HEPATOLOGY, Vol. 61, No. 4, 2015
2
Department of General Surgery and Organ Transplantation Umberto I Hospital Sapienza University Rome, Italy
References 1. Halazun KJ, Patzer RE, Rana AA, Verna EC, Griesemer AD, Parsons RF, et al. Standing the test of time: outcomes of a decade of prioritizing patients with HCC: results of the UNOS natural geographic experiment. HEPATOLOGY 2014;60:1957–1962. 2. Samoylova ML, Dodge JL, Yao FY, Roberts JP. Time-to-transplant as a predictor of hepatocellular carcinoma recurrence after liver transplant. Liver Transpl 2014 [Epub ahead of print]. 3. Lai Q, Avolio AW, Lerut J, Singh G, Chan SC, Berloco PB, et al. Recurrence of hepatocellular cancer after liver transplantation: the role of primary resection and salvage transplantation in East and West. J Hepatol 2012;57:974-979. 4. Lai Q, Lerut JP. Hepatocellular cancer: how to expand safely inclusion criteria for liver transplantation. Curr Opin Organ Transplant. 2014;19:229-234. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27434 Potential conflict of interest: Dr. Lerut received grants from Fresenius Biotech and Takeda.
Reply: We thank the authors for their insightful comments. The lack of uniformity in organ allocation among regions has allowed us and others to examine the vast regional variation in waiting times to discover major differences in the outcomes of hepatocellular carcinoma (HCC) patients on the list, both with respect to recurrence and overall outcome.1,2 It is clear that uniformity among transplant centers in the U.S. and worldwide in both access and treatment of these patients is required, and will allow the transplant community to better understand how to manage patients with HCC on the waiting list. While the Milan criteria are widely accepted despite having no specific tumor biology indices associated with them, many groups including our own have attempted to define markers of poor biology; however, none of these have been universally accepted or applied to organ allocation models.3,4 Currently, it
CORRESPONDENCE
1439
seems that lack of progression of tumor while waiting for a liver transplant, or successful down-staging of tumor, may be the best available biological marker, as demonstrated by the UCSF group and our article.1,2,5 We agree that the development, and universal implementation of appropriate and reproducible biomarkers of tumor biology to allocation models of liver transplantation for HCC is required in order to optimize outcomes for HCC patients. KARIM J. HALAZUN, M.D. ABBAS A. RANA, M.D. RACHEL E. PATZER, PH.D. BENJAMIN SAMSTEIN, M.D. ROBERT S. BROWN, Jr., M.D. JEAN C. EMOND, M.D. Columbia University Center for Liver Disease and Transplantation New York, NY
References 1. Samoylova ML, Dodge JL, Yao FY, Roberts JP. Time-to-transplant as a predictor of hepatocellular carcinoma recurrence after liver transplant. Liver Transpl 2014 [Epub ahead of print]. 2. Halazun KJ, Patzer RE, Rana AA, Verna EC, Griesemer AD, Parsons RF, et al. Standing the test of time: outcomes of a decade of prioritizing patients with HCC, results of the UNOS natural geographic experiment. HEPATOLOGY 2014;60:1957-1962. 3. Halazun KJ, Hardy MA, Rana AA, Woodland DC 4th, Luyten EJ, Mahadev S, et al. Negative impact of neutrophil-lymphocyte ratio on outcome after liver transplantation for hepatocellular carcinoma. Ann Surg 2009;250:141-151. 4. Vibert E1, Azoulay D, Hoti E, Iacopinelli S, Samuel D, Salloum C, et al. Progression of alphafetoprotein before liver transplantation for hepatocellular carcinoma in cirrhotic patients: a critical factor. Am J Transplant 2010;10:129-137. 5. Roberts JP, Venook A, Kerlan R, Yao F. Hepatocellular carcinoma: ablate and wait versus rapid transplantation. Liver Transpl 2010;16:925-929. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27435 Potential conflict of interest: Dr. Brown consults for and received grants from Gilead, AbbVie, Janssen, and Vertex. He consults for Merck, Genentech, and Salix.
