This clinical study plans to include 350 liver cancer patients from 10 tertiary hospitals nationwide, starting from August 1, 2025, at multiple centers such as the affiliated Run Run Shaw Hospital of Zhejiang University School of Medicine (the leading unit). They will be divided into a new typing queue (100 cases) and another typing queue (250 cases) using the 29 gene set algorithm. The study will collect tumor tissue samples obtained from surgical resection or puncture of patients (meeting the requirements of sample size and tumor cell proportion), perform RNA seq transcriptome sequencing, and extract patient baseline data, clinical pathological characteristics, laboratory test results, treatment information, and follow-up data from the hospital medical record system. The main objective of this study is to observe the disease progression time (TTP) and objective response rate (ORR) of patients after receiving targeted combined immunotherapy. The secondary observations include progression free survival (PFS), overall survival (OS), dynamic changes in tumor markers, liver function status, and survival after progression. The aim is to analyze the correlation between the 29 gene based new subtype of liver cancer and the efficacy of immunotherapy, providing a basis for precise diagnosis and treatment of liver cancer.

This is a multicenter cohort study aimed at exploring the correlation between a novel 29-gene-based subtype of primary hepatocellular carcinoma (HCC) and response to immunotherapy, with the goal of verifying that this new subtype exhibits higher responsiveness to immunotherapy.

Study Population: A total of 350 patients with pathologically confirmed primary HCC will be enrolled from 10 tertiary hospitals across China, with Sir Run Run Shaw Hospital, Zhejiang University School of Medicine as the leading center. Eligibility criteria include: age ≥ 18 years; having measurable tumor lesions per RECIST 1.1; receiving targeted therapy combined with immune checkpoint inhibitors (ICI) after sample collection (with ≥4 weeks interval from prior local treatments if any); availability of qualified tumor samples (≥3g frozen surgical tissues or biopsy specimens sufficient for transcriptome sequencing); and provision of informed consent. Patients will be excluded if they have overlapping local treatments (e.g., TACE, ablation, radiotherapy) during or within 4 weeks of target-ICI combination therapy, concurrent active malignancies (except cured basal cell carcinoma, etc.), severe organ dysfunction (e.g., Child-Pugh Class C liver function, NYHA Class Ⅲ-Ⅳ cardiac function), or other factors interfering with efficacy assessment as judged by investigators.

Study Procedures:

Sample Processing and Subtyping: Tumor samples will undergo RNA sequencing using the Illumina HiSeq platform (outsourced to Hangzhou DIAN Diagnostics company). After quality control, alignment, and quantification, hierarchical clustering based on a 29-gene signature will classify patients into a "novel subtype cohort" (unmatchable to TCGA HCC clusters) and an "other subtypes cohort".

Data Collection: Clinical data will be extracted from electronic medical records, including baseline characteristics (age, gender, Child-Pugh classification), treatment details (target-ICI regimens, doses, cycles), imaging evaluations (enhanced CT/MRI for tumor response assessment per mRECIST), and follow-up data (survival status, tumor marker dynamics).

Outcome Measures:

Primary Outcomes: Time to Progression (TTP, defined as the interval from initiation of target-ICI therapy to first progression per mRECIST) and Objective Response Rate (ORR, proportion of patients achieving complete response [CR, disappearance of arterial enhancement in all target lesions] or partial response [PR, ≥30% reduction in sum of target lesion diameters]). ORR will be assessed at Weeks 8, 16, 24, or progression for each line of therapy; if regimens are changed due to poor efficacy, assessment timelines will be reset for the new regimen, with CR/PR status strictly linked to the corresponding regimen.

Secondary Outcomes: Progression-Free Survival (PFS), Overall Survival (OS), dynamic changes in tumor markers (AFP, PIVKA-II), liver function status at progression, and Post-Progression Survival (PPS).

Statistical Analysis: Group differences will be compared using appropriate tests (t-test, ANOVA, Mann-Whitney U test, Chi-square test, or Fisher's exact test as applicable). Survival analyses (TTP, PFS, OS, PPS) will employ the Kaplan-Meier method with Log-rank tests, and Cox proportional hazards models will evaluate the impact of 29-gene subtyping on progression risk. A p-value < 0.05 will be considered statistically significant.

