Molecular tools and hepatocellular carcinoma: Adding help or confusion in liver transplantation?
Authors: Eric Vibert, Didier Samuel
Journal: Journal of Hepatology (2008)

Liver transplantation (LT) is in theory, the best treatment for hepatocellular carcinoma (HCC) in cirrhotic patients since it is a treatment that simultaneously deals with the malignancy and its causes [1Adam R. McMaster P. O’Grady J.G. Castaing D. Klempnauer J.L. Jamieson N. et al.Evolution of liver transplantation in Europe: report of the European Liver Transplant Registry.Liver Transpl. 2003; 9: 1231-1243Crossref PubMed Scopus (491) Google Scholar, 2Yoo H.Y. Patt C.H. Geschwind J.F. Thuluvath P.J. The outcome of liver transplantation in patients with hepatocellular carcinoma in the United States between 1988 and 2001: 5-year survival has improved significantly with time.J Clin Oncol. 2003; 21: 4329-4335Crossref PubMed Scopus (214) Google Scholar, 3El-Serag H.B. Siegel A.B. Davila J.A. Shaib Y.H. Cayton-Woody M. McBride R. et al.Treatment and outcomes of treating of hepatocellular carcinoma among medicare recipients in the United States: a population-based study.J Hepatol. 2006; 44: 158-166Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar]. However, the prognosis is directly related to the risk of HCC recurrence post-transplantation. The aim of preoperative assessment in LT for HCC is therefore to detect existing extra-hepatic metastases and predict extra-hepatic micro-metastases that are responsible for recurrence, graft loss and often, death of the patient.Following the retrospective study by Bismuth et al. [[4]Bismuth H. Chiche L. Adam R. Castaing D. Diamond T. Dennison A. Liver resection versus transplantation for hepatocellular carcinoma in cirrhotic patients.Ann Surg. 1993; 218: 145-151Crossref PubMed Scopus (823) Google Scholar], Mazzaferro et al. prospectively showed that careful selection of patients with a single lesion of <5 cm or three lesions each <3 cm, known as Milan criteria, has resulted in a satisfactory balance between survival (75%) at four years and recurrence rates (8%) [[5]Mazzaferro V. Chun Y.S. Poon R.T. Schwartz M.E. Yao F.Y. Marsh J.W. et al.Liver transplantation for hepatocellular carcinoma.Ann Surg Oncol. 2008; 15: 1001-1007Crossref PubMed Scopus (227) Google Scholar]. The validity of these criteria to predict recurrence-free survival after transplantation has been largely confirmed by several studies, which have demonstrated an overall 10-year survival ranging from 60% to 62% [6Jonas S. Bechstein W.O. Steinmuller T. Herrmann M. Radke C. Berg T. et al.Vascular invasion and histopathologic grading determine outcome after liver transplantation for hepatocellular carcinoma in cirrhosis.Hepatology. 2001; 33: 1080-1086Crossref PubMed Scopus (805) Google Scholar, 7Zavaglia C. De Carlis L. Alberti A.B. Minola E. Belli L.S. Slim A.O. et al.Predictors of long-term survival after liver transplantation for hepatocellular carcinoma.Am J Gastroenterol. 2005; 100: 2708-2716Crossref PubMed Scopus (214) Google Scholar]. Hence, survival of carefully selected patients is not different from survival of patients transplanted for non-malignant indications [[8]Mazzaferro V. Regalia E. Doci R. Andreola S. Pulvirenti A. Bozzetti F. et al.Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis.N Engl J Med. 1996; 334: 693-699Crossref PubMed Scopus (5580) Google Scholar]. Survival analysis by ITT clearly suggested that resection or ablation in patients with compensated cirrhosis are the only alternatives to liver transplantation on the wait list. Moreover there is growing evidence that by limiting OLT only to patients falling within the Milan criteria might represent an unjust penalty for selected patients beyond the Milan criteria who could still have a favourable outcome with OLT.In the United States, around 70% of cirrhotic patients diagnosed with a HCC fall beyond the Milan criteria [[3]El-Serag H.B. Siegel A.B. Davila J.A. Shaib Y.H. Cayton-Woody M. McBride R. et al.Treatment and outcomes of treating of hepatocellular carcinoma among medicare recipients in the United States: a population-based study.J Hepatol. 2006; 44: 158-166Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar]. Survival analysis by ITT clearly suggested that regular ultrasound screening of cirrhotic patients has permitted detection of small unique nodule in Child-Pugh A cirrhotic patients in whom alternatives to transplantation are possible. Second, liver transplantation will provide a major survival gain in some patients with a HCC who fall beyond the Milan criteria. Therefore, several expansions of the Milan criteria have been proposed, with the University of California, San Francisco (UCSF) criteria described by Yao et al. being the most investigated [9Yao F.Y. Ferrell L. Bass N.M. Watson J.J. Bacchetti P. Venook A. et al.Liver transplantation for hepatocellular carcinoma: expansion of the tumor size limits does not adversely impact survival.Hepatology. 2001; 33: 1394-1403Crossref PubMed Scopus (1732) Google Scholar, 10Yao F.Y. Xiao L. Bass N.M. Kerlan R. Ascher N.L. Roberts J.P. Liver transplantation for hepatocellular carcinoma: validation of the UCSF-expanded criteria based on preoperative imaging.Am J Transplant. 