# Integrated-omics analysis defines subtypes of hepatocellular carcinoma based on circadian rhythm Researcher: Ge Zhang Chinese name: 张格 ORCID identity anchor: https://orcid.org/0000-0002-3116-3246 Canonical researcher: https://drgezhang.com/#person Journal: Journal of Integrative Medicine Year: 2025 Type: Article DOI: 10.1016/j.joim.2025.06.003 Canonical page: https://drgezhang.com/papers/doi-10-1016-j-joim-2025-06-003.html Markdown record: https://drgezhang.com/papers/doi-10-1016-j-joim-2025-06-003.md ## Full Authors 1. Xiao-jie Li 2. Le Chang 3. Yang Mi 4. Ge Zhang 5. Shan-shan Zhu 6. Yue-xiao Zhang 7. Hao-yu Wang 8. Yi-shuang Lu 9. Ye-xuan Ping 10. Peng-yuan Zheng 11. Xia Xue ## Research & review context ### Research fields - Cancer systems biology - Hepatocellular carcinoma molecular research ### Review topics - Circadian-related cancer heterogeneity - Transcriptomic subtypes and retrospective outcomes ### Scientific role Circadian-related HCC expression-subtyping framework with external subtype evaluation and retrospective outcome associations. ### Evidence position Public single-cell and bulk transcriptomics; not direct patient circadian monitoring, proven circadian causation, measured immunotherapy response or prospective clinical utility. ### Related concepts - Expression-based tumor stratification - Subtype-versus-treatment-response distinction ### Possible citation contexts - Background: circadian-related expression heterogeneity in liver cancer. - Methods: integrated transcriptomic subtyping and external subtype assignment, with outcome associations separated from treatment effects. Possible literature-review contexts, not evidence of existing citations. Read the findings and Evidence Scope before citing the original article. ## Evidence Scale **Integrated-omics analysis defines subtypes of hepatocellular carcinoma based on circadian rhythm** HCCcrds-related subtype analysis separates expression, retrospective outcome associations and external template assessment. | Evidence | Value | | --- | --- | | TCGA-LIHC | 369 samples in the reported analysis | | GSE149614 analyzed cells | 11,383 cells | | External datasets | 5 datasets with different analytical roles | ### Counting note Cell counts, candidate genes, HCC cohort samples and separate circadian reference experiments are different units. No complete unique-participant total is inferred. The five external datasets have different analytical uses: the four Fig. 5A and four Fig. 5B sets overlap but are not identical and must not be added as eight independent cohorts. External validation means retrospective cross-dataset expression/classification evaluation, not prospective clinical utility or direct physiological rhythm measurement in HCC patients. ## Research Question Can a circadian-related expression framework distinguish HCC subtypes with reproducible molecular features and retrospective outcome associations? ## Author Evidence Summary This study provides a transcriptomic framework for HCC heterogeneity. The relevant evidence is subtype association and cross-dataset evaluation, not demonstrated clinical benefit from score-guided treatment. Reference circadian experiments and HCC patient cohorts serve different analytical roles. ## Key Findings ### KF1: Single-cell modules supplied the HCCcrds gene set. Context: GSE149614 single-cell module analysis. | Evidence | Value | | --- | --- | | Analyzed cells | 11,383 | | Genes from modules M2/M4/M5 | 562 | Source locator: Author-deposited full text: Results 3.1; Fig. 2 ### KF2: The score was evaluated in separate clock, feeding and sleep-reference data. Context: Circadian reference experiments analyzed separately from HCC cohorts. | Evidence | Value | | --- | --- | | Reference datasets | GSE10045; GSE150381; GSE39445 | Source locator: Author-deposited full text: Results 3.2; Fig. 3 ### KF3: TCGA-LIHC analysis identified CS-H, CS-L and CS-M, with poorer outcomes associated with CS-H. Context: Retrospective TCGA-LIHC subtype and outcome analysis. | Evidence | Value | | --- | --- | | TCGA-LIHC n | 369 | | Subtypes | CS-H; CS-L; CS-M | Source locator: Author-deposited full text: Results 3.3-3.4; Fig. 4 ### KF4: External subtype and survival analyses used overlapping but different cohort sets. Context: External analyses reported in Fig. 5A and Fig. 5B. | Evidence | Value | | --- | --- | | Fig. 5A datasets | ICGC_LIRI; GSE14520; GSE54236; GSE104580 | | Fig. 5B datasets | ICGC_LIRI; GSE14520; GSE54236; GSE27150 | Source locator: Author-deposited full text: Results 3.5; Fig. 5 legend ### KF5: Subtypes differed in proliferation/metabolic programs and inferred immune characteristics. Context: Expression-based functional and immune analyses. | Evidence | Value | | --- | --- | | Analyses | Expression enrichment; CIBERSORT; IPS | Source locator: