Assessment of liver cancer biomarkers
Gastroenterology and Hepatology from Bed to Bench,
Liver cancer is the third cause of cancer-related deaths in the world. It is primarily divides into two main types, namely hepatocellular carcinoma (HC) and cholangiocarcinoma (IC). Due to the increasing number of patients with liver cancer and the high mortality rate, early diagnosis of the disease can be helpful in treatment, but most patients are diagnosed atlate stages of HC. The aim of this study is to screen and provide an overview on candidate biomarkers related to primary liver cancer to introduce the critical ones. In this study, various biomarkers related to the diagnosis of primary liver cancer have been studied. Accordingly, biomarkers are divided into different groups as blood biomarkers classified as serum and plasma biomarkers, tissue biomarkers, microRNA biomarkers, proteomic biomarkers and altered genes. Previous researches have focused on liver cells and bile ducts, the surround cellular environment, how cells differentiate, and the types of genes expressed in liver cancer. Some even have focused on the origin of tumor cells and how they differentiate and develop. In all these studies, the expression of specific proteins and genes in liver cancer has been considered. Based on available sources, biomarkers can be considered as candidates to diagnose and prognosis of various types of primary liver cancer, from sources such as blood, tissue, mic-RNA, proteome and genes. However, more investigations are required to introduce a biomarker for precise detection of early liver cancer.
Keywords: Hepatocellular carcinoma, Biomarker, Diagnosis, Prognosis, Proteomics.
(Please cite as: Mansouri V, Razzaghi MR, Nikzamir A, Ahmadzadeh AR, Iranshahi M, Haghazali M, et al. Assessment of liver cancer biomarkers. Gastroenterol Hepatol Bed Bench 2020;13(Suppl.1):S29-S39).
- Hepatocellular carcinoma, Biomarker, diagnosis, prognosis , proteomics
How to Cite
Villanueva A, Schwartz ME, Llovet JM. Liver cancer. In: Oh W, Chari A, Editors. Mount Sinai Expert Guides: Oncology. New York: John Wiley & Sons Ltd; 2019. P.89-100.
Yang JD, Roberts LR. Hepatocellular carcinoma: a global view. Nat Rev Gastroenterol Hepatol 2010;7:448.
Karagozian R, Derdák Z, Baffy G. Obesity-associated mechanisms of hepatocarcinogenesis. Metabolism 2014;63:607-17.
Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378-90.
Llovet JM, Hernandez-Gea V. Hepatocellular carcinoma: reasons for phase III failure and novel perspectives on trial design. Clin Cancer Res 2014;20:2072-9.
Zucman-Rossi J, Villanueva A, Nault JC, Llovet JM. Genetic landscape and biomarkers of hepatocellular carcinoma. Gastroenterology 2015;149:1226-39.
Torrecilla S, Sia D, Harrington AN, Zhang Z, Cabellos L, Cornella H, et al. Trunk mutational events present minimal intra-and inter-tumoral heterogeneity in hepatocellular carcinoma. J Hepatol 2017;67:1222-31.
Chan LK, Ng IOL. Proteomic profiling in liver cancer: another new page. Trans Gastroenterol Hepatol 2019;4.
Lee SC, Tan HT, Chung MCM. Prognostic biomarkers for prediction of recurrence of hepatocellular carcinoma: current status and future prospects. World J Gastroenterol 2014;20:3112.
Jiang Y, Sun A, Zhao Y, Ying W, Sun H, Yang X, et al. Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma. Nature 2019;567:257-61.
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394-424.
Gera S, Ettel M, Acosta-Gonzalez G, Xu R. Clinical features, histology, and histogenesis of combined hepatocellular-cholangiocarcinoma. World J Hepatol 2017;9:300.
Saffroy R, Pham P, Reffas M, Takka M, Lemoine A, Debuire B. New perspectives and strategy research biomarkers for hepatocellular carcinoma. Clin Chem Lab Med 2007;45:1169-79.
