Korean J Gastroenterol.  2023 Jul;82(1):1-9. 10.4166/kjg.2023.076.

Current Status and Prospects of Liquid Biopsy for Hepatocellular Carcinoma

• Kang J
• Kim SS

• KMID: 2544933
• DOI: http://doi.org/10.4166/kjg.2023.076

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths globally. Early detection and treatment response monitoring of HCC are vital for improved outcomes. Traditional diagnostic methods rely on imaging, serum tumor markers, and invasive liver tissue biopsy. The advent of liquid biopsy has revolutionized cancer diagnostics and management. Liquid biopsy involves the detection and analysis of tumor-derived components, such as circulating tumor DNA, circulating tumor cells, and extracellular vesicles, in various body fluids. These components reflect tumor characteristics, genetic alterations, and functional changes, providing valuable information for HCC diagnosis and treatment response monitoring. Liquid biopsy offers several advantages, including its non-invasive nature, potential for repetitive sampling, and real-time monitoring of disease progression and treatment response. However, challenges remain, including the sensitivity of detection methods, and standardization. In this review, we discuss the methods, current status, and prospects of liquid biopsy for HCC, highlighting its potential as a valuable tool in HCC management.

Keyword

Liquid biopsies; Carcinoma, hepatocellular; DNA, circulating tumor; Neoplastic cells, circulating; Extracellular vesicles

Reference

1. Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71:209–249. DOI: 10.3322/caac.21660. PMID: 33538338.

2. Rumgay H, Ferlay J, de Martel C, et al. 2022; Global, regional and national burden of primary liver cancer by subtype. Eur J Cancer. 161:108–118. DOI: 10.1016/j.ejca.2021.11.023. PMID: 34942552.

3. Renzulli M, Pecorelli A, Brandi N, et al. 2022; The feasibility of liver biopsy for undefined nodules in patients under surveillance for hepatocellular carcinoma: Is biopsy really a useful tool? J Clin Med. 11:4399. DOI: 10.3390/jcm11154399. PMID: 35956016. PMCID: PMC9369413.

4. Siravegna G, Marsoni S, Siena S, Bardelli A. 2017; Integrating liquid biopsies into the management of cancer. Nat Rev Clin Oncol. 14:531–548. DOI: 10.1038/nrclinonc.2017.14. PMID: 28252003.

5. Pantel K, Alix-Panabières C. Liquid biopsy in 2016: Circulating tumour cells and cell-free DNA in gastrointestinal cancer. Nat Rev Gastroenterol Hepatol. 2017; 14:73–74. DOI: 10.1038/nrgastro.2016.198. PMID: 28096542.

6. Mody K, Kasi PM, Yang JD, et al. 2019; Feasibility of circulating tumor DNA testing in hepatocellular carcinoma. J Gastrointest Oncol. 10:745–750. DOI: 10.21037/jgo.2019.02.10. PMID: 31392055. PMCID: PMC6657310.

7. van de Stolpe A, Pantel K, Sleijfer S, Terstappen LW, den Toonder JM. 2011; Circulating tumor cell isolation and diagnostics: toward routine clinical use. Cancer Res. 71:5955–5960. DOI: 10.1158/0008-5472.CAN-11-1254. PMID: 21896640.

8. Wu P, Zhang B, Ocansey DKW, Xu W, Qian H. 2021; Extracellular vesicles: A bright star of nanomedicine. Biomaterials. 269:120467. DOI: 10.1016/j.biomaterials.2020.120467. PMID: 33189359.

9. Taft RJ, Pang KC, Mercer TR, Dinger M, Mattick JS. 2010; Non-coding RNAs: regulators of disease. J Pathol. 220:126–139. DOI: 10.1002/path.2638. PMID: 19882673.

10. Roweth HG, Battinelli EM. 2021; Lessons to learn from tumor-educated platelets. Blood. 137:3174–3180. DOI: 10.1182/blood.2019003976. PMID: 33940594. PMCID: PMC8351883.

11. Xu RH, Wei W, Krawczyk M, et al. 2017; Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nat Mater. 16:1155–1161. DOI: 10.1038/nmat4997. PMID: 29035356.

12. Ye Q, Ling S, Zheng S, Xu X. 2019; Liquid biopsy in hepatocellular carcinoma: circulating tumor cells and circulating tumor DNA. Mol Cancer. 18:114. DOI: 10.1186/s12943-019-1043-x. PMID: 31269959. PMCID: PMC6607541.

13. Gao Y, Zhao H, An K, et al. 2022; Whole-genome bisulfite sequencing analysis of circulating tumour DNA for the detection and molecular classification of cancer. Clin Transl Med. 12:e1014. DOI: 10.1002/ctm2.1014.

