Caspase-cleaved keratin 18 as a biomarker for non-alcoholic ...

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generated cytokeratin-18 fragments'' and soluble Fas (sFas). The ... The method to detect caspase-cleaved keratin 18 (M30 Apop- ... Drugs 2002;20:253–259.
Letters to the Editor

Caspase-cleaved keratin 18 as a biomarker for non-alcoholic steatohepatitis (NASH) – The need for correct terminology To the Editor: A recent study by Tamimi et al. [1] reported quantification of the levels of two apoptotic markers in the circulation of patients suffering from non-alcoholic steatohepatitis (NASH): ‘‘caspase 3 generated cytokeratin-18 fragments’’ and soluble Fas (sFas). The combined use of these markers was reported to accurately predict the presence of NASH, ‘‘supporting the potential usefulness of these markers in clinical practice for noninvasive diagnosis of NASH’’. The method to detect caspase-cleaved keratin 18 (M30 ApoptosenseÒ ELISA) in this and other publications was developed by PEVIVA AB (Bromma, Sweden) in collaboration with my laboratory at the Karolinska Institute [2]. The M30 ApoptosenseÒ test was originally developed as a serum biomarker assay to determine therapy response of patients with carcinoma tumors [3,4]. Bantel et al. found the assay to be useful for the detection of liver injury such as that occurring during hepatitis C virus (HCV) infection using patient blood samples [5]. Following this initial discovery by Bantel et al., other investigators have found that also other processes associated with hepatocyte apoptosis are associated with increased levels of circulating caspase-cleaved K18 fragments, including NASH [6] and alcoholic steatohepatitis (ASH) [7]. The M30 ApoptosenseÒ ELISA assay is based on a monoclonal antibody M30. This antibody recognizes a neo-epitope on K18 formed at Asp396 after cleavage by caspases [8]. The M30 antibody is well characterized and shown to be a marker of early apoptosis [8]. Schutte and colleagues [9] demonstrated that the cleavage site at Asp396 is generated by the initiator caspase 9, explaining the cleavage of K18 at Asp396 during early stages of apoptosis. This sequence is also susceptible to cleavage by caspase 3 and 7 [9]. K18 fragments detected by the M30 Apoptosense assay are frequently referred to as ‘‘M30’’ (e.g. ‘‘M30 levels in serum’’) in the literature, which is incorrect since M30 is an antibody and not an antigen. In the study by Tamimi et al. [1], the analyte is referred to as ‘‘caspase 3 generated cytokeratin-18 fragments’’. Since also other caspases cleave K18 at Asp396, this is only partially correct. The fragments can be referred to as ‘‘ccK18’’ (‘‘caspase-cleaved K18’’). Alternatively, since there is a second caspase-cleavage site in the K18 molecule at Asp237 [10], a more precise terminology would be ‘‘K18Asp396’’. The findings by Tamimi et al. and by other investigators in the field, that caspase-cleaved K18/K18Asp396 is a useful biomarker for the diagnosis and management of NASH are of large potential

importance. To avoid confusion in a growing scientific field, the terminology used to describe the biomarker should be as precise as possible.

Conflict of interest The author is a consultant for PEVIVA AB, the manufacturer of the M30 ApoptosenseÒ ELISA.

References [1] Tamimi TI, Elgouhari HM, Alkhouri N, Yerian LM, Berk MP, Lopez R, et al. An apoptosis panel for nonalcoholic steatohepatitis diagnosis. J Hepatol 2011; 54:1224–1229. [2] Hägg M, Biven K, Ueno T, Rydlander L, Björklund P, Wiman KG, et al. A novel high-through-put assay for screening of pro-apoptotic drugs. Invest New Drugs 2002;20:253–259. [3] Kramer G, Erdal H, Mertens HJ, Nap M, Mauermann J, Steiner G, et al. Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18. Cancer Res 2004;64:1751–1756. [4] Olofsson MH, Ueno T, Pan Y, Xu R, Cai F, van der Kuip H, et al. Cytokeratin-18 is a useful serum biomarker for early determination of response of breast carcinomas to chemotherapy. Clin Cancer Res 2007;13:3198–3206. [5] Bantel H, Lugering A, Heidemann J, Volkmann X, Poremba C, Strassburg CP, et al. Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury. Hepatology 2004;40:1078–1087. [6] Wieckowska A, Zein NN, Yerian LM, Lopez AR, McCullough AJ, Feldstein AE. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology 2006;44:27–33. [7] Lavallard VJ, Bonnafous S, Patouraux S, Saint-Paul MC, Rousseau D, Anty R, et al. Serum markers of hepatocyte death and apoptosis are non invasive biomarkers of severe fibrosis in patients with alcoholic liver disease. PLoS One 2011;6:e17599. [8] Leers MP WK, Björklund V, Bergman T, Tribbick G, Persson B, et al. Immunocytochemical detection and mapping of a cytokeratin 18 neoepitope exposed during early apoptosis. J Pathol 1999;187:567–572. [9] Schutte B, Henfling M, Kolgen W, Bouman M, Meex S, Leers MP, et al. Keratin 8/18 breakdown and reorganization during apoptosis. Exp Cell Res 2004;297:11–26. [10] Ku NO, Liao J, Omary MB. Apoptosis generates stable fragments of human type I keratins. J Biol Chem 1997;272:33197–33203.

Journal of Hepatology 2011 vol. 55

Stig Linder Department of Oncology-Pathology, Karolinska Institute, Cancer Center Karolinska R8:00, Karolinska Hospital, 171 76 Stockholm, Sweden Tel.: +46 8 5177 2452 E-mail address: [email protected]

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1467–1472