Zhou YM, Yang JM, Li B, Yin ZF, Xu F, Wang B, et al. Clinicopathologic characteristics of intrahepatic cholangiocarcinoma in patients with positive serum a-fetoprotein. World J Gastroenterol 2008;14:2251.
Yin X, Zhang BH, Qiu SJ, Ren ZG, Zhou J, Chen XH, et al. Combined hepatocellular carcinoma and cholangiocarcinoma: clinical features, treatment modalities, and prognosis. Ann Surg Oncol 2012;19:2869-76.
Yamashita T, Forgues M, Wang W, Kim JW, Ye Q, Jia H, et al. EpCAM and α-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res 2008;68:1451-61.
Li R, Yang D, Tang CL, Cai P, Ma KS, Ding SY, et al. Combined hepatocellular carcinoma and cholangiocarcinoma (biphenotypic) tumors: clinical characteristics, imaging features of contrast-enhanced ultrasound and computed tomography. BMC Cancer 2016;16:158.
Bertino G, Ardiri A, Malaguarnera M, Malaguarnera G, Bertino N, Calvagno GS, et al. Hepatocellualar carcinoma serum markers. Semin Oncol 2012;39:410-33.
Tamura Y, Igarashi M, Kawai H, Suda T, Satomura S, Aoyagi Y. Clinical advantage of highly sensitive on-chip immunoassay for fucosylated fraction of alpha-fetoprotein in patients with hepatocellular carcinoma. Dig Dis Sci 2010;55:3576-83.
Choi J, Kim GA, Han S, Lee W, Chun S, Lim YS. Longitudinal assessment of three serum biomarkers to detect very early‐stage hepatocellular carcinoma. Hepatology 2019;69:1983-94.
Hagiwara S, Kudo M, Kawasaki T, Nagashima M, Minami Y, Chung H, et al. Prognostic factors for portal venous invasion in patients with hepatocellular carcinoma. J Gastroenterol 2006;41:1214-9.
Hu B, Tian X, Sun J, Meng X. Evaluation of individual and combined applications of serum biomarkers for diagnosis of hepatocellular carcinoma: a meta-analysis. Int J Mol Sci 2013;14:23559-80.
Song P, Tobe RG, Inagaki Y, Kokudo N, Hasegawa K, Sugawara Y, et al. The management of hepatocellular carcinoma around the world: a comparison of guidelines from 2001 to 2011. Liver Int 2012;32:1053-63.
Zhang YS, Chu JH, Cui SX, Song ZY, Qu XJ. Des-γ-carboxy prothrombin (DCP) as a potential autologous growth factor for the development of hepatocellular carcinoma. Cell Physiol Biochem 2014;34:903-15.
Chen J, Wu G, Li Y. Evaluation of serum des-gamma-carboxy prothrombin for the diagnosis of hepatitis B virus-related hepatocellular carcinoma: a meta-analysis. Dis Markers 2018;2018: 8906023.
Masuzaki R, Karp SJ, Omata M. New serum markers of hepatocellular carcinoma. Semin Oncol 2012; 39(4):434-9.
Wei C, Yang X, Liu N, Geng J, Tai Y, Sun Z, et al. Tumor microenvironment regulation by the endoplasmic reticulum stress transmission mediator Golgi protein 73 in mice. Hepatology 2019;70:851-70.
Liu Y, Zhang X, Zhou S, Shi J, Xu Y, He J, et al. Knockdown of Golgi phosphoprotein 73 blocks the trafficking of matrix metalloproteinase‐2 in hepatocellular carcinoma cells and inhibits cell invasion. J Cell Mol Med 2019;23:2399-409.
Mao Y, Yang H, Xu H, Lu X, Sang X, Du S, et al. Golgi protein 73 (GOLPH2) is a valuable serum marker for hepatocellular carcinoma. Gut 2010;59:1687-93.
Mintz K, Waidely E, Zhou Y, Peng Z, Al-Youbi AO, Bashammakh AS, et al. Carbon dots and gold nanoparticles based immunoassay for detection of alpha-L-fucosidase. Anal Chim Acta 2018;1041:114-21.