2007; 7: 2587-2596Crossref PubMed Scopus (407) Google Scholar]. The UCSF criteria include a solitary tumor of 6.5 cm or less, no more than three lesions with the largest being 4.5 cm or less, and a total tumor diameter 8 cm or less, without gross vascular invasion. Unfortunately, the 5-year survival of patients beyond Milan and within UCSF criteria ranges from 46% to 93% [9Yao F.Y. Ferrell L. Bass N.M. Watson J.J. Bacchetti P. Venook A. et al.Liver transplantation for hepatocellular carcinoma: expansion of the tumor size limits does not adversely impact survival.Hepatology. 2001; 33: 1394-1403Crossref PubMed Scopus (1732) Google Scholar, 10Yao F.Y. Xiao L. Bass N.M. Kerlan R. Ascher N.L. Roberts J.P. Liver transplantation for hepatocellular carcinoma: validation of the UCSF-expanded criteria based on preoperative imaging.Am J Transplant. 2007; 7: 2587-2596Crossref PubMed Scopus (407) Google Scholar, 11Decaens T. Roudot-Thoraval F. Hadni-Bresson S. Meyer C. Gugenheim J. Durand F. et al.Impact of UCSF criteria according to pre- and post-OLT tumor features: analysis of 479 patients listed for HCC with a short waiting time.Liver Transpl. 2006; 12: 1761-1769Crossref PubMed Scopus (160) Google Scholar, 12Duffy J.P. Vardanian A. Benjamin E. Watson M. Farmer D.G. Ghobrial R.M. et al.Liver transplantation criteria for hepatocellular carcinoma should be expanded: a 22-year experience with 467 patients at UCLA.Ann Surg. 2007; 246: 502-509Crossref PubMed Scopus (2) Google Scholar]. The resulting discrepancies between studies which used UCSF criteria are related to the time of application of the criteria (preoperative/radiological and postoperative/pathological). Interestingly, a French multicentric study of 479 patients demonstrated a higher rate of understaging (48%) if UCSF criteria is used to list patients for LT as compared to the understaging rate of 34% when the Milan criteria is used [[11]Decaens T. Roudot-Thoraval F. Hadni-Bresson S. Meyer C. Gugenheim J. Durand F. et al.Impact of UCSF criteria according to pre- and post-OLT tumor features: analysis of 479 patients listed for HCC with a short waiting time.Liver Transpl. 2006; 12: 1761-1769Crossref PubMed Scopus (160) Google Scholar]. Indeed, the 5-year survival of UCSF excluding Milan patients was 45% compared to 60% in Milan patients [[11]Decaens T. Roudot-Thoraval F. Hadni-Bresson S. Meyer C. Gugenheim J. Durand F. et al.Impact of UCSF criteria according to pre- and post-OLT tumor features: analysis of 479 patients listed for HCC with a short waiting time.Liver Transpl. 2006; 12: 1761-1769Crossref PubMed Scopus (160) Google Scholar]. Heterogeneous survival results in studies using UCSF criteria emphasized that macroscopic parameters like size and number would probably lose their efficacy as surrogate criteria to accurately predict the risk of tumor recurrence in comparison to tumoral volume exceeding the Milan criteria.Several studies have identified vascular invasion as the strongest independent predictor factor of recurrence, a pathological parameter also associated with other poor prognostic factors like a poor differentiation of the tumor, the presence of satellite nodules and a high level of α-fetoprotein [6Jonas S. Bechstein W.O. Steinmuller T. Herrmann M. Radke C. Berg T. et al.Vascular invasion and histopathologic grading determine outcome after liver transplantation for hepatocellular carcinoma in cirrhosis.Hepatology. 2001; 33: 1080-1086Crossref PubMed Scopus (805) Google Scholar, 13Pawlik T.M. Delman K.A. Vauthey J.N. Nagorney D.M. Ng I.O. Ikai I. et al.Tumor size predicts vascular invasion and histologic grade: implications for selection of surgical treatment for hepatocellular carcinoma.Liver Transpl. 2005; 11: 1086-1092Crossref PubMed Scopus (489) Google Scholar, 14Parfitt J.R. Marotta P. Alghamdi M. Wall W. Khakhar A. Suskin N.G. et al.Recurrent hepatocellular carcinoma after transplantation: use of a pathological score on explanted livers to predict recurrence.Liver Transpl. 2007; 13: 543-551Crossref PubMed Scopus (132) Google Scholar]. Unfortunately, except for the latter, all these parameters are not easily available in the preoperative setting and particularly at listing.In 2003, based on the knowledge that HCC display molecular alterations Marsh et al., classified HCC according to the level of gene damage [[15]Marsh J.W. Finkelstein S.D. Demetris A.J. Swalsky P.A. Sasatomi E. Bandos A. et al.Genotyping of hepatocellular carcinoma in liver transplant recipients adds predictive power for determining recurrence-free survival.Liver Transpl. 2003; 9: 664-671Crossref PubMed Scopus (120) Google Scholar]. The goal of this paper was to determine whether a panel of tumor suppressor gene markers could be useful to predict recurrence after LT for HCC. By microdissection, non-neoplastic and neoplastic tissue samples for each patient transplanted with a minimum of 5 years follow-up were evaluated for informative status of 18 genes located on microsatellite. Absence of microsatellites or loss of heterozygoty (LOH) corresponded to the loss of specific tumor suppressor genes such as APC, CDKN2A, p53, and p34. Among 18 microsatellites tested, 9 were significantly correlated with the post-transplant recurrence-free survival but none achieved perfect discrimination alone. As a test, two types of simplified panel of LOH that were the most accurate to predict recurrence were defined. Thanks to this method and with the help of an artificial neural network, an accurate response was obtained in 91 of the 103 patients tested (88%). The prediction of recurrence was accurate in 89% of cases.Very recently, the same team has performed a similar analysis on a larger cohort of patients transplanted for HCC with more than 5 years of follow-up (183 patients from Mount Sinai and University of Pittsburgh Medical Center) [[16]Dvorchik I. Schwartz M. Fiel M.I. Finkelstein S.D. Marsh J.W. Fractional allelic imbalance could allow for the development of an equitable transplant selection policy for patients with hepatocellular carcinoma.Liver Transpl. 