Dear Editor,
We commend Kim et al. for their comprehensive profiling of telomerase reverse transcriptase (TERT) alterations and hepatitis B virus (HBV) integration patterns in HBV-related hepatocellular carcinoma, with particular attention to sexspecific differences among younger individuals [
1]. However, several methodological assumptions embedded in the study design may limit the interpretation of sex disparity as a primary biological phenomenon. Below, we highlight three core areas warranting deeper methodological scrutiny and suggest strategies for future refinement.
The authors report higher HBV-TERT integration frequency and read counts in younger male tumors, interpreting this as evidence of male-biased genomic susceptibility. Yet, no adjustment was made for tumor purity, necrosis, or immune infiltration, which can differ substantially by sex and influence the detectability of HBV-host chimeric reads. Prior studies have shown that immune surveillance exerts sex-differential pressure on viral persistence and clonal architecture in chronic HBV infection [
2]. Without correcting for these confounders using tumor microenvironment deconvolution tools or integration site clonality metrics, the apparent male predominance may be conflated with technical enrichment or immunological bottlenecks, rather than intrinsic integration bias. This raises the need for quantitative modeling of HBV integration clonality across immune phenotypes, stratified by sex.
Although the authors distinguish between HBV-TERT integration and TERT promoter mutation in some analyses, their core conclusion is built on a composite variable (“TERT alteration”). This simplification disregards evidence that these alterations are not only mutually exclusive, but may occur at distinct stages of tumor evolution. For instance, HBV integration is typically an early, often pre-malignant event, while TERT promoter mutations may emerge under later selective pressure during telomere crisis [
3,
4]. Collapsing these two into a single category risks misinterpreting age or sex effects—for example, younger males may simply harbor more HBV-TERT early events, while older females may accumulate TERT mutations later under telomere erosion. Without a phylogenetic framework or lineage tracing (e.g., from matched cirrhotic nodules), it is methodologically unsound to infer shared risk pathways or draw conclusions about hormonal regulation of “TERT alterations” as a unified group.
The study highlights preferential HBV integration into the TERT locus, promoters, and CpG islands in males. However, the analytical framework presumes that these sites are equally accessible across sexes. This assumption is problematic, given that chromatin accessibility, nucleosome positioning, and transcription factor occupancy at the TERT locus are regulated by sex hormones [
5], including androgendriven chromatin remodeling via HNF4α or GABP recruitment [
6]. Without profiling epigenomic landscapes (e.g., ATAC-seq, ChIP-seq for AR/ER/HNF4α), one cannot determine whether the observed male-enriched integration sites reflect stochastic viral insertion or preferential selection into pre-opened chromatin domains. This has major implications: if sex-specific chromatin accessibility governs integration bias, the mechanism is not about HBV insertional randomness, but about the co-option of host transcriptional machinery in a hormone-dependent fashion. Future work could integrate chromatin state maps from male and female HBV-infected livers to test this hypothesis directly.
In sum, while the study by Kim et al. offers valuable observations, its interpretation of male-biased TERT alterations as a biological predisposition would be significantly strengthened by (1) controlling for tumor-intrinsic and microenvironmental variability, (2) temporally resolving mutation versus integration events, and (3) directly probing sexspecific epigenomic landscapes that may shape integration site preference. By critically revisiting these foundational assumptions, future work may uncover deeper, mechanistically grounded explanations for sex disparities in HBV-related hepatocarcinogenesis.
FOOTNOTES
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Authors’ contributions
Qicong Mai and Jianming Zheng wrote the manuscript, Meishi Tang and Yubin Liu provided methodological and revised the manuscript.
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Acknowledgements
Henan Sunshine Healthcare Development Fund Project (HKP20250011). Beijing Medical and Health Public Welfare Foundation Medical Science Research Fund Project (YWJKJJHKYJJ-ZH25002).
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Conflicts of Interest
The authors have no conflicts to disclose.
Abbreviations
telomerase reverse transcriptase
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