El-Tayeh SF, Hussein TD, El-Houseini ME, Amer MA, El-Sherbini M, Elshemey WM. Serological biomarkers of hepatocellular carcinoma in Egyptian patients. Dis Markers 2012;32:255-63.
Waidely E, Al-Youbi AO, Bashammakh AS, El-Shahawi MS, Leblanc RM. Alpha-l-fucosidase immunoassay for early detection of hepatocellular carcinoma. Anal Chem 2017;89:9459-66.
Yamada T, Nakanishi Y, Okamura K, Tsuchikawa T, Nakamura T, Noji T, et al. Impact of serum carbohydrate antigen 19‐9 level on prognosis and prediction of lymph node metastasis in patients with intrahepatic cholangiocarcinoma. J Gastroenterol Hepatol 2018;33:1626-33.
Wang Y, Li J, Xia Y, Gong R, Wang K, Yan Z, et al. Prognostic nomogram for intrahepatic cholangiocarcinoma after partial hepatectomy. J Clin Oncol 2013;31:1188-95.
Ying X, Zhao Y, Wang JL, Zhou X, Zhao J, He CC, et al. Serum anti-osteopontin autoantibody as a novel diagnostic and prognostic biomarker in patients with hepatocellular carcinoma. Oncol Rep 2014;32:1550-6.
Shang S, Plymoth A, Ge S, Feng Z, Rosen HR, Sangrajrang S, et al. Identification of osteopontin as a novel marker for early hepatocellular carcinoma. Hepatology 2012;55:483-90.
Zhu Y, Yang J, Xu D, Gao XM, Zhang Z, Hsu JL, et al. Disruption of tumour-associated macrophage trafficking by the osteopontin-induced colony-stimulating factor-1 signalling sensitises hepatocellular carcinoma to anti-PD-L1 blockade. Gut 2019;68:1653-66.
Carr BI, Kanke F, Wise M, Satomura S. Clinical evaluation of Lens culinaris agglutinin-reactive α-fetoprotein and des-γ-carboxy prothrombin in histologically proven hepatocellular carcinoma in the United States. Dig Dis Sci 2007;52:776-82.
Wang C, Zhang Y, Guo K, Wang N, Jin H, Liu Y, et al. Heat shock proteins in hepatocellular carcinoma: Molecular mechanism and therapeutic potential. Int J Cancer 2016;138:1824-34.
Chuma M, Sakamoto M, Yamazaki K, Ohta T, Ohki M, Asaka M, et al. Expression profiling in multistage hepatocarcinogenesis: identification of HSP70 as a molecular marker of early hepatocellular carcinoma. Hepatology 2003;37:198-207.
Kang GH, Lee BS, Lee ES, Kim SH, Lee HY, Kang DY. Prognostic significance of p53, mTOR, c-Met, IGF-1R, and HSP70 overexpression after the resection of hepatocellular carcinoma. Gut liver 2014;8:79.
Shin E, Ryu HS, Kim SH, Jung H, Jang JJ, Lee K. The clinicopathological significance of heat shock protein 70 and glutamine synthetase expression in hepatocellular carcinoma. J Hepato-biliary-pancreatic Sci 2011;18:544-50.
Ibrahim TR, Abdel-Raouf SM. Immunohistochemical study of Glypican-3 and HepPar-1 in differentiating hepatocellular carcinoma from metastatic carcinomas in FNA of the liver. Pathol Oncol Res 2015;21:379-87.
SY Leong A, Sormunen RT, Tsui W, Liew C. Hep Par 1 and selected antibodies in the immunohistological distinction of hepatocellular carcinoma from cholangiocarcinoma, combined tumours and metastatic carcinoma. Histopathology 1998;33:318-24.
Kakar S, Gown AM, Goodman ZD, Ferrell LD. Best practices in diagnostic immunohistochemistry: hepatocellular carcinoma versus metastatic neoplasms. Arch Pathol Lab Med 2007;131:1648-54.
Nishida T, Kataoka H. Glypican 3-targeted therapy in hepatocellular carcinoma. Cancers 2019;11:1339.