2008; 14: 443-450Crossref PubMed Scopus (41) Google Scholar]. In this study, a fractional allelic imbalance (FAI) rate was calculated for each patient. This index was defined as the number of mutated markers divided by the total number of informative markers. These markers corresponded to the 9 microsatellites retrieved as correlated with the post-transplant recurrence free survival in their initial work [[15]Marsh J.W. Finkelstein S.D. Demetris A.J. Swalsky P.A. Sasatomi E. Bandos A. et al.Genotyping of hepatocellular carcinoma in liver transplant recipients adds predictive power for determining recurrence-free survival.Liver Transpl. 2003; 9: 664-671Crossref PubMed Scopus (120) Google Scholar]. Of note, all these 9 markers were not always informative, i.e present, in each patient. The rate of mutation, i.e. FAI, was then dependent on the number of informative markers. Hence, the FAI represents a rough estimate of cumulative mutational damage in tumor compared to non-neoplastic tissue. FAI in the 9-gene panel ranged from 0% to 89% (mean 31%). Among the patients tested, FAI and vascular invasion were the most important independent factors of tumor-free survival. Patients with FAI > 40% compared to those with FAI ⩽ 20% have an increased risk of recurrence by a factor of 19.5. Comparatively, patients with macrovascular invasion compared with those without vascular invasion have an increased risk of recurrence of 5.9. This difference emphasized the power of the FAI even comparatively with the more predictive pathological factor, vascular invasion [6Jonas S. Bechstein W.O. Steinmuller T. Herrmann M. Radke C. Berg T. et al.Vascular invasion and histopathologic grading determine outcome after liver transplantation for hepatocellular carcinoma in cirrhosis.Hepatology. 2001; 33: 1080-1086Crossref PubMed Scopus (805) Google Scholar, 13Pawlik T.M. Delman K.A. Vauthey J.N. Nagorney D.M. Ng I.O. Ikai I. et al.Tumor size predicts vascular invasion and histologic grade: implications for selection of surgical treatment for hepatocellular carcinoma.Liver Transpl. 2005; 11: 1086-1092Crossref PubMed Scopus (489) Google Scholar, 14Parfitt J.R. Marotta P. Alghamdi M. Wall W. Khakhar A. Suskin N.G. et al.Recurrent hepatocellular carcinoma after transplantation: use of a pathological score on explanted livers to predict recurrence.Liver Transpl. 2007; 13: 543-551Crossref PubMed Scopus (132) Google Scholar]. Interestingly, a correlation with the Milan, UCSF criteria and FAI has been described in this paper. For patients outside the Milan criteria, the risk of recurrence increased 4.5-fold compared to patients within, while a FAI > 40% increased this risk 14-fold compared to FAI ⩽ 20%. For patients beyond UCSF criteria, the risk of recurrence increased 6.0-fold compared to patients within, while a FAI > 40% increased this risk 17.4-fold compared to FAI ⩽ 20%. In summary, this second paper has demonstrated the much more powerful correlation with recurrence of FAI compared with pathological data and macroscopic usual classification.Hence, the paper by Schwartz et al. which appears in this issue of the Journal of Hepatology [[17]Schwartz M. Dvorchik I. Roayaie S. Fiel M.I. Finkelstein S. Marsh J.W. et al.Liver transplantation for hepatocellular carcinoma: Extension of indications based on molecular markers.J Hepatol. 2008; 49: 581-588Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar] focused on the potential applicability of the FAI score in patients beyond the Milan criteria with a favourable outcome of LT. With the same methods of allelic imbalance analysis of HCC in transplanted patients, they have compared tumoral and non-tumoral liver tissue for 18 genes markers to determine the FAI score. Statistically, a FAI score of 0.27 was determined as the more reliable cut-off value to predict recurrence or not. Univariate and multivariate analysis of prognostic factors of recurrence were performed with 70 patients transplanted for HCC with at least 5 years of follow-up. The FAI score and macroscopic vascular invasion were the 2 independent factors associated with recurrence. Interestingly, FAI predicted recurrence in 86% compared to 76% using Milan criteria. Unfortunately, the statistical significance if any of this difference was not reported in the paper. Besides, it was probably due to the low rate of recurrence in Milan criteria (3/35) patients that this paper focused on patients beyond Milan criteria. In the subgroup of patients beyond Milan criteria, the same predictive factors of recurrence were confirmed but, in this group, a FAI ⩾ 0.27 was predictive of recurrence with a sensitivity of 83%, a specificity of 91% and overall accuracy of 89%. Thus, this paper emphasized the utility of FAI analysis only in patients beyond the Milan criteria. Besides, among the 35 patients within the Milan criteria, 4 of 6 of those who were predicted to have recurrence according to FAI did not.Even if it was useful in patients beyond Milan criteria, the major drawback of this technique was the necessity of full pathological examination. To date, all papers that explored evaluation of FAI have used surgical specimens to predict recurrence. As underlined by Marsh et al. in 2003 [[15]Marsh J.W. Finkelstein S.D. Demetris A.J. Swalsky P.A. Sasatomi E. Bandos A. et al.Genotyping of hepatocellular carcinoma in liver transplant recipients adds predictive power for determining recurrence-free survival.Liver Transpl. 