Understanding the Pathophysiological Regulatory Role of MicroRNAs in Acute Liver Failure To the Editor: We read with great interest the recent article by John et al. in HEPATOLOGY that described a potential role for microRNAs (miRNAs) in spontaneous recovery from acute liver failure (ALI).1 These potentially important data demonstrate elevated circulating miRNAs, especially miR-122, in human subjects with ALI, which confirms published work from our group and elsewhere.2 However, we have significant concerns about a number of the findings reported in this article. The investigators report that higher circulating and liver levels of miR-122 relate to spontaneous survival and contribute to regeneration and enhanced proliferation. These conclusions directly conflict with the well-established, replicated findings that miR-122 knockout mice and mice with deletions in miR-122 regulators, such as Dicer1, have enhanced levels of hepatocyte proliferation,3 that the loss of miR-122 from hepato-
cytes results in reduced differentiation and enhanced proliferation,4 and that the levels of miR-122 are decreased in liver, yet increased in blood, of rodent models of ALI.5 Furthermore, we feel that robust conclusions cannot be based on liver miR-122 expression data from a very low number of patients (n 5 4 and n 5 6 for recovery and nonrecovery, respectively). We appreciate that liver tissue cannot always be obtained; but for the small patient cohort where liver samples were collected, it would be very interesting to determine the intrapatient correlation between hepatic and circulating miR-122 expression levels in order to determine the nature of this relationship and how it relates to patient outcome, rather than drawing conclusions based on averages between the heterogeneous groups. The circulating miR-122 data presented, although from a larger cohort, could have been heavily influenced by a few potential outliers. Indeed, this is reflected by the weak association from the receiver operating characteristic analysis.
1440
CORRESPONDENCE
The observations published by John et al. are in conflict with our results published in a larger, well-defined cohort of patients with ALI induced by acetaminophen that show a trend toward increased circulating levels in patients with worse prognosis and outcome.2 We are concerned that the conclusions from this present study are based on patient cohorts with etiologies of ALI that are very diverse and differ substantially between survivors and nonsurvivors, which further complicates the interpretation of liver miR122 expression data. It is possible, even probable, that the natural histories of these underlying disease processes are diverse enough to explain differences in miRNA expression, and the conclusions of this study should be interpreted in light of this. In addition, we have shown that the kinetics of miR-122 are significantly different, compared with other biomarkers, and that miR-122 is raised earlier in blood, compared with more established biomarkers.6 The current conclusions on the quantifiable level of circulating miR122 reflecting patient outcome would be easier to interpret if these variables had been presented (i.e., time taken from biomarker measurement to outcome for each group). In summary, we welcome the important contributions of this current study. Once our concerns can be addressed, the field can move forward together to define the context of use of miRNAs as biomarkers of liver injury with the possibility of their application in patient stratification. DANIEL J. ANTOINE, PH.D.1 JAMES W. DEAR, PH.D.2,3 CHRISTOPHER E. GOLDRING, PH.D.1 B. KEVIN PARK, PH.D.1 1 MRC Center for Drug Safety Science Department of Molecular and Clinical Pharmacology University of Liverpool Liverpool, UK 2 University/BHF Center for Cardiovascular Science Edinburgh University Edinburgh, UK 3 NPIS Edinburgh Scottish Poisons Information Bureau Royal Infirmary of Edinburgh Edinburgh, UK
References 1. John K, Hadem J, Krech T, Wahl K, Manns M, Dooley S, et al. MicroRNAs play a role for spontaneous recovery from acute liver failure. HEPATOLOGY 2014;60:1346-1355. 2. Starkey Lewis PJ, Dear J, Platt V, Simpson KJ, Craig DG, Antoine DJ, et al. Circulating microRNAs as potential markers of human druginduced liver injury. HEPATOLOGY 2011;54:1767-1776. 3. Sekine S, Ogawa R, Ito R, Hiraoka N, McManus MT, Kanai Y, Hebrok M. Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis. Gastroenterology 2009;136:23042315.e1-4. 4. Hsu SH, Wang B, Kota J, Yu J, Costinean S, Kutay H, et al. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR122 in liver. J Clin Invest 2012;122:2871-2883. 5. Wang K, Zhang S, Marzolf B, Troisch P, Brightman A, Hu Z, Hood LE, et al. Circulating microRNAs, potential biomarkers for druginduced liver injury. Proc Natl Acad Sci U S A 2009;106:4402-4407. 6. Antoine DJ, Dear JW, Lewis PS, Platt V, Coyle J, Masson M, et al. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. HEPATOLOGY 2013;58:777-787.