This study aims to provide evidence for precision immunotherapy in HCC by clarifying the association between the 29-gene-based subtype and immunotherapeutic efficacy.

Inclusion Criteria:

• ≥18 years old.
• Patients who visited multiple centers including Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (the leading center) and were diagnosed with primary hepatocellular carcinoma by surgical pathological histology.
• They received targeted drug combined with immune checkpoint inhibitor treatment after the specimen collection time point (post - surgery or post - puncture) (that is: local treatments such as TACE, ablation, etc. are allowed before the start of targeted - immunotherapy combination treatment, but an interval of ≥4 weeks from the targeted - immunotherapy treatment is required).
• Had measurable tumor lesions at the observation start point (according to RECIST 1.1 criteria), and the tumor response status (including complete response [CR], partial response [PR], stable disease [SD] or progressive disease [PD]) could be judged by imaging examination during the observation (such as post - treatment evaluation).
• Had accessible frozen tumor tissue samples from surgical resection (sample volume ≥3g); or tumor tissue samples obtained by puncture (puncture samples need to meet the pathological diagnosis requirements, and the sample quality can support subsequent transcriptome sequencing and analysis).
• Voluntarily participate in this study and sign the informed consent form. If the subject is unable to read and sign the informed consent form due to incapacity and other reasons, their guardian shall act as the agent for the informed process and sign the informed consent form. If the subject is unable to read the informed consent form (e.g., illiterate subject), a witness shall witness the informed process and sign the informed consent form.

Exclusion Criteria:

• Received any local treatment during the targeted - immunotherapy combination treatment (including during treatment and within 4 weeks after treatment interruption), including:

Transcatheter arterial chemoembolization (TACE), transcatheter arterial embolization (TAE), drug - eluting bead transcatheter arterial chemoembolization (DEB - TACE) and other vascular interventional therapies; Radiofrequency ablation, microwave ablation, cryoablation, laser ablation and other ablation therapies; Stereotactic body radiation therapy (SBRT), three - dimensional conformal radiation therapy, intensity - modulated radiation therapy and other local radiation therapies; Intratumoral injection therapy, high - intensity focused ultrasound (HIFU) and other local physical/chemical therapies.

• Local treatment was initiated before the start of targeted - immunotherapy combination treatment, and the local treatment continued into the targeted - immunotherapy treatment period (that is, there was an overlap between local treatment and targeted - immunotherapy treatment or the interval was < 4 weeks).
• Complicated with other active malignant tumors (except for those with cured basal cell carcinoma of the skin, carcinoma in situ of the cervix, and papillary thyroid carcinoma with no recurrence for more than 5 years after surgery).
• Severe impairment of important organ functions:

Liver function: Child - Pugh class C, or total bilirubin > 3×ULN; Renal function: Serum creatinine > 1.5×ULN and estimated glomerular filtration rate (eGFR) < 50ml/min; Cardiac function: New York Heart Association (NYHA) cardiac function class Ⅲ - Ⅳ, or left ventricular ejection fraction (LVEF) < 50%.

• Had uncontrolled active infections, including:

Hepatitis B virus DNA > 2000IU/ml (without receiving antiviral treatment); Hepatitis C virus RNA positive and without receiving antiviral treatment; Active tuberculosis infection, septicemia, etc.

• Had a history of autoimmune diseases and currently still needed to use systemic glucocorticoids (prednisone > 10mg/day) or other immunosuppressive therapies.
• Pregnant or lactating women, or patients planning to become pregnant during the study.
• Had a definite history of allergy to the targeted drugs or immune checkpoint inhibitors involved in the study.
• The researcher believed that there were other factors that might affect the judgment of treatment response (such as uncontrolled hypertension, coagulation dysfunction, simultaneous use of other antineoplastic drugs during targeted - immunotherapy treatment, etc.).

Adult patients (≥18 years) with pathologically confirmed primary hepatocellular carcinoma, with accessible tumor samples (surgical/frozen biopsy tissues ≥3g or qualified puncture specimens). They receive targeted-immunotherapy after sample collection, with measurable lesions (RECIST 1.1). Local treatments are allowed if not overlapping with target-immunotherapy (interval ≥4 weeks). Excluded: active malignancies/organ dysfunction/uncontrolled infections/autoimmune diseases/pregnancy. All provide informed consent.