Chen C, Huang X, Ying Z, Wu D, Yu Y, Wang X, et al. Can glypican-3 be a disease-specific biomarker? Clin Trans Med 2017;6:18.
Shirakawa H, Kuronuma T, Nishimura Y, Hasebe T, Nakano M, Gotohda N, et al. Glypican-3 is a useful diagnostic marker for a component of hepatocellular carcinoma in human liver cancer. Int J Oncol 2009;34:649-56.
Kolluri A, Ho M. The role of glypican-3 in regulating Wnt, YAP and hedgehog in liver cancer. Front Oncol 2019;9:708.
Liu H, Li P, Zhai Y, Qu CF, Zhang LJ, Tan YF, et al. Diagnostic value of glypican-3 in serum and liver for primary hepatocellular carcinoma. World J Gastroenterol 2010;16:4410.
Lagana SM, Moreira RK, Remotti HE, Bao F. Glutamine synthetase, heat shock protein-70, and glypican-3 in intrahepatic cholangiocarcinoma and tumors metastatic to liver. Appl Immunohistochem Mol Morphol 2013;21:254-7.
Yan BC, Gong C, Song J, Krausz T, Tretiakova M, Hyjek E, et al. Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms. Am J Surg Pathol 2010;34:1147.
Fujiwara M, Kwok S, Yano H, Pai RK. Arginase‐1 is a more sensitive marker of hepatic differentiation than HepPar‐1 and glypican‐3 in fine‐needle aspiration biopsies. Cancer Cytopathol 2012;120:230-7.
Timek DT, Shi J, Liu H, Lin F. Arginase-1, HepPar-1, and Glypican-3 are the most effective panel of markers in distinguishing hepatocellular carcinoma from metastatic tumor on fine-needle aspiration specimens. Am J Clin Pathol 2012;138:203-10.
Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol 2008;129:705.
Ryu HS, Lee K, Shin E, Kim SH, Jing J, Jung HY, et al. Comparative analysis of immunohistochemical markers for differential diagnosis of hepatocelluar carcinoma and cholangiocarcinoma. Tumori Journal 2012;98:478-84.
Liu LZ, Yang LX, Zheng BH, Dong PP, Liu XY, Wang ZC, et al. CK7/CK19 index: a potential prognostic factor for postoperative intrahepatic cholangiocarcinoma patients. J Surg Oncol 2018;117:1531-9.
Dal Bello B, Rosa L, Campanini N, Tinelli C, Viera FT, D'Ambrosio G, et al. Glutamine synthetase immunostaining correlates with pathologic features of hepatocellular carcinoma and better survival after radiofrequency thermal ablation. Clin Cancer Res 2010;16:2157-66.
Nguyen TB, Roncalli M, Di Tommaso L, Kakar S. Combined use of heat-shock protein 70 and glutamine synthetase is useful in the distinction of typical hepatocellular adenoma from atypical hepatocellular neoplasms and well-differentiated hepatocellular carcinoma. Mod Pathol 2016;29:283-92.
Totoki Y, Tatsuno K, Yamamoto S, Arai Y, Hosoda F, Ishikawa S, et al. High-resolution characterization of a hepatocellular carcinoma genome. Nat Genet 2011;43:464-9.
Sung W-K, Zheng H, Li S, Chen R, Liu X, Li Y, et al. Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma. Nat Genet 2012;44:765-9.
Kan Z, Zheng H, Liu X, Li S, Barber TD, Gong Z, et al. Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma. Genome Res 2013;23:1422-33.
Cancer Genome Atlas Research Network. Electronic address: email@example.com; Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma. Cell 2017;169:1327-41.
Schulze K, Imbeaud S, Letouzé E, Alexandrov LB, Calderaro J, Rebouissou S, et al. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Gen 2015;47:505-11.
Wheeler DA, Roberts LR, Network CGAR. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell 2017;169:1327.
Ho DW, Chan LK, Chiu YT, Xu IM, Poon RT, Cheung TT, et al. TSC1/2 mutations define a molecular subset of HCC with aggressive behaviour and treatment implication. Gut 2017;66:1496-506.