HEPATOLOGY, April 2015
Author names in bold designate shared co-first authorship. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27349 Potential conflict of interest: Nothing to report.
Reply: We thank Dr. Antoine and colleagues for their comment on our publication by John et al.1 Our study aimed at investigating the role of certain microRNAs (miRNAs), which have been implicated in liver cell death or regeneration, in sera from patients revealing either spontaneous (SR) or nonspontaneous recovery (NSR) from acute liver failure (ALF; n 5 63). The time interval between acquisition of serum samples and the fulfillment of ALF criteria was 1-2 days (n 5 46; 66.7% of SR and 76.2% of NSR patients) or 3-12 days (n 5 17). We demonstrated that ALF patients with SR revealed significantly higher levels of the proregenerative miR-21 and 2221 compared to NSR. Compared to NSR patients, we also found increased miR-122 serum levels in SR patients, although the role of this miRNA in liver regeneration remains less clear compared to miR-21 and -221.1 Antoine et al. mention that deletion of Dicer—a regulator of miRNA processing—is associated with enhanced hepatocyte proliferation. As reported by others, however, elimination of Dicer results in overwhelming apoptosis and therefore primarily impairs cell survival, which was not only observed in hepatocytes but also other cell types.2,3 Likewise, deletion of miR-122 first results in liver damage, hepatitis, and steatosis, even though under certain circumstances, i.e., the presence of oncogenic stimuli, Dicer or miR-122 deletion might trigger compensatory processes promoting hepatocarcinogenesis.2,4 Similarly, hepatocyte-specific deletion of another component of miRNA processing (DGCR8) resulted in delayed liver regeneration.5 This is in line with our observation that increased miR-122 levels in sera of spontaneous survivors are associated with downregulated target genes that impair liver regeneration.1 It has to be noticed, however, that conclusions from genetically manipulated, i.e., miRNA-deficient, mice remain limited and that target genes of various miRNAs might differ between mice and humans. Antoine et al. found elevated miR-122 serum levels in patients with acetaminophen-mediated acute liver injury.6 Acute liver injury is a strong trigger for liver regeneration and the duration or intensity of the cytotoxic stimulus might determine whether cell death overwhelms the regenerative capacity of the liver. Thus, the enhanced miR-122 serum levels in patients with acetaminophen-induced liver injury might either reflect liver cell death or regeneration. In a mouse model of acetaminophen intoxication, enhanced miR-122 serum levels were associated with a reciprocal decrease in liver tissues following 24 hours of acetaminophen application.7 The changes of miR-122 levels in that study were dependent on the dose and duration of drug exposure, albeit the intoxication outcome was not reported. In our study, we found significantly decreased levels of miR-221 and 221 in liver tissues of SR compared to NSR patients, whereas miR-122 levels were not reduced in the SR patients.1 Differences in the kinetic and release of miRNAs might explain this observation. Nevertheless, the time between sampling of serum and liver tissues did not exceed more than 1 day in all of our SR patients and 50% of the NSR patients. Only from two NSR patients biopsies were taken 5 or 6 days later than the sera, and in just one NSR patient the sampling of the serum and biopsy differed by 4 weeks. We agree that analysis of a larger number of liver tissues would be desirable, although this remains difficult considering the higher risk of liver biopsy in critically ill patients. It is well documented that ALF outcome is influenced by etiology.8 Thus, it is not surprising that etiologies differ between
2
Department of General Surgery and Organ Transplantation Umberto I Hospital Sapienza University Rome, Italy
References 1. Halazun KJ, Patzer RE, Rana AA, Verna EC, Griesemer AD, Parsons RF, et al. Standing the test of time: outcomes of a decade of prioritizing patients with HCC: results of the UNOS natural geographic experiment. HEPATOLOGY 2014;60:1957–1962. 2. Samoylova ML, Dodge JL, Yao FY, Roberts JP. Time-to-transplant as a predictor of hepatocellular carcinoma recurrence after liver transplant. Liver Transpl 2014 [Epub ahead of print]. 3. Lai Q, Avolio AW, Lerut J, Singh G, Chan SC, Berloco PB, et al. Recurrence of hepatocellular cancer after liver transplantation: the role of primary resection and salvage transplantation in East and West. J Hepatol 2012;57:974-979. 4. Lai Q, Lerut JP. Hepatocellular cancer: how to expand safely inclusion criteria for liver transplantation. Curr Opin Organ Transplant. 2014;19:229-234. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27434 Potential conflict of interest: Dr. Lerut received grants from Fresenius Biotech and Takeda.
