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Original article
Integrated multiomic analysis reveals comprehensive tumour heterogeneity and novel immunophenotypic classification in hepatocellular carcinomas
  1. Qi Zhang1,2,3,
  2. Yu Lou1,2,3,
  3. Jiaqi Yang1,2,3,
  4. Junli Wang1,2,3,
  5. Jie Feng4,
  6. Yali Zhao5,
  7. Lin Wang2,
  8. Xing Huang2,
  9. Qihan Fu2,3,6,
  10. Mao Ye1,2,3,
  11. Xiaozhen Zhang1,2,3,
  12. Yiwen Chen1,2,3,
  13. Ce Ma5,
  14. Hongbin Ge1,2,3,
  15. Jianing Wang1,2,3,
  16. Jiangchao Wu1,2,3,
  17. Tao Wei1,2,3,
  18. Qi Chen1,2,3,
  19. Junqing Wu7,
  20. Chengxuan Yu7,
  21. Yanyu Xiao7,
  22. Xinhua Feng8,
  23. Guoji Guo7,
  24. Tingbo Liang1,2,3,
  25. Xueli Bai1,2,3
  1. 1 Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
  2. 2 Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
  3. 3 The Innovation Center for the Study of Pancreatic Diseases of Zhejiang Province, Hangzhou, China
  4. 4 Beijing Neurosurgical Institute, Beijing Tiantan Hospital, Capital Medical University, Beijing, China
  5. 5 Novogene Biotechnology Inc, Beijing, China
  6. 6 Department of Medical Oncology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
  7. 7 Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, China
  8. 8 Life Sciences Institute Zhejiang University, Hangzhou, China
  1. Correspondence to Prof Tingbo Liang and Dr Xueli Bai, Department of Hepatobiliary and Pancreatic Surgery Zhejiang University School of Medicine First Affiliated Hospital, Hangzhou, China ; liangtingbo{at}, shirleybai{at}


Objective Hepatocellular carcinoma (HCC) is heterogeneous, especially in multifocal tumours, which decreases the efficacy of clinical treatments. Understanding tumour heterogeneity is critical when developing novel treatment strategies. However, a comprehensive investigation of tumour heterogeneity in HCC is lacking, and the available evidence regarding tumour heterogeneity has not led to improvements in clinical practice.

Design We harvested 42 samples from eight HCC patients and evaluated tumour heterogeneity using whole-exome sequencing, RNA sequencing, mass spectrometry-based proteomics and metabolomics, cytometry by time-of-flight, and single-cell analysis. Immunohistochemistry and quantitative polymerase chain reactions were performed to confirm the expression levels of genes. Three independent cohorts were further used to validate the findings.

Results Tumour heterogeneity is considerable with regard to the genomes, transcriptomes, proteomes, and metabolomes of lesions and tumours. The immune status of the HCC microenvironment was relatively less heterogenous. Targeting local immunity could be a suitable intervention with balanced precision and practicability. By clustering immune cells in the HCC microenvironment, we identified three distinctive HCC subtypes with immunocompetent, immunodeficient, and immunosuppressive features. We further revealed the specific metabolic features and cytokine/chemokine expression levels of the different subtypes. Determining the expression levels of CD45 and Foxp3 using immunohistochemistry facilitated the correct classification of HCC patients and the prediction of their prognosis.

Conclusion There is comprehensive intratumoral and intertumoral heterogeneity in all dimensions of HCC. Based on the results, we propose a novel immunophenotypic classification of HCCs that facilitates prognostic prediction and may support decision making with regard to the choice of therapy.

  • hepatocellular carcinoma
  • cancer immunobiology
  • immunogenetics
  • immunotherapy
  • energy metabolism

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  • QZ, YL and JY contributed equally.

  • Contributors Study concept and design: QZ, TL, XB; Acquisition of data: QZ, YL, JY, JW, QF, MY, XZ, HG, QC, JW, JW, YC, TW; Data analysis: QZ, YL, JY, CM, JF, JW, LW, XH, YZ, CY, YX; Obtained funding: QZ, TL, XB; Study supervision: GJ, TL, XB; Drafting of manuscript: QZ, YL, YZ; Critical revision of manuscript: JY, XF, GJ, TL, XB.

  • Funding This work was supported by Key Program of the National Natural Science Foundation of China (81830089), the National Natural Science Foundation of China (81871320, 81472212), the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Patient consent for publication Not required.

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