Moon S-H, Huang C-H, Houlihan SL, Regunath K, Freed-Pastor WA, Morris IV JP, et al. p53 represses the mevalonate pathway to mediate tumor suppression. Cell 2019;176:564-80.
Che L, Chi W, Qiao Y, Zhang J, Song X, Liu Y, et al. Cholesterol biosynthesis supports the growth of hepatocarcinoma lesions depleted of fatty acid synthase in mice and humans. Gut 2020;69:177-86.
Gao Q, Zhu H, Dong L, Shi W, Chen R, Song Z, et al. Integrated proteogenomic characterization of HBV-related hepatocellular carcinoma. Cell 2019;179:561-77.
Ding J, Kuo ML, Su L, Xue L, Luh F, Zhang H, et al. Human mitochondrial pyrroline-5-carboxylate reductase 1 promotes invasiveness and impacts survival in breast cancers. Carcinogenesis 2017;38:519-31.
Molotkov A, Deltour L, Foglio MH, Cuenca AE, Duester G. Distinct retinoid metabolic functions for alcohol dehydrogenase genes Adh1 and Adh4 in protection against vitamin A toxicity or deficiency revealed in double null mutant mice. J Biol Chem 2002;277:13804-11.
Teng L, Wang K, Liu Y, Ma Y, Chen W, Bi L. Based on integrated bioinformatics analysis identification of biomarkers in hepatocellular carcinoma patients from different regions. BioMed Res Int 2019;2019.
Liu L, Wu J, Guo Y, Xie W, Chen B, Zhang Y, et al. Overexpression of FoxM1 predicts poor prognosis of intrahepatic cholangiocarcinoma. Aging 2018;10:4120.
Dibb M, Han N, Choudhury J, Hayes S, Valentine H, West C, et al. The FOXM1-PLK1 axis is commonly upregulated in oesophageal adenocarcinoma. Br J Cancer 2012;107:1766-75.
Zeng J, Wang L, Li Q, Li W, Björkholm M, Jia J, et al. FoxM1 is up‐regulated in gastric cancer and its inhibition leads to cellular senescence, partially dependent on p27kip1. J Pathol 2009;218:419-27.
Hu G, Yan Z, Zhang C, Cheng M, Yan Y, Wang Y, et al. FOXM1 promotes hepatocellular carcinoma progression by regulating KIF4A expression. J Exp Clin Cancer Res 2019;38:188.
Seimiya M, Tomonaga T, Matsushita K, Sunaga M, Oh‐ishi M, Kodera Y, et al. Identification of novel immunohistochemical tumor markers for primary hepatocellular carcinoma; clathrin heavy chain and formiminotransferase cyclodeaminase. Hepatology 2008;48:519-30.
Yeoman AD, Al‐Chalabi T, Karani JB, Quaglia A, Devlin J, Mieli‐Vergani G, et al. Evaluation of risk factors in the development of hepatocellular carcinoma in autoimmune hepatitis: implications for follow‐up and screening. Hepatology 2008;48:863-70.
Sheng Y, Wei J, Zhang Y, Gao X, Wang Z, Yang J, et al. Mutated EPHA2 is a target for combating lymphatic metastasis in intrahepatic cholangiocarcinoma. Int J Cancer 2019;144:2440-52.
Singh G, Yoshida EM, Rathi S, Marquez V, Kim P, Erb SR, et al. Biomarkers for hepatocellular cancer. World J Hepatol 2020;12:558.
Huang W. MicroRNAs: biomarkers, diagnostics, and therapeutics. In: Huang J, Borchert GM, Dou D, Huan J, Lan W, Tan M, et al, Editors. Bioinformatics in MicroRNA Research. Totowa, New Jersey, United States: Humana Press; 2017.
Zhou J, Yu L, Gao X, Hu J, Wang J, Dai Z, et al. Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J Clin Oncol 2011;29:4781-8.