2003; 9: 664-671Crossref PubMed Scopus (120) Google Scholar], the two principal limitations in extrapolating this work to the preoperative area were: (1) All tumors are not recognized preoperatively. (2) It is not practical or feasible to biopsy all detected tumors multiple times in the preoperative setting. Indeed, Pawlik et al. have analyzed the concordance between histological data using preoperative needle core biopsy (14–18 gauges) of HCC and final pathological analysis [[18]Pawlik T.M. Gleisner A.L. Anders R.A. Assumpcao L. Maley W. Choti M.A. Preoperative assessment of hepatocellular carcinoma tumor grade using needle biopsy. Implications for transplant eligibility.Ann Surg. 2007; 245: 435-442Crossref PubMed Scopus (168) Google Scholar]. In this study that included 120 patients, preoperative biopsy misclassified tumor grade in 55% of cases. The poor diagnostic accuracy of preoperative biopsy was related to the very heterogeneous pathological and molecular alteration of HCC, even in small tumors. Incidence of grading heterogeneity was reported in 18–46% [19Kenmochi K. Sugihara S. Kojiro M. Relationship of histologic grade of hepatocellular carcinoma (HCC) to tumor size, and demonstration of tumor cells of multiple different grades in single small HCC.Liver. 1987; 7: 18-26Crossref PubMed Scopus (232) Google Scholar, 20Nakashima O. Sugihara S. Kage M. Kojiro M. Pathomorphologic characteristics of small hepatocellular carcinoma: a special reference to small hepatocellular carcinoma with indistinct margins.Hepatology. 1995; 22: 101-105PubMed Google Scholar, 21An F.Q. Matsuda M. Fujii H. Tang R.F. Amemiya H. Dai Y.M. et al.Tumor heterogeneity in small hepatocellular carcinoma: analysis of tumor cell proliferation, expression and mutation of p53 and beta-catenin.Int J Cancer. 2001; 93: 468-474Crossref PubMed Scopus (61) Google Scholar] of HCC smaller than 2 cm and 36–43% in 2–3 cm nodules [19Kenmochi K. Sugihara S. Kojiro M. Relationship of histologic grade of hepatocellular carcinoma (HCC) to tumor size, and demonstration of tumor cells of multiple different grades in single small HCC.Liver. 1987; 7: 18-26Crossref PubMed Scopus (232) Google Scholar, 21An F.Q. Matsuda M. Fujii H. Tang R.F. Amemiya H. Dai Y.M. et al.Tumor heterogeneity in small hepatocellular carcinoma: analysis of tumor cell proliferation, expression and mutation of p53 and beta-catenin.Int J Cancer. 2001; 93: 468-474Crossref PubMed Scopus (61) Google Scholar]. This heterogeneity was due to sequential development of HCC often displaying the nodule-in-nodule pattern where a subpopulation with a more malignant potential develops in a pre-existing nodule. Interestingly, as grading heterogeneity, molecular alterations, notably mutation of p53 and β-catenin, were also retrieved inside small HCCs [[21]An F.Q. Matsuda M. Fujii H. Tang R.F. Amemiya H. Dai Y.M. et al.Tumor heterogeneity in small hepatocellular carcinoma: analysis of tumor cell proliferation, expression and mutation of p53 and beta-catenin.Int J Cancer. 2001; 93: 468-474Crossref PubMed Scopus (61) Google Scholar].As reported in the paper by Schwartz et al. [[17]Schwartz M. Dvorchik I. Roayaie S. Fiel M.I. Finkelstein S. Marsh J.W. et al.Liver transplantation for hepatocellular carcinoma: Extension of indications based on molecular markers.J Hepatol. 2008; 49: 581-588Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar], the value of FAI was dependent on the number of mutated genes to the total number of informative genes. It was the variability of informative gene numbers that has imposed the calculation of an index. Indeed, a recent paper has focused on the genetic diversity of human HCC [[22]Boyault S. Rickman D.S. de Reynies A. Balabaud C. Rebouissou S. Jeannot E. et al.Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.Hepatology. 