14. Browne CD, Mattmann ME, Wycoco MJ, et al. Abstract 2758: Comparison of cell-free DNA blood collection tubes. Cancer Res. 2017; 77(13 Suppl):2758. DOI: 10.1158/1538-7445.AM2017-2758.

15. Pantel K, Alix-Panabieres C. 2013; Real-time liquid biopsy in cancer patients: fact or fiction? Cancer Res. 73:6384–6388. DOI: 10.1158/0008-5472.CAN-13-2030. PMID: 24145355.

16. Diehl F, Schmidt K, Choti MA, et al. 2008; Circulating mutant DNA to assess tumor dynamics. Nat Med. 14:985–990. DOI: 10.1038/nm.1789. PMID: 18670422. PMCID: PMC2820391.

17. Chan KC, Jiang P, Zheng YW, et al. 2013; Cancer genome scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing. Clin Chem. 59:211–224. DOI: 10.1373/clinchem.2012.196014. PMID: 23065472.

18. Newman AM, Bratman SV, To J, et al. 2014; An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med. 20:548–554. DOI: 10.1038/nm.3519. PMID: 24705333. PMCID: PMC4016134.

19. Taly V, Pekin D, Benhaim L, et al. 2013; Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin Chem. 59:1722–1731. DOI: 10.1373/clinchem.2013.206359. PMID: 23938455.

20. Kinde I, Wu J, Papadopoulos N, Kinzler KW, Vogelstein B. 2011; Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci U S A. 108:9530–9535. DOI: 10.1073/pnas.1105422108. PMID: 21586637. PMCID: PMC3111315.

21. Yan L, Chen Y, Zhou J, Zhao H, Zhang H, Wang G. 2018; Diagnostic value of circulating cell-free DNA levels for hepatocellular carcinoma. Int J Infect Dis. 67:92–97. DOI: 10.1016/j.ijid.2017.12.002. PMID: 29229500.

22. Kisiel JB, Dukek BA, R VSRK, et al. 2019; Hepatocellular carcinoma detection by plasma methylated DNA: Discovery, phase I pilot, and phase II clinical validation. Hepatology. 69:1180–1192. DOI: 10.1002/hep.30244. PMID: 30168613. PMCID: PMC6429916.

23. Xu W, Lu J, Zhao Q, et al. 2019; Genome-wide plasma cell-free DNA methylation profiling identifies potential biomarkers for lung cancer. Dis Markers. 2019:4108474. DOI: 10.1155/2019/4108474. PMID: 30867848. PMCID: PMC6379867.

24. Chalasani NP, Porter K, Bhattacharya A, et al. 2022; Validation of a novel multitarget blood test shows high sensitivity to detect early stage hepatocellular carcinoma. Clin Gastroenterol Hepatol. 20:173–182.e7. DOI: 10.1016/j.cgh.2021.08.010. PMID: 34391922.

25. Ono A, Fujimoto A, Yamamoto Y, et al. 2015; Circulating tumor DNA analysis for liver cancers and its usefulness as a liquid biopsy. Cell Mol Gastroenterol Hepatol. 1:516–534. DOI: 10.1016/j.jcmgh.2015.06.009. PMID: 28210698. PMCID: PMC5301414.

26. Ng CKY, Di Costanzo GG, Tosti N, et al. 2018; Genetic profiling using plasma-derived cell-free DNA in therapy-naive hepatocellular carcinoma patients: a pilot study. Ann Oncol. 29:1286–1291. DOI: 10.1093/annonc/mdy083. PMID: 29509837.

27. Howell J, Atkinson SR, Pinato DJ, et al. 2019; Identification of mutations in circulating cell-free tumour DNA as a biomarker in hepatocellular carcinoma. Eur J Cancer. 116:56–66. DOI: 10.1016/j.ejca.2019.04.014. PMID: 31173963.

28. Qu C, Wang Y, Wang P, et al. 2019; Detection of early-stage hepatocellular carcinoma in asymptomatic HBsAg-seropositive individuals by liquid biopsy. Proc Natl Acad Sci U S A. 116:6308–6312. DOI: 10.1073/pnas.1819799116. PMID: 30858324. PMCID: PMC6442629.

29. Cohen JD, Li L, Wang Y, et al. 2018; Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science. 359:926–930. DOI: 10.1126/science.aar3247. PMID: 29348365. PMCID: PMC6080308.

30. Chan KC, Jiang P, Chan CW, et al. 2013; Noninvasive detection of cancer-associated genome-wide hypomethylation and copy number aberrations by plasma DNA bisulfite sequencing. Proc Natl Acad Sci U S A. 110:18761–18768. DOI: 10.1073/pnas.1313995110. PMID: 24191000. PMCID: PMC3839703.