Reply: We thank the authors for their insightful comments. The lack of uniformity in organ allocation among regions has allowed us and others to examine the vast regional variation in waiting times to discover major differences in the outcomes of hepatocellular carcinoma (HCC) patients on the list, both with respect to recurrence and overall outcome.1,2 It is clear that uniformity among transplant centers in the U.S. and worldwide in both access and treatment of these patients is required, and will allow the transplant community to better understand how to manage patients with HCC on the waiting list. While the Milan criteria are widely accepted despite having no specific tumor biology indices associated with them, many groups including our own have attempted to define markers of poor biology; however, none of these have been universally accepted or applied to organ allocation models.3,4 Currently, it
CORRESPONDENCE
1439
seems that lack of progression of tumor while waiting for a liver transplant, or successful down-staging of tumor, may be the best available biological marker, as demonstrated by the UCSF group and our article.1,2,5 We agree that the development, and universal implementation of appropriate and reproducible biomarkers of tumor biology to allocation models of liver transplantation for HCC is required in order to optimize outcomes for HCC patients. KARIM J. HALAZUN, M.D. ABBAS A. RANA, M.D. RACHEL E. PATZER, PH.D. BENJAMIN SAMSTEIN, M.D. ROBERT S. BROWN, Jr., M.D. JEAN C. EMOND, M.D. Columbia University Center for Liver Disease and Transplantation New York, NY
References 1. Samoylova ML, Dodge JL, Yao FY, Roberts JP. Time-to-transplant as a predictor of hepatocellular carcinoma recurrence after liver transplant. Liver Transpl 2014 [Epub ahead of print]. 2. Halazun KJ, Patzer RE, Rana AA, Verna EC, Griesemer AD, Parsons RF, et al. Standing the test of time: outcomes of a decade of prioritizing patients with HCC, results of the UNOS natural geographic experiment. HEPATOLOGY 2014;60:1957-1962. 3. Halazun KJ, Hardy MA, Rana AA, Woodland DC 4th, Luyten EJ, Mahadev S, et al. Negative impact of neutrophil-lymphocyte ratio on outcome after liver transplantation for hepatocellular carcinoma. Ann Surg 2009;250:141-151. 4. Vibert E1, Azoulay D, Hoti E, Iacopinelli S, Samuel D, Salloum C, et al. Progression of alphafetoprotein before liver transplantation for hepatocellular carcinoma in cirrhotic patients: a critical factor. Am J Transplant 2010;10:129-137. 5. Roberts JP, Venook A, Kerlan R, Yao F. Hepatocellular carcinoma: ablate and wait versus rapid transplantation. Liver Transpl 2010;16:925-929. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27435 Potential conflict of interest: Dr. Brown consults for and received grants from Gilead, AbbVie, Janssen, and Vertex. He consults for Merck, Genentech, and Salix.