2007; 45: 42-52Crossref PubMed Scopus (842) Google Scholar]. In this work, a transcriptome analysis with genotype and phenotype correlation has been performed in a surgical series of 120 HCCs and 3 hepatocellular adenomas. The transcriptome analysis identified 6 subgroups (G1–G6) of HCCs associated with clinical and genetic characteristics. HCC of G1 and G2 group were associated with HBV infection. G3 HCC mainly included p53 mutation but without HBV infection. G4 HCC was a heterogeneous group of HCCs.G5 and G6 were highly related to β-catenin activation. Interestingly, HCC groups G1 to G3 were associated with a high rate of chromosomal instability in contrast to G5 and G6 where no chromosome deletion, then no LOH was identified. Hence, claims that FAI, largely correlated with LOH, in these subgroups of HCC is questionable and emphasized the fact that the FAI ratio is only a rough measure of cumulative mutational damage. It should be emphasized that the correlation of FAI and viral hepatitis was not assessed in the paper by Schwartz and colleagues.Despite these limitations, FAI represents a promising new tool to predict recurrence after LT, even if it is only of benefit postoperatively in patients falling beyond the Milan criteria. In the post-transplant setting, FAI could provide useful information to adapt immunosuppressive therapy and adjuvant chemotherapy thus attenuating the risk of recurrence [[23]Zimmerman M.A. Trotter J.F. Wachs M. Bak T. Campsen J. Skibba A. et al.Sirolimus-based immunosuppression following liver transplantation for hepatocellular carcinoma.Liver Transpl. 2008; 14: 633-638Crossref PubMed Scopus (171) Google Scholar]. In the pre-transplant setting, the application of molecular markers needs further evaluation before being used as selection criteria for liver transplantation. Liver transplantation (LT) is in theory, the best treatment for hepatocellular carcinoma (HCC) in cirrhotic patients since it is a treatment that simultaneously deals with the malignancy and its causes [1Adam R. McMaster P. O’Grady J.G. Castaing D. Klempnauer J.L. Jamieson N. et al.Evolution of liver transplantation in Europe: report of the European Liver Transplant Registry.Liver Transpl. 2003; 9: 1231-1243Crossref PubMed Scopus (491) Google Scholar, 2Yoo H.Y. Patt C.H. Geschwind J.F. Thuluvath P.J. The outcome of liver transplantation in patients with hepatocellular carcinoma in the United States between 1988 and 2001: 5-year survival has improved significantly with time.J Clin Oncol. 2003; 21: 4329-4335Crossref PubMed Scopus (214) Google Scholar, 3El-Serag H.B. Siegel A.B. Davila J.A. Shaib Y.H. Cayton-Woody M. McBride R. et al.Treatment and outcomes of treating of hepatocellular carcinoma among medicare recipients in the United States: a population-based study.J Hepatol. 2006; 44: 158-166Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar]. However, the prognosis is directly related to the risk of HCC recurrence post-transplantation. The aim of preoperative assessment in LT for HCC is therefore to detect existing extra-hepatic metastases and predict extra-hepatic micro-metastases that are responsible for recurrence, graft loss and often, death of the patient. Following the retrospective study by Bismuth et al. [[4]Bismuth H. Chiche L. Adam R. Castaing D. Diamond T. Dennison A. Liver resection versus transplantation for hepatocellular carcinoma in cirrhotic patients.Ann Surg. 1993; 218: 145-151Crossref PubMed Scopus (823) Google Scholar], Mazzaferro et al. prospectively showed that careful selection of patients with a single lesion of <5 cm or three lesions each <3 cm, known as Milan criteria, has resulted in a satisfactory balance between survival (75%) at four years and recurrence rates (8%) [[5]Mazzaferro V. Chun Y.S. Poon R.T. Schwartz M.E. Yao F.Y. Marsh J.W. et al.Liver transplantation for hepatocellular carcinoma.Ann Surg Oncol. 2008; 15: 1001-1007Crossref PubMed Scopus (227) Google Scholar]. The validity of these criteria to predict recurrence-free survival after transplantation has been largely confirmed by several studies, which have demonstrated an overall 10-year survival ranging from 60% to 62% [6Jonas S. Bechstein W.O. Steinmuller T. Herrmann M. Radke C. Berg T. et al.Vascular invasion and histopathologic grading determine outcome after liver transplantation for hepatocellular carcinoma in cirrhosis.Hepatology. 2001; 33: 1080-1086Crossref PubMed Scopus (805) Google Scholar, 7Zavaglia C. De Carlis L. Alberti A.B. Minola E. Belli L.S. Slim A.O. et al.Predictors of long-term survival after liver transplantation for hepatocellular carcinoma.Am J Gastroenterol. 2005; 100: 2708-2716Crossref PubMed Scopus (214) Google Scholar]. Hence, survival of carefully selected patients is not different from survival of patients transplanted for non-malignant indications [[8]Mazzaferro V. Regalia E. Doci R. Andreola S. Pulvirenti A. Bozzetti F. et al.Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis.N Engl J Med. 1996; 334: 693-699Crossref PubMed Scopus (5580) Google Scholar]. Survival analysis by ITT clearly suggested that resection or ablation in patients with compensated cirrhosis are the only alternatives to liver transplantation on the wait list. Moreover there is growing evidence that by limiting OLT only to patients falling within the Milan criteria might represent an unjust penalty for selected patients beyond the Milan criteria who could still have a favourable outcome with OLT. In the United States, around 70% of cirrhotic patients diagnosed with a HCC fall beyond the Milan criteria [[3]El-Serag H.B. Siegel A.B. Davila J.A. Shaib Y.H. Cayton-Woody M. McBride R. et al.Treatment and outcomes of treating of hepatocellular carcinoma among medicare recipients in the United States: a population-based study.J Hepatol. 