31. Cai ZX, Chen G, Zeng YY, et al. 2017; Circulating tumor DNA profiling reveals clonal evolution and real-time disease progression in advanced hepatocellular carcinoma. Int J Cancer. 141:977–985. DOI: 10.1002/ijc.30798. PMID: 28543104.

32. Cai Z, Chen G, Zeng Y, et al. 2019; Comprehensive liquid profiling of circulating tumor DNA and protein biomarkers in long-term follow-up patients with hepatocellular carcinoma. Clin Cancer Res. 25:5284–5294. DOI: 10.1158/1078-0432.CCR-18-3477. PMID: 31217202.

33. Ren N, Qin LX, Tu H, Liu YK, Zhang BH, Tang ZY. 2006; The prognostic value of circulating plasma DNA level and its allelic imbalance on chromosome 8p in patients with hepatocellular carcinoma. J Cancer Res Clin Oncol. 132:399–407. DOI: 10.1007/s00432-005-0049-5. PMID: 16502316.

34. Chan KC, Lai PB, Mok TS, et al. 2008; Quantitative analysis of circulating methylated DNA as a biomarker for hepatocellular carcinoma. Clin Chem. 54:1528–1536. DOI: 10.1373/clinchem.2008.104653. PMID: 18653827.

35. Kim SS, Eun JW, Choi JH, et al. 2020; MLH1 single-nucleotide variant in circulating tumor DNA predicts overall survival of patients with hepatocellular carcinoma. Sci Rep. 10:17862. DOI: 10.1038/s41598-020-74494-y. PMID: 33082400. PMCID: PMC7576198.

36. Zhou L, Xu Y, Wang D, et al. 2020; Perioperative circulating tumor DNA analysis to predict patient prognosis in liver cancer. J Clin Oncol. 38:4593. DOI: 10.1200/JCO.2020.38.15_suppl.4593.

37. Meng S, Tripathy D, Frenkel EP, et al. 2004; Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res. 10:8152–8162. DOI: 10.1158/1078-0432.CCR-04-1110. PMID: 15623589.

38. Okajima W, Komatsu S, Ichikawa D, et al. 2017; Liquid biopsy in patients with hepatocellular carcinoma: Circulating tumor cells and cell-free nucleic acids. World J Gastroenterol. 23:5650–5668. DOI: 10.3748/wjg.v23.i31.5650. PMID: 28883691. PMCID: PMC5569280.

39. Strilic B, Offermanns S. 2017; Intravascular survival and extravasation of tumor cells. Cancer Cell. 32:282–293. DOI: 10.1016/j.ccell.2017.07.001. PMID: 28898694.

40. Vona G, Estepa L, Beroud C, et al. 2004; Impact of cytomorphological detection of circulating tumor cells in patients with liver cancer. Hepatology. 39:792–797. DOI: 10.1002/hep.20091. PMID: 14999698.

41. Patil MA, Gutgemann I, Zhang J, et al. 2005; Array-based comparative genomic hybridization reveals recurrent chromosomal aberrations and Jab1 as a potential target for 8q gain in hepatocellular carcinoma. Carcinogenesis. 26:2050–2057. DOI: 10.1093/carcin/bgi178. PMID: 16000397.

42. Thompson EW, Haviv I. 2011; The social aspects of EMT-MET plasticity. Nat Med. 17:1048–1049. DOI: 10.1038/nm.2437. PMID: 21900919.

43. de Boer CJ, van Krieken JH, Janssen-van Rhijn CM, Litvinov SV. 1999; Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J Pathol. 188:201–206. DOI: 10.1002/(SICI)1096-9896(199906)188:2<201::AID-PATH339>3.0.CO;2-8.

44. Went PT, Lugli A, Meier S, et al. 2004; Frequent EpCam protein expression in human carcinomas. Hum Pathol. 35:122–128. DOI: 10.1016/j.humpath.2003.08.026. PMID: 14745734.

45. Yu X, Wang B, Zhang N, et al. 2015; Capture and release of cancer cells by combining on-chip purification and off-chip enzymatic treatment. ACS Appl Mater Interfaces. 7:24001–24007. DOI: 10.1021/acsami.5b06791. PMID: 26488449.

46. Sun C, Liao W, Deng Z, et al. 2017; The diagnostic value of assays for circulating tumor cells in hepatocellular carcinoma: A meta-analysis. Medicine (Baltimore). 96:e7513. DOI: 10.1097/MD.0000000000007513. PMID: 28723763. PMCID: PMC5521903.