Understanding the Pathophysiological Regulatory Role of MicroRNAs in Acute Liver Failure To the Editor: We read with great interest the recent article by John et al. in HEPATOLOGY that described a potential role for microRNAs (miRNAs) in spontaneous recovery from acute liver failure (ALI).1 These potentially important data demonstrate elevated circulating miRNAs, especially miR-122, in human subjects with ALI, which confirms published work from our group and elsewhere.2 However, we have significant concerns about a number of the findings reported in this article. The investigators report that higher circulating and liver levels of miR-122 relate to spontaneous survival and contribute to regeneration and enhanced proliferation. These conclusions directly conflict with the well-established, replicated findings that miR-122 knockout mice and mice with deletions in miR-122 regulators, such as Dicer1, have enhanced levels of hepatocyte proliferation,3 that the loss of miR-122 from hepato-
cytes results in reduced differentiation and enhanced proliferation,4 and that the levels of miR-122 are decreased in liver, yet increased in blood, of rodent models of ALI.5 Furthermore, we feel that robust conclusions cannot be based on liver miR-122 expression data from a very low number of patients (n 5 4 and n 5 6 for recovery and nonrecovery, respectively). We appreciate that liver tissue cannot always be obtained; but for the small patient cohort where liver samples were collected, it would be very interesting to determine the intrapatient correlation between hepatic and circulating miR-122 expression levels in order to determine the nature of this relationship and how it relates to patient outcome, rather than drawing conclusions based on averages between the heterogeneous groups. The circulating miR-122 data presented, although from a larger cohort, could have been heavily influenced by a few potential outliers. Indeed, this is reflected by the weak association from the receiver operating characteristic analysis.
1440
CORRESPONDENCE
The observations published by John et al. are in conflict with our results published in a larger, well-defined cohort of patients with ALI induced by acetaminophen that show a trend toward increased circulating levels in patients with worse prognosis and outcome.2 We are concerned that the conclusions from this present study are based on patient cohorts with etiologies of ALI that are very diverse and differ substantially between survivors and nonsurvivors, which further complicates the interpretation of liver miR122 expression data. It is possible, even probable, that the natural histories of these underlying disease processes are diverse enough to explain differences in miRNA expression, and the conclusions of this study should be interpreted in light of this. In addition, we have shown that the kinetics of miR-122 are significantly different, compared with other biomarkers, and that miR-122 is raised earlier in blood, compared with more established biomarkers.6 The current conclusions on the quantifiable level of circulating miR122 reflecting patient outcome would be easier to interpret if these variables had been presented (i.e., time taken from biomarker measurement to outcome for each group). In summary, we welcome the important contributions of this current study. Once our concerns can be addressed, the field can move forward together to define the context of use of miRNAs as biomarkers of liver injury with the possibility of their application in patient stratification. DANIEL J. ANTOINE, PH.D.1 JAMES W. DEAR, PH.D.2,3 CHRISTOPHER E. GOLDRING, PH.D.1 B. KEVIN PARK, PH.D.1 1 MRC Center for Drug Safety Science Department of Molecular and Clinical Pharmacology University of Liverpool Liverpool, UK 2 University/BHF Center for Cardiovascular Science Edinburgh University Edinburgh, UK 3 NPIS Edinburgh Scottish Poisons Information Bureau Royal Infirmary of Edinburgh Edinburgh, UK
References 1. John K, Hadem J, Krech T, Wahl K, Manns M, Dooley S, et al. MicroRNAs play a role for spontaneous recovery from acute liver failure. HEPATOLOGY 2014;60:1346-1355. 2. Starkey Lewis PJ, Dear J, Platt V, Simpson KJ, Craig DG, Antoine DJ, et al. Circulating microRNAs as potential markers of human druginduced liver injury. HEPATOLOGY 2011;54:1767-1776. 3. Sekine S, Ogawa R, Ito R, Hiraoka N, McManus MT, Kanai Y, Hebrok M. Disruption of Dicer1 induces dysregulated fetal gene expression and promotes hepatocarcinogenesis. Gastroenterology 2009;136:23042315.e1-4. 4. Hsu SH, Wang B, Kota J, Yu J, Costinean S, Kutay H, et al. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR122 in liver. J Clin Invest 2012;122:2871-2883. 5. Wang K, Zhang S, Marzolf B, Troisch P, Brightman A, Hu Z, Hood LE, et al. Circulating microRNAs, potential biomarkers for druginduced liver injury. Proc Natl Acad Sci U S A 2009;106:4402-4407. 6. Antoine DJ, Dear JW, Lewis PS, Platt V, Coyle J, Masson M, et al. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. HEPATOLOGY 2013;58:777-787.