2006; 44: 158-166Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar]. Survival analysis by ITT clearly suggested that regular ultrasound screening of cirrhotic patients has permitted detection of small unique nodule in Child-Pugh A cirrhotic patients in whom alternatives to transplantation are possible. Second, liver transplantation will provide a major survival gain in some patients with a HCC who fall beyond the Milan criteria. Therefore, several expansions of the Milan criteria have been proposed, with the University of California, San Francisco (UCSF) criteria described by Yao et al. being the most investigated [9Yao F.Y. Ferrell L. Bass N.M. Watson J.J. Bacchetti P. Venook A. et al.Liver transplantation for hepatocellular carcinoma: expansion of the tumor size limits does not adversely impact survival.Hepatology. 2001; 33: 1394-1403Crossref PubMed Scopus (1732) Google Scholar, 10Yao F.Y. Xiao L. Bass N.M. Kerlan R. Ascher N.L. Roberts J.P. Liver transplantation for hepatocellular carcinoma: validation of the UCSF-expanded criteria based on preoperative imaging.Am J Transplant. 2007; 7: 2587-2596Crossref PubMed Scopus (407) Google Scholar]. The UCSF criteria include a solitary tumor of 6.5 cm or less, no more than three lesions with the largest being 4.5 cm or less, and a total tumor diameter 8 cm or less, without gross vascular invasion. Unfortunately, the 5-year survival of patients beyond Milan and within UCSF criteria ranges from 46% to 93% [9Yao F.Y. Ferrell L. Bass N.M. Watson J.J. Bacchetti P. Venook A. et al.Liver transplantation for hepatocellular carcinoma: expansion of the tumor size limits does not adversely impact survival.Hepatology. 2001; 33: 1394-1403Crossref PubMed Scopus (1732) Google Scholar, 10Yao F.Y. Xiao L. Bass N.M. Kerlan R. Ascher N.L. Roberts J.P. Liver transplantation for hepatocellular carcinoma: validation of the UCSF-expanded criteria based on preoperative imaging.Am J Transplant. 2007; 7: 2587-2596Crossref PubMed Scopus (407) Google Scholar, 11Decaens T. Roudot-Thoraval F. Hadni-Bresson S. Meyer C. Gugenheim J. Durand F. et al.Impact of UCSF criteria according to pre- and post-OLT tumor features: analysis of 479 patients listed for HCC with a short waiting time.Liver Transpl. 2006; 12: 1761-1769Crossref PubMed Scopus (160) Google Scholar, 12Duffy J.P. Vardanian A. Benjamin E. Watson M. Farmer D.G. Ghobrial R.M. et al.Liver transplantation criteria for hepatocellular carcinoma should be expanded: a 22-year experience with 467 patients at UCLA.Ann Surg. 2007; 246: 502-509Crossref PubMed Scopus (2) Google Scholar]. The resulting discrepancies between studies which used UCSF criteria are related to the time of application of the criteria (preoperative/radiological and postoperative/pathological). Interestingly, a French multicentric study of 479 patients demonstrated a higher rate of understaging (48%) if UCSF criteria is used to list patients for LT as compared to the understaging rate of 34% when the Milan criteria is used [[11]Decaens T. Roudot-Thoraval F. Hadni-Bresson S. Meyer C. Gugenheim J. Durand F. et al.Impact of UCSF criteria according to pre- and post-OLT tumor features: analysis of 479 patients listed for HCC with a short waiting time.Liver Transpl. 2006; 12: 1761-1769Crossref PubMed Scopus (160) Google Scholar]. Indeed, the 5-year survival of UCSF excluding Milan patients was 45% compared to 60% in Milan patients [[11]Decaens T. Roudot-Thoraval F. Hadni-Bresson S. Meyer C. Gugenheim J. Durand F. et al.Impact of UCSF criteria according to pre- and post-OLT tumor features: analysis of 479 patients listed for HCC with a short waiting time.Liver Transpl. 2006; 12: 1761-1769Crossref PubMed Scopus (160) Google Scholar]. Heterogeneous survival results in studies using UCSF criteria emphasized that macroscopic parameters like size and number would probably lose their efficacy as surrogate criteria to accurately predict the risk of tumor recurrence in comparison to tumoral volume exceeding the Milan criteria. Several studies have identified vascular invasion as the strongest independent predictor factor of recurrence, a pathological parameter also associated with other poor prognostic factors like a poor differentiation of the tumor, the presence of satellite nodules and a high level of α-fetoprotein [6Jonas S. Bechstein W.O. Steinmuller T. Herrmann M. Radke C. Berg T. et al.Vascular invasion and histopathologic grading determine outcome after liver transplantation for hepatocellular carcinoma in cirrhosis.Hepatology. 2001; 33: 1080-1086Crossref PubMed Scopus (805) Google Scholar, 13Pawlik T.M. Delman K.A. Vauthey J.N. Nagorney D.M. Ng I.O. Ikai I. et al.Tumor size predicts vascular invasion and histologic grade: implications for selection of surgical treatment for hepatocellular carcinoma.Liver Transpl. 2005; 11: 1086-1092Crossref PubMed Scopus (489) Google Scholar, 14Parfitt J.R. Marotta P. Alghamdi M. Wall W. Khakhar A. Suskin N.G. et al.Recurrent hepatocellular carcinoma after transplantation: use of a pathological score on explanted livers to predict recurrence.Liver Transpl. 2007; 13: 543-551Crossref PubMed Scopus (132) Google Scholar]. Unfortunately, except for the latter, all these parameters are not easily available in the preoperative setting and particularly at listing. In 2003, based on the knowledge that HCC display molecular alterations Marsh et al., classified HCC according to the level of gene damage [[15]Marsh J.W. Finkelstein S.D. Demetris A.J. Swalsky P.A. Sasatomi E. Bandos A. et al.Genotyping of hepatocellular carcinoma in liver transplant recipients adds predictive power for determining recurrence-free survival.Liver Transpl. 