Huang JT, Liu SM, Ma H, Yang Y, Zhang X, Sun H, et al. Systematic review and meta‐analysis: Circulating miRNAs for diagnosis of hepatocellular carcinoma. J Cell Physiol 2016;231:328-35.
Tsai WC, Hsu SD, Hsu CS, Lai TC, Chen SJ, Shen R, et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J Clin Invest 2012;122:2884-97.
Amr KS, Atia HAE, Elbnhawy RAE, Ezzat WM. Early diagnostic evaluation of miR-122 and miR-224 as biomarkers for hepatocellular carcinoma. Gen Dis 2017;4:215-21.
Moshiri F, Salvi A, Gramantieri L, Sangiovanni A, Guerriero P, De Petro G, et al. Circulating miR-106b-3p, miR-101-3p and miR-1246 as diagnostic biomarkers of hepatocellular carcinoma. Oncotarget 2018;9:15350.
Tian XP, Wang CY, Jin XH, Li M, Wang FW, Huang WJ, et al. Acidic microenvironment up-regulates exosomal miR-21 and miR-10b in early-stage hepatocellular carcinoma to promote cancer cell proliferation and metastasis. Theranostics 2019;9:1965.
Takigawa Y, Brown A. Wnt signaling in liver cancer. Curr Drug Targ 2008;9:1013-24.
Armengol C, Cairo S, Fabre M, Buendia M. Wnt signaling and hepatocarcinogenesis: the hepatoblastoma model. Int J Biochem Cell Biol 2011;43:265-70.
Austinat M, Dunsch R, Wittekind C, Tannapfel A, Gebhardt R, Gaunitz F. Correlation between β-catenin mutations and expression of Wnt-signaling target genes in hepatocellular carcinoma. Mol Cancer 2008;7:1-9.
Cao H, Chen X, Wang Z, Wang L, Xia Q, Zhang W. The role of MDM2–p53 axis dysfunction in the hepatocellular carcinoma transformation. Cell Death Discov 2020;6:1-14.
García-Vilas JA, Medina MÁ. Updates on the hepatocyte growth factor/c-Met axis in hepatocellular carcinoma and its therapeutic implications. World J Gastroenterol 2018;24:3695.
Gao W, Kim H, Ho M. Human monoclonal antibody targeting the heparan sulfate chains of glypican-3 inhibits HGF-mediated migration and motility of hepatocellular carcinoma cells. PloS one 2015;10:e0137664.
Sherman M. How to improve HCC surveillance outcomes. JHEP Rep 2019;1:460-7.
Tzartzeva K, Singal AG. Testing for AFP in combination with ultrasound improves early liver cancer detection. Expert Rev Gastroenterol Hepatol 2018;12:947-49.
Han C, Gao L, Zhao L, Sheng Q, Zhang C, An Z, et al. Immunohistochemistry detects increased expression of aldo-keto reductase family 1 member b10 (AKR1B10) in early-stage hepatocellular carcinoma. Int Med J Exp Clin Res 2018;24:7414.
Wan HG, Xu H, Gu YM, Wang H, Xu W, Zu MH. Comparison osteopontin vs AFP for the diagnosis of HCC: a meta-analysis. Clin Res Hepatol Gastroenterol 2014;38:706-14.
Park JW, Chen M, Colombo M, Roberts LR, Schwartz M, Chen PJ, et al. Global patterns of hepatocellular carcinoma management from diagnosis to death: the BRIDGE Study. Liver Int 2015;35:2155-66.
Gao YX, Yang TW, Yin JM, Yang PX, Kou BX, Chai MY, et al. Progress and prospects of biomarkers in primary liver cancer. Int J Oncol 2020;57:54-66.
Calderaro J, Couchy G, Imbeaud S, Amaddeo G, Letouzé E, Blanc JF, et al. Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification. J Hepatol 2017;67:727-38.
Wang P, Dong Q, Zhang C, Kuan PF, Liu Y, Jeck WR, et al. Mutations in isocitrate dehydrogenase 1 and 2 occur frequently in intrahepatic cholangiocarcinomas and share hypermethylation targets with glioblastomas. Oncogene 2013;32:3091-100.
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