HEPATOLOGY, April 2015
Author names in bold designate shared co-first authorship. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27349 Potential conflict of interest: Nothing to report.
Reply: We thank Dr. Antoine and colleagues for their comment on our publication by John et al.1 Our study aimed at investigating the role of certain microRNAs (miRNAs), which have been implicated in liver cell death or regeneration, in sera from patients revealing either spontaneous (SR) or nonspontaneous recovery (NSR) from acute liver failure (ALF; n 5 63). The time interval between acquisition of serum samples and the fulfillment of ALF criteria was 1-2 days (n 5 46; 66.7% of SR and 76.2% of NSR patients) or 3-12 days (n 5 17). We demonstrated that ALF patients with SR revealed significantly higher levels of the proregenerative miR-21 and 2221 compared to NSR. Compared to NSR patients, we also found increased miR-122 serum levels in SR patients, although the role of this miRNA in liver regeneration remains less clear compared to miR-21 and -221.1 Antoine et al. mention that deletion of Dicer—a regulator of miRNA processing—is associated with enhanced hepatocyte proliferation. As reported by others, however, elimination of Dicer results in overwhelming apoptosis and therefore primarily impairs cell survival, which was not only observed in hepatocytes but also other cell types.2,3 Likewise, deletion of miR-122 first results in liver damage, hepatitis, and steatosis, even though under certain circumstances, i.e., the presence of oncogenic stimuli, Dicer or miR-122 deletion might trigger compensatory processes promoting hepatocarcinogenesis.2,4 Similarly, hepatocyte-specific deletion of another component of miRNA processing (DGCR8) resulted in delayed liver regeneration.5 This is in line with our observation that increased miR-122 levels in sera of spontaneous survivors are associated with downregulated target genes that impair liver regeneration.1 It has to be noticed, however, that conclusions from genetically manipulated, i.e., miRNA-deficient, mice remain limited and that target genes of various miRNAs might differ between mice and humans. Antoine et al. found elevated miR-122 serum levels in patients with acetaminophen-mediated acute liver injury.6 Acute liver injury is a strong trigger for liver regeneration and the duration or intensity of the cytotoxic stimulus might determine whether cell death overwhelms the regenerative capacity of the liver. Thus, the enhanced miR-122 serum levels in patients with acetaminophen-induced liver injury might either reflect liver cell death or regeneration. In a mouse model of acetaminophen intoxication, enhanced miR-122 serum levels were associated with a reciprocal decrease in liver tissues following 24 hours of acetaminophen application.7 The changes of miR-122 levels in that study were dependent on the dose and duration of drug exposure, albeit the intoxication outcome was not reported. In our study, we found significantly decreased levels of miR-221 and 221 in liver tissues of SR compared to NSR patients, whereas miR-122 levels were not reduced in the SR patients.1 Differences in the kinetic and release of miRNAs might explain this observation. Nevertheless, the time between sampling of serum and liver tissues did not exceed more than 1 day in all of our SR patients and 50% of the NSR patients. Only from two NSR patients biopsies were taken 5 or 6 days later than the sera, and in just one NSR patient the sampling of the serum and biopsy differed by 4 weeks. We agree that analysis of a larger number of liver tissues would be desirable, although this remains difficult considering the higher risk of liver biopsy in critically ill patients. It is well documented that ALF outcome is influenced by etiology.8 Thus, it is not surprising that etiologies differ between