2003; 9: 664-671Crossref PubMed Scopus (120) Google Scholar]. The goal of this paper was to determine whether a panel of tumor suppressor gene markers could be useful to predict recurrence after LT for HCC. By microdissection, non-neoplastic and neoplastic tissue samples for each patient transplanted with a minimum of 5 years follow-up were evaluated for informative status of 18 genes located on microsatellite. Absence of microsatellites or loss of heterozygoty (LOH) corresponded to the loss of specific tumor suppressor genes such as APC, CDKN2A, p53, and p34. Among 18 microsatellites tested, 9 were significantly correlated with the post-transplant recurrence-free survival but none achieved perfect discrimination alone. As a test, two types of simplified panel of LOH that were the most accurate to predict recurrence were defined. Thanks to this method and with the help of an artificial neural network, an accurate response was obtained in 91 of the 103 patients tested (88%). The prediction of recurrence was accurate in 89% of cases. Very recently, the same team has performed a similar analysis on a larger cohort of patients transplanted for HCC with more than 5 years of follow-up (183 patients from Mount Sinai and University of Pittsburgh Medical Center) [[16]Dvorchik I. Schwartz M. Fiel M.I. Finkelstein S.D. Marsh J.W. Fractional allelic imbalance could allow for the development of an equitable transplant selection policy for patients with hepatocellular carcinoma.Liver Transpl. 2008; 14: 443-450Crossref PubMed Scopus (41) Google Scholar]. In this study, a fractional allelic imbalance (FAI) rate was calculated for each patient. This index was defined as the number of mutated markers divided by the total number of informative markers. These markers corresponded to the 9 microsatellites retrieved as correlated with the post-transplant recurrence free survival in their initial work [[15]Marsh J.W. Finkelstein S.D. Demetris A.J. Swalsky P.A. Sasatomi E. Bandos A. et al.Genotyping of hepatocellular carcinoma in liver transplant recipients adds predictive power for determining recurrence-free survival.Liver Transpl. 2003; 9: 664-671Crossref PubMed Scopus (120) Google Scholar]. Of note, all these 9 markers were not always informative, i.e present, in each patient. The rate of mutation, i.e. FAI, was then dependent on the number of informative markers. Hence, the FAI represents a rough estimate of cumulative mutational damage in tumor compared to non-neoplastic tissue. FAI in the 9-gene panel ranged from 0% to 89% (mean 31%). Among the patients tested, FAI and vascular invasion were the most important independent factors of tumor-free survival. Patients with FAI > 40% compared to those with FAI ⩽ 20% have an increased risk of recurrence by a factor of 19.5. Comparatively, patients with macrovascular invasion compared with those without vascular invasion have an increased risk of recurrence of 5.9. This difference emphasized the power of the FAI even comparatively with the more predictive pathological factor, vascular invasion [6Jonas S. Bechstein W.O. Steinmuller T. Herrmann M. Radke C. Berg T. et al.Vascular invasion and histopathologic grading determine outcome after liver transplantation for hepatocellular carcinoma in cirrhosis.Hepatology. 2001; 33: 1080-1086Crossref PubMed Scopus (805) Google Scholar, 13Pawlik T.M. Delman K.A. Vauthey J.N. Nagorney D.M. Ng I.O. Ikai I. et al.Tumor size predicts vascular invasion and histologic grade: implications for selection of surgical treatment for hepatocellular carcinoma.Liver Transpl. 2005; 11: 1086-1092Crossref PubMed Scopus (489) Google Scholar, 14Parfitt J.R. Marotta P. Alghamdi M. Wall W. Khakhar A. Suskin N.G. et al.Recurrent hepatocellular carcinoma after transplantation: use of a pathological score on explanted livers to predict recurrence.Liver Transpl. 2007; 13: 543-551Crossref PubMed Scopus (132) Google Scholar]. Interestingly, a correlation with the Milan, UCSF criteria and FAI has been described in this paper. For patients outside the Milan criteria, the risk of recurrence increased 4.5-fold compared to patients within, while a FAI > 40% increased this risk 14-fold compared to FAI ⩽ 20%. For patients beyond UCSF criteria, the risk of recurrence increased 6.0-fold compared to patients within, while a FAI > 40% increased this risk 17.4-fold compared to FAI ⩽ 20%. In summary, this second paper has demonstrated the much more powerful correlation with recurrence of FAI compared with pathological data and macroscopic usual classification. Hence, the paper by Schwartz et al. which appears in this issue of the Journal of Hepatology [[17]Schwartz M. Dvorchik I. Roayaie S. Fiel M.I. Finkelstein S. Marsh J.W. et al.Liver transplantation for hepatocellular carcinoma: Extension of indications based on molecular markers.J Hepatol. 