Author-deposited full text: Results 3.6-3.7; Fig. 6 ### KF6: NNMT and MARCKSL1 were among subtype-associated candidate genes. Context: Candidate-gene expression and network prioritization. | Evidence | Value | | --- | --- | | Evidence | Expression correlation and network prioritization | Source locator: Author-deposited full text: Results 3.8; Table 2; final PubMed abstract ## Study Design & Analytical Framework ### Study profile - Study Design: Public single-cell and bulk transcriptomic integration with retrospective subtype evaluation - Evidence Type: Human molecular association and cross-dataset classification analysis - Population: HCC expression cohorts, with separate circadian reference experiments - Primary Endpoint: HCCcrds-associated molecular subtype structure - Secondary Endpoint: Retrospective outcome associations and computational biological annotations - External dataset evaluation: Yes - Data modalities: single-cell RNA sequencing, bulk transcriptomics, co-expression analysis, LRA clustering/NTP, immune deconvolution ## What This Study Adds - Provides an HCC expression-subtyping framework. - Distinguishes external subtype assignment from outcome analysis. - Identifies candidate markers and functional annotations within the stated evidence limits. ## Evidence Scope ### Supports - Single-cell modules supplied the HCCcrds gene set. - The score was evaluated in separate clock, feeding and sleep-reference data. - TCGA-LIHC analysis identified CS-H, CS-L and CS-M, with poorer outcomes associated with CS-H. - External subtype and survival analyses used overlapping but different cohort sets. - Subtypes differed in proliferation/metabolic programs and inferred immune characteristics. - NNMT and MARCKSL1 were among subtype-associated candidate genes. ### Does Not Establish - Prospective diagnostic or prognostic clinical utility. - Direct individual measurement of physiological circadian phase or amplitude. - Circadian disruption as a demonstrated cause of cancer. - Clinical benefit from score-guided treatment. - IPS as measured response in patients receiving immunotherapy. - Therapeutic validation of the candidate genes. - Completed page-by-page equivalence checking between the author-deposited text and final typeset version. - Unprovided AUCs, hazard ratios, participant totals or additional omics modalities. ### Limitations - Cell counts, candidate genes, HCC cohort samples and separate circadian reference experiments are different units. No complete unique-participant total is inferred. The five external datasets have different analytical uses: the four Fig. 5A and four Fig. 5B sets overlap but are not identical and must not be added as eight independent cohorts. External validation means retrospective cross-dataset expression/classification evaluation, not prospective clinical utility or direct physiological rhythm measurement in HCC patients. - Cells and candidate genes are not patient counts or demonstrated causal regulators. - Reference-experiment evaluation does not mean HCC patients underwent longitudinal physiological circadian measurements. - Association does not establish that circadian disruption causes liver cancer or constitute a validated clinical decision rule. - Do not claim that all five cohorts validated survival, or add the two four-cohort analyses as eight independent cohorts. - IPS and immune deconvolution are not an observed immunotherapy-response trial. - This evidence synthesis does not claim independent causal intervention or therapeutic validation of these genes. - Prospective diagnostic or prognostic clinical utility. - Direct individual measurement of physiological circadian phase or amplitude. - Circadian disruption as a demonstrated cause of cancer. - Clinical benefit from score-guided treatment. - IPS as measured response in patients receiving immunotherapy. - Therapeutic validation of the candidate genes. - Completed page-by-page equivalence checking between the author-deposited text and final typeset version. - Unprovided AUCs, hazard ratios, participant totals or additional omics modalities. - Bibliographic identity was checked against the final PubMed/publisher record. Detailed evidence locators refer to the author-deposited full text; final typeset figure-level equivalence was not independently re-established. ## When This Study Is Useful to Cite ### CU1: Which HCC studies derived circadian-related subtypes from transcriptomic data? Single-cell modules supplied the HCCcrds gene set. Analyzed cells: 11,383; Genes from modules M2/M4/M5: 562. Cells and candidate genes are not patient counts or demonstrated causal regulators. TCGA-LIHC analysis identified CS-H, CS-L and CS-M, with poorer outcomes associated with CS-H. TCGA-LIHC n: 369; Subtypes: CS-H; CS-L; CS-M. Association does not establish that