological diagnosis of HCC; 2) presence of at least one measurable evaluated lesion according to the RECIST 1.1; 3) age 18 to 75 years and ECOG score of 0–2. 4) disease progression according to RECIST 1.1 or intolerance after first-line targeted therapy; 5) Child-Pugh A or B; 6) normal bone marrow function: neutrophils > 1.5x 109/L or platelet > 75 x109/L; 7) adequate renal reserve: creatinine < 130 µmol/L; 8) absence of cardiac insufficiency, chest pain (medically uncontrollable), or myocardial infarction within 12 months before study initiation; and 9) estimated survival time ≥ 3 months.
The exclusion criteria were as follows: 1) previous use of PD-1 inhibitor or anlotinib; 2) secondary malignancies or other neoplasms (except superficial skin cancer and localized low-grade malignancies) occurring in the 3 years before study initiation; 3) presence of brain or meningeal metastasis; 4) tumor radiographically suggesting the involvement of important blood vessels or likely to cause fatal bleeding during follow-up as assessed by the investigator; 5) bleeding events at any site ≥ CTCAE grade 3, unhealed wounds, ulcers or fractures occurring 4 weeks before medication; 6) history of arteriovenous thrombosis such as cerebrovascular accident, deep vein thrombosis, and pulmonary embolism within 6 months before enrollment; 7) untreated intestinal obstruction or sub-obstruction that prevents eating or affects systemic medication delivery; 8) inflammatory infections during the active period of infection or presence of disabilities requiring planned treatment; 9) history of uncontrolled substance abuse or mental disorders; 10) presence of concomitant diseases that, in the judgment of the investigator, may seriously endanger patient safety or interfere with study completion; 11) participation in other clinical trials; and 12) pregnancy or lactation.
Treatment Protocol and Adjustment
All enrolled patients received toripalimab: 3 mg/kg, Q2W (or 240 mg, Q3W) intravenously for 1 year or until disease progression occurred or adverse reactions were impossible to tolerate. Anlotinib 10 mg orally was taken before breakfast, QD, for 2 consecutive weeks and stopped for 1 week as a 3-week treatment cycle until disease progression occurred or adverse reactions could not be tolerated. If the dose was missed and the time before the following medication was confirmed to be less than 12 h, no makeup medication was to be taken.
Adverse reactions were closely monitored during the administration of toripalimab and anlotinib. Toripalimab was administered according to the principles of immunotherapy-related adverse reactions, and the anlotinib dose was adjusted according to the adverse reactions. The drug was discontinued if the dosage was reduced to 8 mg and could not be tolerated.
DNA Extraction and Sequencing
DNA was extracted from fresh tumor and paracancerous tissues using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. DNA concentrations were determined using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). A total of 1 g of DNA was fragmented into 200 250 bp segments using a Covaris S2 instrument (Woburn, MA, USA). Sequencing libraries were prepared using the KAPA DNA Library Preparation Kit (Kapa Biosystems, Boston, MA, USA), according to the manufacturer’s protocol. In brief, the ends of the fragments were repaired before A addition, adapter ligation, amplification, and hybridization to the SeqCap EZ library. The captured DNA was recovered using Streptavidin Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) and amplified by PCR. Sequencing was performed using the Geneplus-2000 sequencing platform (GenePlus, Beijing, China).
Whole-exome Sequencing (WES) Analysis
BWA (version 0.7.12-r1039, Heng Li and Richard Durbin, Cambridge, UK) was used to align clean reads to the reference human genome (hg19). Picard (version 1.98, Broad Institute, Cambridge, MA, USA) was used to mark the PCR duplicates. Realignment and recalibration were performed using GATK version 3.4-46-gbc02625 (Broad Institute, Cambridge, MA, USA). Single nucleotide variants were identified using MuTect (version 1.1.4, Broad Institute, Cambridge, MA, USA). Small insertions and deletions (indels) were identified using GATK. Somatic copy number alterations were identified using CONTRA (v2.0.8, Jason Li et al., Melbourne, Australia). A mutation was considered a candidate somatic mutation only when it 1) was detected in at least five high-quality reads, 2) had a variant allele frequency > 0.01, 3) was absent in > 1% of the population in the 1000 Genomes Project (version phase 3) or dbSNP databases ( Single Nucleotide Polymorphism Database, version dbSNP 137); and 4) was absent from a local database of normal samples. MSI status was defined as MSI-H or microsatellite stable (MSS) using an MSI sensor (v0.2). Tumors with an MSI score of ≥ 10 were classified as MSI-H. Patients with ≥ 20 mutations/megabases (Mb) were classified as TMB-H, while those with < 20 mutations/Mb were classified as TMB-low (TMB-L).
Evaluation
CT/MRI examinations were performed every 6–8 weeks until the progression or treatment was intolerable. Routine blood, liver function, kidney function, electrolytes, coagulation function, tumor indicators, and immune-related indicators were reviewed. The ORR was assessed using RECIST v1.1 and reviewed by independent radiologists.
Statistical Analysis
Statistical analyses were performed using Prism version 8.0.2 (GraphPad). COX analysis was performed using SPSS version 26.0.0. A two-sided p-value of < 0.05 was considered statistically significant.