47. Sun YF, Xu Y, Yang XR, et al. 2013; Circulating stem cell-like epithelial cell adhesion molecule-positive tumor cells indicate poor prognosis of hepatocellular carcinoma after curative resection. Hepatology. 57:1458–1468. DOI: 10.1002/hep.26151. PMID: 23175471.

48. Zhou Y, Wang B, Wu J, et al. 2016; Association of preoperative EpCAM Circulating Tumor Cells and peripheral Treg cell levels with early recurrence of hepatocellular carcinoma following radical hepatic resection. BMC Cancer. 16:506. DOI: 10.1186/s12885-016-2526-4. PMID: 27439521. PMCID: PMC4955266.

49. Wang Z, Luo L, Cheng Y, et al. 2018; Correlation between postoperative early recurrence of hepatocellular carcinoma and mesenchymal circulating tumor cells in peripheral blood. J Gastrointest Surg. 22:633–639. DOI: 10.1007/s11605-017-3619-3. PMID: 29159757. PMCID: PMC5869875.

50. Yu JJ, Xiao W, Dong SL, et al. 2018; Effect of surgical liver resection on circulating tumor cells in patients with hepatocellular carcinoma. BMC Cancer. 18:835. DOI: 10.1186/s12885-018-4744-4. PMID: 30126375. PMCID: PMC6102841.

51. Kelley RK, Magbanua MJ, Butler TM, et al. 2015; Circulating tumor cells in hepatocellular carcinoma: a pilot study of detection, enumeration, and next-generation sequencing in cases and controls. BMC Cancer. 15:206. DOI: 10.1186/s12885-015-1195-z. PMID: 25884197. PMCID: PMC4399150.

52. Thery C, Witwer KW, Aikawa E, et al. 2018; Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 7:1535750. DOI: 10.1080/20013078.2018.1535750. PMID: 30637094. PMCID: PMC6322352.

53. Gyorgy B, Szabo TG, Pasztoi M, et al. 2011; Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci. 68:2667–2688. DOI: 10.1007/s00018-011-0689-3. PMID: 21560073. PMCID: PMC3142546.

54. Arraud N, Linares R, Tan S, et al. 2014; Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration. J Thromb Haemost. 12:614–627. DOI: 10.1111/jth.12554. PMID: 24618123.

55. Brennan K, Martin K, FitzGerald SP, et al. 2020; A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum. Sci Rep. 10:1039. DOI: 10.1038/s41598-020-57497-7. PMID: 31974468. PMCID: PMC6978318.

56. Théry C, Amigorena S, Raposo G, Clayton A. 2006; Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 3:Unit 3.22. DOI: 10.1002/0471143030.cb0322s30. PMID: 18228490.

57. Xu R, Greening DW, Zhu HJ, Takahashi N, Simpson RJ. 2016; Extracellular vesicle isolation and characterization: toward clinical application. J Clin Invest. 126:1152–1162. DOI: 10.1172/JCI81129. PMID: 27035807. PMCID: PMC4811150.

58. Sidhom K, Obi PO, Saleem A. 2020; A review of exosomal isolation methods: Is size exclusion chromatography the best option? Int J Mol Sci. 21:6466. DOI: 10.3390/ijms21186466. PMID: 32899828. PMCID: PMC7556044.

59. Inglis HC, Danesh A, Shah A, Lacroix J, Spinella PC, Norris PJ. 2015; Techniques to improve detection and analysis of extracellular vesicles using flow cytometry. Cytometry A. 87:1052–1063. DOI: 10.1002/cyto.a.22649. PMID: 25847910. PMCID: PMC4876854.

60. Soo CY, Song Y, Zheng Y, et al. 2012; Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells. Immunology. 136:192–197. DOI: 10.1111/j.1365-2567.2012.03569.x. PMID: 22348503. PMCID: PMC3403268.

61. Kogej K, Bozic D, Kobal B, Herzog M, Cerne K. 2021; Application of dynamic and static light scattering for size and shape characterization of small extracellular nanoparticles in plasma and ascites of ovarian cancer patients. Int J Mol Sci. 22:12946. DOI: 10.3390/ijms222312946. PMID: 34884751. PMCID: PMC8657631.

62. Wang W, Li H, Zhou Y, Jie S. 2013; Peripheral blood microvesicles are potential biomarkers for hepatocellular carcinoma. Cancer Biomark. 13:351–357. DOI: 10.3233/CBM-130370. PMID: 24440975.

63. Julich-Haertel H, Urban SK, Krawczyk M, et al. 2017; Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma. J Hepatol. 67:282–292. DOI: 10.1016/j.jhep.2017.02.024. PMID: 28267620.

