Tracking HCC pathogenesis but not the tumor cells after biopsy
Authors: Scott L. Friedman
Journal: Journal of Hepatology (2014)

Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagyJournal of HepatologyVol. 61Issue 3PreviewAutophagy is an intracellular lysosomal degradation process that plays an important role in regulating normal physiological functions of the liver. The purpose of the present study was to investigate the mechanism(s) by which the loss of hepatic autophagy leads to liver inflammation, fibrosis and tumorigenesis. Full-Text PDF Preoperative tumour biopsy does not affect the oncologic course of patients with transplantable HCCJournal of HepatologyVol. 61Issue 3PreviewPreoperative fine-needle aspiration biopsy (PFNAB) allows obtaining reliable hepatocellular carcinoma (HCC) diagnosis before liver transplantation (LT) in doubtful situations, but may result in higher recurrence rates following LT. This study aimed to evaluate whether PFNAB actually jeopardized the outcome of patients with transplantable HCC. Full-Text PDF Two articles in this month’s issue of the Journal reveal new insights into intracellular pathways in hepatocytes that contribute to tumorigenesis in an animal model, and provide reassurance about the safety of percutaneous fine needle biopsy in patients with suspected HCC being evaluated for liver transplantation.The first article, by Ni et al., investigates the consequences of blocking autophagy, a vital intracellular degradative pathway, in hepatocytes of experimental mice, and uncovers Nrf2 as an important mediator that contributes to the resulting fibrosis and tumorigenesis. As reviewed in one of my earlier Focus pieces, autophagy is a well-regulated pathway that helps preserve energy homeostasis by degrading intracellular substrates when the cell is stressed or starved [1Czaja M.J. Ding W.X. Donohue Jr., T.M. Friedman S.L. Kim J.S. Komatsu M. et al.Functions of autophagy in normal and diseased liver.Autophagy. 2013; 9: 1131-1158Crossref PubMed Scopus (328) Google Scholar, 2Choi A.M. Ryter S.W. Levine B. Autophagy in human health and disease.N Engl J Med. 2013; 368: 651-662Crossref PubMed Scopus (962) Google Scholar]. A growing number of mediator proteins in the autophagy cascade have been uncovered, and two in particular, Atg5 and Atg7, have been knocked out in several studies by using a cre-lox strategy, in which inserted LoxP sites flank a portion of the coding region – when these ‘floxed’ mice are crossed with a mouse expressing the albumin promoter driving Cre recombinase, the gene is specifically deleted in hepatocytes. Using this approach, Ni and colleagues have characterized the phenotype of a hepatocyte-specific Atg5 knockout mice. As reported previously with a knockout of Atg7 [[3]Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. et al.Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1882) Google Scholar], mice with loss of Atg5 have gross hepatic morphologic abnormalities, including cell death, inflammation, fibrosis and then hepatic tumors.A model of this type provides important opportunities to determine novel pathways of disease pathogenesis in rodents that can be used to illuminate our understanding of the human disease, and the authors have exploited this opportunity to uncover a role for the protein Nrf2 (nuclear factor erythroid 2–related factor 2) in mediating the phenotype. Nrf2 is a master transcription factor that drives induction of a range of antioxidant and detoxification genes [4Ma Q. Role of Nrf2 in oxidative stress and toxicity.Annu Rev Pharmacol Toxicol. 2013; 53: 401-426Crossref PubMed Scopus (2404) Google Scholar, 5Suzuki T. Motohashi H. Yamamoto M. Toward clinical application of the Keap1-Nrf2 pathway.Trends Pharmacol Sci. 2013; 34: 340-346Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar]. Its activity is normally restrained by cytoplasmic sequestration through binding to the intracellular protein Keap1 (kelch-like ECH-associated protein 1), and thus therapeutic strategies that dissociate Keap1 from Nrf2 will enhance its nuclear localization and lead to increased expression of antioxidant defense genes [[5]Suzuki T. Motohashi H. Yamamoto M. Toward clinical application of the Keap1-Nrf2 pathway.Trends Pharmacol Sci. 2013; 34: 340-346Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar]. Interestingly, earlier studies demonstrated that when autophagy is