Caspase-cleaved keratin 18 as a biomarker for non-alcoholic steatohepatitis (NASH) – The need for correct terminology
Authors: Stig Linder
Journal: Journal of Hepatology (2011)

An apoptosis panel for nonalcoholic steatohepatitis diagnosisJournal of HepatologyVol. 54Issue 6PreviewThe extrinsic death receptor-mediated pathway of apoptosis is involved in nonalcoholic steatohepatitis (NASH) development. Our aims were to create and validate a noninvasive prediction model for NASH diagnosis based on specific circulating markers of apoptosis. Full-Text PDF A recent study by Tamimi et al. [[1]Tamimi T.I. Elgouhari H.M. Alkhouri N. Yerian L.M. Berk M.P. Lopez R. et al.An apoptosis panel for nonalcoholic steatohepatitis diagnosis.J Hepatol. 2011; 54: 1224-1229Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar] 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 Apoptosense® ELISA) in this and other publications was developed by PEVIVA AB (Bromma, Sweden) in collaboration with my laboratory at the Karolinska Institute [[2]Hägg M. Biven K. Ueno T. Rydlander L. Björklund P. Wiman K.G. et al.A novel high-through-put assay for screening of pro-apoptotic drugs.Invest New Drugs. 2002; 20: 253-259Crossref PubMed Scopus (109) Google Scholar]. The M30 Apoptosense® test was originally developed as a serum biomarker assay to determine therapy response of patients with carcinoma tumors [3Kramer G. Erdal H. Mertens H.J. 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: 1751-1756Crossref PubMed Scopus (242) Google Scholar, 4Olofsson M.H. 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: 3198-3206Crossref PubMed Scopus (120) Google Scholar]. 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]Bantel H. Lugering A. Heidemann J. Volkmann X. Poremba C. Strassburg C.P. et al.Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury.Hepatology. 2004; 40: 1078-1087Crossref PubMed Scopus (219) Google Scholar]. 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]Wieckowska A. Zein N.N. Yerian L.M. Lopez A.R. McCullough A.J. Feldstein A.E. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease.Hepatology. 2006; 44: 27-33Crossref PubMed Scopus (570) Google Scholar] and alcoholic steatohepatitis (ASH) [[7]Lavallard V.J. Bonnafous S. Patouraux S. Saint-Paul M.C. 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: e17599Crossref PubMed Scopus (51) Google Scholar].The M30 Apoptosense® 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]Leers MP W.K. Björklund V. Bergman T. Tribbick G. Persson B. et al.Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis.J Pathol. 1999; 187: 567-572Crossref PubMed Scopus (567) Google Scholar]. The M30 antibody is well characterized and shown to be a marker of early apoptosis [[8]Leers MP W.K. Björklund V. Bergman T. Tribbick G. Persson B. et al.Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis.J Pathol. 1999; 187: 567-572Crossref PubMed Scopus (567) Google Scholar]. Schutte and colleagues [[9]Schutte B. Henfling M. Kolgen W. Bouman M. Meex S. Leers M.P. et al.Keratin 8/18 breakdown and reorganization during apoptosis.Exp Cell Res. 2004; 297: 11-26Crossref PubMed Scopus (168) Google Scholar] 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]Schutte B. Henfling M. Kolgen W. Bouman M. Meex S. Leers M.P. et al.Keratin 8/18 breakdown and reorganization during apoptosis.Exp Cell Res. 2004; 297: 11-26Crossref PubMed Scopus (168) Google Scholar].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]Tamimi T.I. Elgouhari H.M. Alkhouri N. Yerian L.M. Berk M.P. Lopez R. et al.An apoptosis panel for nonalcoholic steatohepatitis diagnosis.J Hepatol. 2011; 54: 1224-1229Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar], 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]Ku N.O. Liao J. Omary M.B. Apoptosis generates stable fragments of human type I keratins.J Biol Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar], 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 interestThe author is a consultant for PEVIVA AB, the manufacturer of the M30 Apoptosense® ELISA. A recent study by Tamimi et al. [[1]Tamimi T.I. Elgouhari H.M. Alkhouri N. Yerian L.M. Berk M.P. Lopez R. et al.An apoptosis panel for nonalcoholic steatohepatitis diagnosis.J Hepatol. 2011; 54: 1224-1229Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar] 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 Apoptosense® ELISA) in this and other publications was developed by PEVIVA AB (Bromma, Sweden) in collaboration with my laboratory at the Karolinska Institute [[2]Hägg M. Biven K. Ueno T. Rydlander L. Björklund P. Wiman K.G. et al.A novel high-through-put assay for screening of pro-apoptotic drugs.Invest New Drugs. 2002; 20: 253-259Crossref PubMed Scopus (109) Google Scholar]. The M30 Apoptosense® test was originally developed as a serum biomarker assay to determine therapy response of patients with carcinoma tumors [3Kramer G. Erdal H. Mertens H.J. 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: 1751-1756Crossref PubMed Scopus (242) Google Scholar, 4Olofsson M.H. 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: 3198-3206Crossref PubMed Scopus (120) Google Scholar]. 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]Bantel H. Lugering A. Heidemann J. Volkmann X. Poremba C. Strassburg C.P. et al.Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury.Hepatology. 2004; 40: 1078-1087Crossref PubMed Scopus (219) Google Scholar]. 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]Wieckowska A. Zein N.N. Yerian L.M. Lopez A.R. McCullough A.J. Feldstein A.E. In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease.Hepatology. 2006; 44: 27-33Crossref PubMed Scopus (570) Google Scholar] and alcoholic steatohepatitis (ASH) [[7]Lavallard V.J. Bonnafous S. Patouraux S. Saint-Paul M.C. 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: e17599Crossref PubMed Scopus (51) Google Scholar]. The M30 Apoptosense® 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]Leers MP W.K. Björklund V. Bergman T. Tribbick G. Persson B. et al.Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis.J Pathol. 1999; 187: 567-572Crossref PubMed Scopus (567) Google Scholar]. The M30 antibody is well characterized and shown to be a marker of early apoptosis [[8]Leers MP W.K. Björklund V. Bergman T. Tribbick G. Persson B. et al.Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis.J Pathol. 1999; 187: 567-572Crossref PubMed Scopus (567) Google Scholar]. Schutte and colleagues [[9]Schutte B. Henfling M. Kolgen W. Bouman M. Meex S. Leers M.P. et al.Keratin 8/18 breakdown and reorganization during apoptosis.Exp Cell Res. 2004; 297: 11-26Crossref PubMed Scopus (168) Google Scholar] 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]Schutte B. Henfling M. Kolgen W. Bouman M. Meex S. Leers M.P. et al.Keratin 8/18 breakdown and reorganization during apoptosis.Exp Cell Res. 2004; 297: 11-26Crossref PubMed Scopus (168) Google Scholar]. 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]Tamimi T.I. Elgouhari H.M. Alkhouri N. Yerian L.M. Berk M.P. Lopez R. et al.An apoptosis panel for nonalcoholic steatohepatitis diagnosis.J Hepatol. 2011; 54: 1224-1229Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar], 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]Ku N.O. Liao J. Omary M.B. Apoptosis generates stable fragments of human type I keratins.J Biol Chem. 1997; 272: 33197-33203Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar], 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 interestThe author is a consultant for PEVIVA AB, the manufacturer of the M30 Apoptosense® ELISA. The author is a consultant for PEVIVA AB, the manufacturer of the M30 Apoptosense® ELISA.