Tumour Mutation Burden Does Not Predict Immune Cell Frequency or Risk of Recurrence After Surgery in Hepatocellular Carcinoma

Introduction/Objectives: Understanding the genetic and immune heterogeneity in hepatocellular carcinoma (HCC) is critical for accurate pathological characterisation and treatment planning, as therapies advance towards personalisation. Tumour mutational burden (TMB) has been proposed as a biomarker of response to immune checkpoint blockade but its relationship with immune infiltration in HCC remains unclear, particularly across different aetiologies and within multifocal disease. Methods: This study characterises both the inter- and intra-patient heterogeneity in HCC, integrating whole-exome sequencing with detailed histopathologic and immune cell profiling, to identify implications for diagnosis and response to treatment. Samples of 54 tumours from 26 patients undergoing surgery at the Leeds Teaching Hospital NHS Trust were examined, including 10 patients with multifocal disease. The cohort includes the major UK aetiological groups. Whole-exome sequencing of tumours and matched background liver identified somatic variants. Histopathological and immunohistochemical evaluations were performed to assess tumour features and characterise the tumour immune microenvironment. Results: Marked inter-individual heterogeneity was observed across aetiological groups with variation in tumour mutation burden, genes containing somatic mutations and immune infiltration. Multifocal tumours frequently demonstrated discordant genetic and immune profiles irrespective of clonal relatedness. NeoTMB was not associated with recurrence following surgery, whereas recurrent cases demonstrated significantly higher intratumoural FoxP3+ T-cell frequencies. Conclusions: This study demonstrates substantial genetic and immune heterogeneity in HCC, including marked discordance between multifocal tumours within the same patient. Tumour mutation burden was not related to immune cell infiltration or risk of recurrence after surgery, whereas elevated intratumoural FoxP3+ regulatory T-cell frequencies were associated with recurrence. These findings suggest that the immunological composition of the tumour microenvironment may be more relevant to clinical outcome than mutational burden alone and highlight the importance of comprehensive tumour profiling to inform personalised therapeutic strategies.