inhibited, a specific protein called p62 accumulates. This protein targets intracellular substrates for autophagy, and earlier studies demonstrated that when p62 accumulates in this setting it binds to Keap1, and thus liberates Nrf2 for nuclear translocation and activation of target genes [[6]Komatsu M. Kurokawa H. Waguri S. Taguchi K. Kobayashi A. Ichimura Y. et al.The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.Nat Cell Biol. 2010; 12: 213-223Crossref PubMed Scopus (1562) Google Scholar]. Therefore, these earlier studies indicate that the excessive activation of Nrf2 arising as a result of autophagy inhibition might contribute to the phenotype of livers that lack Atg5 or Atg7.Ni and colleagues have addressed this possibility elegantly and definitively by simply crossing the mice whose livers lack autophagy with animals that additionally lack Nrf2, creating a ‘double knockout’ mouse. Remarkably, the abnormalities evident in the Atg5 knockout livers were abrogated when Nrf2 was absent, clearly indicating that the major defects in these livers lacking autophagy are attributable to Nrf2; in other words, loss of Nrf2 rescues the Atg5 knockout phenotype. The authors also demonstrate that Atg5 is intact in hepatic stellate cells, suggesting that the fibrosis that occurs in Atg5 hepatocyte knockout mice probably results from stellate cell activation following release of mediators from damaged hepatocytes, not from altered autophagy in stellate cells. Indeed, autophagy is required for stellate cells to activate [[7]Hernandez-Gea V. Ghiassi-Nejad Z. Rozenfeld R. Gordon R. Fiel M.I. Yue Z. et al.Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.Gastroenterology. 2012; 142: 938-946Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar]. Moreover, since tumor development is also abrogated when Nrf2 is lost, the findings further suggest that without the severe damage to hepatocytes, combined with elimination of the fibrotic microenvironment, the risk of neoplasia is mitigated.The surprising but clear implication of these findings is that Nrf2, whose role is to enhance antioxidant and detoxification pathways, also has a dark side when its activity is unrestrained. The authors propose several credible explanations for this paradoxical behavior of Nrf2, which should be tested experimentally. The broader implication is that there is an optimal level of activity of key autophagic and antioxidant mediators in hepatocytes required in order to maintain healthy intracellular pathways of energy and substrate homeostasis. The findings of Ni et al. yield valuable insight into these pathways, and also offer a cautionary tale about the potential deleterious impact of manipulating them therapeutically without fully understanding the consequences.Both our understanding of HCC pathogenesis and advances in its management have been greatly accelerated by uncovering specific patterns of liver gene expression that correlate with clinical outcomes [8Hoshida Y. Moeini A. Alsinet C. Kojima K. Villanueva A. Gene signatures in the management of hepatocellular carcinoma.Semin Oncol. 2012; 39: 473-485Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Nault J.C. De Reynies A. Villanueva A. Calderaro J. Rebouissou S. Couchy G. et al.A hepatocellular carcinoma 5-gene score associated with survival of patients after liver resection.Gastroenterology. 2013; 145: 176-187Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar]. Indeed, we are entering an era where comprehensive assessment of gene expression patterns both in the tumor and in the surrounding stroma of HCC will help personalize therapy and refine the prediction of recurrence after surgery or transplantation. For example, a ‘favorable’ gene signature in the stroma or tumor in a patient whose HCC exceeds Milan criteria could reinstate the possibility of liver transplantation. Of course, biopsy is required to obtain these gene expression signatures preoperatively. So it is ironic that genetic information assessed from biopsies is becoming very valuable at a time when the definitive diagnosis of HCC no longer requires tissue confirmation, making routine biopsies less common in diagnosing this tumor. Concerns about performing directed biopsy of suspicious lesions have included the possibility of not accessing the tumor and of bleeding or other complications. Another concern has been the risk of tracking tumor cells along