64. Sun N, Zhang C, Lee YT, et al. 2023; HCC EV ECG score: An extracellular vesicle-based protein assay for detection of early-stage hepatocellular carcinoma. Hepatology. 77:774–788. DOI: 10.1002/hep.32692. PMID: 35908246.

65. Sun N, Lee YT, Zhang RY, et al. 2020; Purification of HCC-specific extracellular vesicles on nanosubstrates for early HCC detection by digital scoring. Nat Commun. 11:4489. DOI: 10.1038/s41467-020-18311-0. PMID: 32895384. PMCID: PMC7477161.

66. Abbate V, Marcantoni M, Giuliante F, et al. 2017; HepPar1-positive circulating microparticles are increased in subjects with hepatocellular carcinoma and predict early recurrence after liver resection. Int J Mol Sci. 18:1043. DOI: 10.3390/ijms18051043. PMID: 28498353. PMCID: PMC5454955.

67. Wang Y, Zhang C, Zhang P, et al. 2018; Serum exosomal microRNAs combined with alpha-fetoprotein as diagnostic markers of hepatocellular carcinoma. Cancer Med. 7:1670–1679. DOI: 10.1002/cam4.1390. PMID: 29573235. PMCID: PMC5943469.

68. Wang H, Hou L, Li A, Duan Y, Gao H, Song X. 2014; Expression of serum exosomal microRNA-21 in human hepatocellular carcinoma. Biomed Res Int. 2014:864894. DOI: 10.1155/2014/864894. PMID: 24963487. PMCID: PMC4052145.

69. Tian XP, Wang CY, Jin XH, et al. 2019; Acidic microenvironment up-regulates exosomal miR-21 and miR-10b in early-stage hepatocellular carcinoma to promote cancer cell proliferation and metastasis. Theranostics. 9:1965–1979. DOI: 10.7150/thno.30958. PMID: 31037150. PMCID: PMC6485281.

70. Kim SS, Baek GO, Ahn HR, et al. 2020; Serum small extracellular vesicle-derived LINC00853 as a novel diagnostic marker for early hepatocellular carcinoma. Mol Oncol. 14:2646–2659. DOI: 10.1002/1878-0261.12745. PMID: 32525601. PMCID: PMC7530776.

71. Huang X, Sun L, Wen S, et al. 2020; RNA sequencing of plasma exosomes revealed novel functional long noncoding RNAs in hepatocellular carcinoma. Cancer Sci. 111:3338–3349. DOI: 10.1111/cas.14516. PMID: 32506598. PMCID: PMC7469810.

72. Ghosh S, Bhowmik S, Majumdar S, et al. 2020; The exosome encapsulated microRNAs as circulating diagnostic marker for hepatocellular carcinoma with low alpha-fetoprotein. Int J Cancer. 147:2934–2947. DOI: 10.1002/ijc.33111. PMID: 32441313.

73. Nilsson RJ, Balaj L, Hulleman E, et al. 2011; Blood platelets contain tumor-derived RNA biomarkers. Blood. 118:3680–3683. DOI: 10.1182/blood-2011-03-344408. PMID: 21832279. PMCID: PMC7224637.

74. Asghar S, Waqar W, Umar M, Manzoor S. 2020; Tumor educated platelets, a promising source for early detection of hepatocellular carcinoma: Liquid biopsy an alternative approach to tissue biopsy. Clin Res Hepatol Gastroenterol. 44:836–844. DOI: 10.1016/j.clinre.2020.03.023. PMID: 32312598.

75. Umu SU, Langseth H, Bucher-Johannessen C, et al. 2018; A comprehensive profile of circulating RNAs in human serum. RNA Biol. 15:242–250. DOI: 10.1080/15476286.2017.1403003. PMID: 29219730. PMCID: PMC5798962.

76. Koustas E, Trifylli EM, Sarantis P, et al. 2023; An insight into the arising role of microRNAs in hepatocellular carcinoma: Future diagnostic and therapeutic approaches. Int J Mol Sci. 24:7168. DOI: 10.3390/ijms24087168. PMID: 37108330. PMCID: PMC10138911.

77. Danielson KM, Rubio R, Abderazzaq F, Das S, Wang YE. 2017; High throughput sequencing of extracellular RNA from human plasma. PLoS One. 12:e0164644. DOI: 10.1371/journal.pone.0164644. PMID: 28060806. PMCID: PMC5218574.

78. Wan JCM, Massie C, Garcia-Corbacho J, et al. 2017; Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 17:223–238. DOI: 10.1038/nrc.2017.7. PMID: 28233803.