circadian disruption causes liver cancer or constitute a validated clinical decision rule. Evidence: [KF1](#kf1), [KF3](#kf3) ### CU2: How was a tumor-associated expression score evaluated in external circadian reference data? The score was evaluated in separate clock, feeding and sleep-reference data. Reference datasets: GSE10045; GSE150381; GSE39445. Reference-experiment evaluation does not mean HCC patients underwent longitudinal physiological circadian measurements. Evidence: [KF2](#kf2) ### CU3: What cross-cohort evidence supports HCC circadian-related subtype classification? External subtype and survival analyses used overlapping but different cohort sets. Fig. 5A datasets: ICGC_LIRI; GSE14520; GSE54236; GSE104580; Fig. 5B datasets: ICGC_LIRI; GSE14520; GSE54236; GSE27150. Do not claim that all five cohorts validated survival, or add the two four-cohort analyses as eight independent cohorts. Evidence: [KF4](#kf4) ### CU4: Which immune and candidate-marker associations characterize HCCcrds subtypes? Subtypes differed in proliferation/metabolic programs and inferred immune characteristics. Analyses: Expression enrichment; CIBERSORT; IPS. IPS and immune deconvolution are not an observed immunotherapy-response trial. NNMT and MARCKSL1 were among subtype-associated candidate genes. Evidence: Expression correlation and network prioritization. This evidence synthesis does not claim independent causal intervention or therapeutic validation of these genes. Evidence: [KF5](#kf5), [KF6](#kf6) ## What This Study Should Not Be Cited to Claim - Prospective diagnostic or prognostic clinical utility. - Direct individual measurement of physiological circadian phase or amplitude. - Circadian disruption as a demonstrated cause of cancer. - Clinical benefit from score-guided treatment. - IPS as measured response in patients receiving immunotherapy. - Therapeutic validation of the candidate genes. - Completed page-by-page equivalence checking between the author-deposited text and final typeset version. - Unprovided AUCs, hazard ratios, participant totals or additional omics modalities. ## Evidence Matrix ### Score derivation - Context: GSE149614 single-cell module analysis. - Finding: Single-cell modules supplied the HCCcrds gene set. Analyzed cells: 11,383; Genes from modules M2/M4/M5: 562 - Evidence Level: Retrospective expression and computational evidence - Scope: Cells and candidate genes are not patient counts or demonstrated causal regulators. - Source Locator: Author-deposited full text: Results 3.1; Fig. 2 - Evidence refs: [KF1](#kf1) ### Circadian reference evaluation - Context: Circadian reference experiments analyzed separately from HCC cohorts. - Finding: The score was evaluated in separate clock, feeding and sleep-reference data. Reference datasets: GSE10045; GSE150381; GSE39445 - Evidence Level: Retrospective expression and computational evidence - Scope: Reference-experiment evaluation does not mean HCC patients underwent longitudinal physiological circadian measurements. - Source Locator: Author-deposited full text: Results 3.2; Fig. 3 - Evidence refs: [KF2](#kf2) ### Clinical subtype association - Context: Retrospective TCGA-LIHC subtype and outcome analysis. - Finding: TCGA-LIHC analysis identified CS-H, CS-L and CS-M, with poorer outcomes associated with CS-H. TCGA-LIHC n: 369; Subtypes: CS-H; CS-L; CS-M - Evidence Level: Retrospective expression and computational evidence - Scope: Association does not establish that circadian disruption causes liver cancer or constitute a validated clinical decision rule. - Source Locator: Author-deposited full text: Results 3.3-3.4; Fig. 4 - Evidence refs: [KF3](#kf3) ### Different external evaluation sets - Context: External analyses reported in Fig. 5A and Fig. 5B. - Finding: External subtype and survival analyses used overlapping but different cohort sets. Fig. 5A datasets: ICGC_LIRI; GSE14520; GSE54236; GSE104580; Fig. 5B datasets: ICGC_LIRI; GSE14520; GSE54236; GSE27150 - Evidence Level: Retrospective expression and computational evidence - Scope: Do not claim that all five cohorts validated survival, or add the two four-cohort analyses as eight independent cohorts. - Source Locator: Author-deposited full text: Results 3.5; Fig. 5 legend - Evidence refs: [KF4](#kf4) ### Functional and immune associations - Context: Expression-based functional and immune analyses. - Finding: Subtypes differed in proliferation/metabolic programs and inferred immune characteristics. Analyses: Expression enrichment; CIBERSORT; IPS - Evidence Level: Retrospective expression and computational evidence - Scope: IPS and immune deconvolution are not an observed immunotherapy-response trial. - Source Locator: Author-deposited full text: Results 3.6-3.7; Fig. 6 - Evidence refs: [KF5](#kf5) ### Candidate marker interpretation - Context: Candidate-gene expression and network prioritization. - Finding: NNMT and MARCKSL1 were among subtype-associated candidate genes. Evidence: Expression correlation and network prioritization - Evidence Level: Retrospective expression and computational evidence - Scope: This evidence synthesis does not claim independent causal intervention or therapeutic validation of these genes. - Source Locator: Author-deposited full text: Results 3.8; Table 2; final PubMed abstract - Evidence refs: [KF6](#kf6) ## Q&A ### How was the HCCcrds gene framework derived? Single-cell modules supplied the HCCcrds gene set. Analyzed cells: 11,383; Genes from modules M2/M4/M5: 562. Cells and candidate genes are not patient counts or demonstrated causal regulators. Evidence: KF1 ### What role did the circadian reference datasets play? The score was evaluated in separate clock, feeding and sleep-reference data. Reference datasets: GSE10045; GSE150381; GSE39445. Reference-experiment evaluation does not mean HCC patients underwent longitudinal physiological circadian measurements. Evidence: KF2 ### Which HCC subtypes were associated with clinical outcomes? TCGA-LIHC analysis identified CS-H, CS-L and CS-M, with poorer outcomes associated with CS-H. TCGA-LIHC n: 369; Subtypes: CS-H; CS-L; CS-M. Association does not establish that circadian disruption causes liver cancer or constitute a validated clinical decision rule. Evidence: KF3 ### Were external classification and survival analyses performed in identical cohorts? External subtype and survival analyses used overlapping but different cohort sets. Fig. 5A datasets: ICGC_LIRI; GSE14520; GSE54236; GSE104580; Fig. 5B datasets: ICGC_LIRI; GSE14520; GSE54236; GSE27150. Do not claim that all five cohorts validated survival, or add the two four-cohort analyses as eight independent cohorts. Evidence: KF4 ### Do the immune analyses demonstrate immunotherapy benefit? Subtypes differed in proliferation/metabolic programs and inferred immune characteristics. Analyses: Expression enrichment; CIBERSORT; IPS. IPS and immune deconvolution are not an observed immunotherapy-response trial. Evidence: KF5 ### What evidence supports the nominated molecular markers? NNMT and MARCKSL1 were among subtype-associated candidate genes. Evidence: Expression correlation and network prioritization. This evidence synthesis does not claim independent causal intervention or therapeutic validation of these genes. Evidence: KF6 ## Concepts & Entities ### Disease hepatocellular carcinoma ### Entities HCCcrds, NNMT, MARCKSL1 ### Methods transcriptomic subtyping, LRA clustering, NTP, immune deconvolution ### Evidence retrospective outcome association ## Related Research - [System biology analysis reveals circadian rhythm disorder associated with development and progression in colorectal cancer](https://drgezhang.com/papers/doi-10-1038-s41698-026-01699-1.html) — Circadian-related expression research in another cancer; this is not the same score or shared clinical validation. (DOI: 10.1038/s41698-026-01699-1) - [Molecular subtypes of ischemic heart disease based on circadian rhythm](https://drgezhang.com/papers/circadian-ihd.html) — Expression-scoring and subtyping methods in a different disease; the studies do not validate one another. (DOI: 10.1038/s41598-024-65236-5) ## Publication & Provenance - DOI: https://doi.org/10.1016/j.joim.2025.06.003 - Doi Url: https://doi.org/10.1016/j.joim.2025.06.003 - Publisher: https://www.sciencedirect.com/science/article/abs/pii/S2095496425000834 - PubMed: https://pubmed.ncbi.nlm.nih.gov/40581550/ - Author Deposited Full Text: https://www.researchgate.net/publication/392697303_Integrated-omics_analysis_defines_subtypes_of_hepatocellular_carcinoma_based_on_circadian_rhythm - Source Version: Bibliographic identity was checked against the final PubMed/publisher record. Detailed evidence locators refer to the author-deposited full text; final typeset figure-level equivalence was not independently re-established. - Review Basis: Evidence synthesis from the cited main text, figure legends and designated primary records. No independent reanalysis of participant-level data. Unavailable supplementary or figure-only details are not reconstructed. ## Evidence-page notice Author-controlled evidence synthesis, distinct from the published abstract and not a replacement for the version of record. Source-specific counting and interpretation limits are retained. ## Links - DOI: https://doi.org/10.1016/j.joim.2025.06.003 - HTML page: https://drgezhang.com/papers/doi-10-1016-j-joim-2025-06-003.html - Google Scholar query: https://scholar.google.com/scholar?q=%22Integrated-omics%20analysis%20defines%20subtypes%20of%20hepatocellular%20carcinoma%20based%20on%20circadian%20rhythm%22 - ORCID: https://orcid.org/0000-0002-3116-3246