the needle path, thereby worsening the prognosis prior to attempts at curative therapy. Studies estimate a risk of needle track seeding of 2.3–2.7% when performing liver biopsy to diagnose HCC [10Silva M.A. Hegab B. Hyde C. Guo B. Buckels J.A. Mirza D.F. Needle track seeding following biopsy of liver lesions in the diagnosis of hepatocellular cancer: a systematic review and meta-analysis.Gut. 2008; 57: 1592-1596Crossref PubMed Scopus (348) Google Scholar, 11Stigliano R. Marelli L. Yu D. Davies N. Patch D. Burroughs A.K. Seeding following percutaneous diagnostic and therapeutic approaches for hepatocellular carcinoma. What is the risk and the outcome? Seeding risk for percutaneous approach of HCC.Cancer Treat Rev. 2007; 33: 437-447Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar].The article by Fuks et al. from Hopital Beaujon in Paris in this issue of the Journal examines the risk of biopsy in a different, but very sensible way by asking, “Do patients who have a percutaneous needle biopsy for HCC diagnosis prior to listing for liver transplantation have a poorer outcome than those who did not have a biopsy?”. The authors address this question by comparing the outcomes among 309 patients listed for liver transplantation, 80 of whom underwent pre-operative percutaneous fine needle aspiration biopsy, and 229 patients of whom did not, with each group matched for several key variables that could influence outcomes, although they were not matched for tumor stage. Among the 309 patients, 278 were eventually transplanted, and the survival and rates of tumor recurrence were not affected by whether the patient had a biopsy in either transplanted or non-transplanted patients.While the study is relatively small and from a single center, the findings nonetheless suggest that percutaneous biopsy does not alter the outcome in patients with suspected HCC who are being evaluated for liver transplantation. Although the risk of tumor cell tracking by biopsy is real, its overall impact is likely to be modest in this setting. Moreover, as the value of genetic and diagnostic information available from biopsies continues to increase, investigators should not be dissuaded from incorporating biopsy into prognostic and management algorithms if it will greatly improve risk stratification or decision-making. As they do so, larger data sets should be accumulated to confirm the safety of biopsy in the preoperative management of liver transplantation candidates with suspected HCC, while also tracking whether the additional information gained from biopsy improves patient selection and outcomes.Conflict of interestThe authors declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Two articles in this month’s issue of the Journal reveal new insights into intracellular pathways in hepatocytes that contribute to tumorigenesis in an animal model, and provide reassurance about the safety of percutaneous fine needle biopsy in patients with suspected HCC being evaluated for liver transplantation. The first article, by Ni et al., investigates the consequences of blocking autophagy, a vital intracellular degradative pathway, in hepatocytes of experimental mice, and uncovers Nrf2 as an important mediator that contributes to the resulting fibrosis and tumorigenesis. As reviewed in one of my earlier Focus pieces, autophagy is a well-regulated pathway that helps preserve energy homeostasis by degrading intracellular substrates when the cell is stressed or starved [1Czaja M.J. Ding W.X. Donohue Jr., T.M. Friedman S.L. Kim J.S. Komatsu M. et al.Functions of autophagy in normal and diseased liver.Autophagy. 2013; 9: 1131-1158Crossref PubMed Scopus (328) Google Scholar, 2Choi A.M. Ryter S.W. Levine B. Autophagy in human health and disease.N Engl J Med. 2013; 368: 651-662Crossref PubMed Scopus (962) Google Scholar]. A growing number of mediator proteins in the autophagy cascade have been uncovered, and two in particular, Atg5 and Atg7, have been knocked out in several studies by using a cre-lox strategy, in which inserted LoxP sites flank a portion of the coding region – when these ‘floxed’ mice are crossed with a mouse expressing the albumin promoter driving Cre recombinase, the gene is specifically deleted in hepatocytes. Using this approach, Ni and colleagues have characterized the phenotype of a hepatocyte-specific Atg5 knockout mice. As reported previously with a knockout of Atg7 [[3]Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. et al.Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1882) Google Scholar], mice with loss of Atg5 have gross hepatic morphologic abnormalities, including cell death, inflammation, fibrosis and then hepatic tumors. A model of this type provides important opportunities to determine novel pathways of disease pathogenesis in rodents that can be used to illuminate our understanding of the human disease, and the authors have exploited this opportunity to uncover a role for the protein Nrf2 (nuclear factor erythroid 2–related factor 2) in mediating the phenotype. Nrf2 is a master transcription factor that drives induction of a range of antioxidant and detoxification genes [4Ma Q. Role of Nrf2 in oxidative stress and toxicity.Annu Rev Pharmacol Toxicol. 2013; 53: 401-426Crossref PubMed Scopus (2404) Google Scholar, 5Suzuki T. Motohashi H. Yamamoto M. Toward clinical application of the Keap1-Nrf2 pathway.Trends Pharmacol Sci. 2013; 34: 340-346Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar]. Its activity is normally restrained by cytoplasmic sequestration through binding to the intracellular protein Keap1 (kelch-like ECH-associated protein 1), and thus therapeutic strategies that dissociate Keap1 from Nrf2 will enhance its nuclear localization and lead to increased expression of antioxidant defense genes [[5]Suzuki T. Motohashi H. Yamamoto M. Toward clinical application of the Keap1-Nrf2 pathway.Trends Pharmacol Sci. 2013; 34: 340-346Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar]. Interestingly, earlier studies demonstrated that when autophagy is inhibited, a specific protein called p62 accumulates. This protein targets intracellular substrates for autophagy, and earlier studies demonstrated that when p62 accumulates in this setting it binds to Keap1, and thus liberates Nrf2 for nuclear translocation and activation of target genes [[6]Komatsu M. Kurokawa H. Waguri S. Taguchi K. Kobayashi A. Ichimura Y. et al.The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1.Nat Cell Biol. 2010; 12: 213-223Crossref PubMed Scopus (1562) Google Scholar]. Therefore, these earlier studies indicate that the excessive activation of Nrf2 arising as a result of autophagy inhibition might contribute to the phenotype of livers that lack Atg5 or Atg7. Ni and colleagues have addressed this possibility elegantly and definitively by simply crossing the mice whose livers lack autophagy with animals that additionally lack Nrf2, creating a ‘double knockout’ mouse. Remarkably, the abnormalities evident in the Atg5 knockout livers were abrogated when Nrf2 was absent, clearly indicating that the major defects in these livers lacking autophagy are attributable to Nrf2; in other words, loss of Nrf2 rescues the Atg5 knockout phenotype. The authors also demonstrate that Atg5 is intact in hepatic stellate cells, suggesting that the fibrosis that occurs in Atg5 hepatocyte knockout mice probably results from stellate cell activation following release of mediators from damaged hepatocytes, not from altered autophagy in stellate cells. Indeed, autophagy is required for stellate cells to activate [[7]Hernandez-Gea V. Ghiassi-Nejad Z. Rozenfeld R. Gordon R. Fiel M.I. Yue Z. et al.Autophagy releases lipid that promotes fibrogenesis by activated hepatic stellate cells in mice and in human tissues.Gastroenterology. 2012; 142: 938-946Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar]. Moreover, since tumor development is also abrogated when Nrf2 is lost, the findings further suggest that without the severe damage to hepatocytes, combined with elimination of the fibrotic microenvironment, the risk of neoplasia is mitigated. The surprising but clear implication of these findings is that Nrf2, whose role is to enhance antioxidant and detoxification pathways, also has a dark side when its activity is unrestrained. The authors propose several credible explanations for this paradoxical behavior of Nrf2, which should be tested experimentally. The broader implication is that there is an optimal level of activity of key autophagic and antioxidant mediators in hepatocytes required in order to maintain healthy intracellular pathways of energy and substrate homeostasis. The findings of Ni et al. yield valuable insight into these pathways, and also offer a cautionary tale about the potential deleterious impact of manipulating them therapeutically without fully understanding the consequences. Both our understanding of HCC pathogenesis and advances in its management have been greatly accelerated by uncovering specific patterns of liver gene expression that correlate with clinical outcomes [8Hoshida Y. Moeini A. Alsinet C. Kojima K. Villanueva A. Gene signatures in the management of hepatocellular carcinoma.Semin Oncol. 2012; 39: 473-485Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Nault J.C. De Reynies A. Villanueva A. Calderaro J. Rebouissou S. Couchy G. et al.A hepatocellular carcinoma 5-gene score associated with survival of patients after liver resection.Gastroenterology. 2013; 145: 176-187Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar]. Indeed, we are entering an era where comprehensive assessment of gene expression patterns both in the tumor and in the surrounding stroma of HCC will help personalize therapy and refine the prediction of recurrence after surgery or transplantation. For example, a ‘favorable’ gene signature in the stroma or tumor in a patient whose HCC exceeds Milan criteria could reinstate the possibility of liver transplantation. Of course, biopsy is required to obtain these gene expression signatures preoperatively. So it is ironic that genetic information assessed from biopsies is becoming very valuable at a time when the definitive diagnosis of HCC no longer requires tissue confirmation, making routine biopsies less common in diagnosing this tumor. Concerns about performing directed biopsy of suspicious lesions have included the possibility of not accessing the tumor and of bleeding or other complications. Another concern has been the risk of tracking tumor cells along the needle path, thereby worsening the prognosis prior to attempts at curative therapy. Studies estimate a risk of needle track seeding of 2.3–2.7% when performing liver biopsy to diagnose HCC [10Silva M.A. Hegab B. Hyde C. Guo B. Buckels J.A. Mirza D.F. Needle track seeding following biopsy of liver lesions in the diagnosis of hepatocellular cancer: a systematic review and meta-analysis.Gut. 2008; 57: 1592-1596Crossref PubMed Scopus (348) Google Scholar, 11Stigliano R. Marelli L. Yu D. Davies N. Patch D. Burroughs A.K. Seeding following percutaneous diagnostic and therapeutic approaches for hepatocellular carcinoma. What is the risk and the outcome? Seeding risk for percutaneous approach of HCC.Cancer Treat Rev. 2007; 33: 437-447Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar]. The article by Fuks et al. from Hopital Beaujon in Paris in this issue of the Journal examines the risk of biopsy in a different, but very sensible way by asking, “Do patients who have a percutaneous needle biopsy for HCC diagnosis prior to listing for liver transplantation have a poorer outcome than those who did not have a biopsy?”. The authors address this question by comparing the outcomes among 309 patients listed for liver transplantation, 80 of whom underwent pre-operative percutaneous fine needle aspiration biopsy, and 229 patients of whom did not, with each group matched for several key variables that could influence outcomes, although they were not matched for tumor stage. Among the 309 patients, 278 were eventually transplanted, and the survival and rates of tumor recurrence were not affected by whether the patient had a biopsy in either transplanted or non-transplanted patients. While the study is relatively small and from a single center, the findings nonetheless suggest that percutaneous biopsy does not alter the outcome in patients with suspected HCC who are being evaluated for liver transplantation. Although the risk of tumor cell tracking by biopsy is real, its overall impact is likely to be modest in this setting. Moreover, as the value of genetic and diagnostic information available from biopsies continues to increase, investigators should not be dissuaded from incorporating biopsy into prognostic and management algorithms if it will greatly improve risk stratification or decision-making. As they do so, larger data sets should be accumulated to confirm the safety of biopsy in the preoperative management of liver transplantation candidates with suspected HCC, while also tracking whether the additional information gained from biopsy improves patient selection and outcomes. Conflict of interestThe authors declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.