2008; 49: 581-588Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar] focused on the potential applicability of the FAI score in patients beyond the Milan criteria with a favourable outcome of LT. With the same methods of allelic imbalance analysis of HCC in transplanted patients, they have compared tumoral and non-tumoral liver tissue for 18 genes markers to determine the FAI score. Statistically, a FAI score of 0.27 was determined as the more reliable cut-off value to predict recurrence or not. Univariate and multivariate analysis of prognostic factors of recurrence were performed with 70 patients transplanted for HCC with at least 5 years of follow-up. The FAI score and macroscopic vascular invasion were the 2 independent factors associated with recurrence. Interestingly, FAI predicted recurrence in 86% compared to 76% using Milan criteria. Unfortunately, the statistical significance if any of this difference was not reported in the paper. Besides, it was probably due to the low rate of recurrence in Milan criteria (3/35) patients that this paper focused on patients beyond Milan criteria. In the subgroup of patients beyond Milan criteria, the same predictive factors of recurrence were confirmed but, in this group, a FAI ⩾ 0.27 was predictive of recurrence with a sensitivity of 83%, a specificity of 91% and overall accuracy of 89%. Thus, this paper emphasized the utility of FAI analysis only in patients beyond the Milan criteria. Besides, among the 35 patients within the Milan criteria, 4 of 6 of those who were predicted to have recurrence according to FAI did not. Even if it was useful in patients beyond Milan criteria, the major drawback of this technique was the necessity of full pathological examination. To date, all papers that explored evaluation of FAI have used surgical specimens to predict recurrence. As underlined by Marsh et al. in 2003 [[15]Marsh J.W. Finkelstein S.D. Demetris A.J. Swalsky P.A. Sasatomi E. Bandos A. et al.Genotyping of hepatocellular carcinoma in liver transplant recipients adds predictive power for determining recurrence-free survival.Liver Transpl. 2003; 9: 664-671Crossref PubMed Scopus (120) Google Scholar], the two principal limitations in extrapolating this work to the preoperative area were: (1) All tumors are not recognized preoperatively. (2) It is not practical or feasible to biopsy all detected tumors multiple times in the preoperative setting. Indeed, Pawlik et al. have analyzed the concordance between histological data using preoperative needle core biopsy (14–18 gauges) of HCC and final pathological analysis [[18]Pawlik T.M. Gleisner A.L. Anders R.A. Assumpcao L. Maley W. Choti M.A. Preoperative assessment of hepatocellular carcinoma tumor grade using needle biopsy. Implications for transplant eligibility.Ann Surg. 2007; 245: 435-442Crossref PubMed Scopus (168) Google Scholar]. In this study that included 120 patients, preoperative biopsy misclassified tumor grade in 55% of cases. The poor diagnostic accuracy of preoperative biopsy was related to the very heterogeneous pathological and molecular alteration of HCC, even in small tumors. Incidence of grading heterogeneity was reported in 18–46% [19Kenmochi K. Sugihara S. Kojiro M. Relationship of histologic grade of hepatocellular carcinoma (HCC) to tumor size, and demonstration of tumor cells of multiple different grades in single small HCC.Liver. 1987; 7: 18-26Crossref PubMed Scopus (232) Google Scholar, 20Nakashima O. Sugihara S. Kage M. Kojiro M. Pathomorphologic characteristics of small hepatocellular carcinoma: a special reference to small hepatocellular carcinoma with indistinct margins.Hepatology. 1995; 22: 101-105PubMed Google Scholar, 21An F.Q. Matsuda M. Fujii H. Tang R.F. Amemiya H. Dai Y.M. et al.Tumor heterogeneity in small hepatocellular carcinoma: analysis of tumor cell proliferation, expression and mutation of p53 and beta-catenin.Int J Cancer. 2001; 93: 468-474Crossref PubMed Scopus (61) Google Scholar] of HCC smaller than 2 cm and 36–43% in 2–3 cm nodules [19Kenmochi K. Sugihara S. Kojiro M. Relationship of histologic grade of hepatocellular carcinoma (HCC) to tumor size, and demonstration of tumor cells of multiple different grades in single small HCC.Liver. 1987; 7: 18-26Crossref PubMed Scopus (232) Google Scholar, 21An F.Q. Matsuda M. Fujii H. Tang R.F. Amemiya H. Dai Y.M. et al.Tumor heterogeneity in small hepatocellular carcinoma: analysis of tumor cell proliferation, expression and mutation of p53 and beta-catenin.Int J Cancer. 2001; 93: 468-474Crossref PubMed Scopus (61) Google Scholar]. This heterogeneity was due to sequential development of HCC often displaying the nodule-in-nodule pattern where a subpopulation with a more malignant potential develops in a pre-existing nodule. Interestingly, as grading heterogeneity, molecular alterations, notably mutation of p53 and β-catenin, were also retrieved inside small HCCs [[21]An F.Q. Matsuda M. Fujii H. Tang R.F. Amemiya H. Dai Y.M. et al.Tumor heterogeneity in small hepatocellular carcinoma: analysis of tumor cell proliferation, expression and mutation of p53 and beta-catenin.Int J Cancer. 2001; 93: 468-474Crossref PubMed Scopus (61) Google Scholar]. As reported in the paper by Schwartz et al. [[17]Schwartz M. Dvorchik I. Roayaie S. Fiel M.I. Finkelstein S. Marsh J.W. et al.Liver transplantation for hepatocellular carcinoma: Extension of indications based on molecular markers.J Hepatol. 2008; 49: 581-588Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar], the value of FAI was dependent on the number of mutated genes to the total number of informative genes. It was the variability of informative gene numbers that has imposed the calculation of an index. Indeed, a recent paper has focused on the genetic diversity of human HCC [[22]Boyault S. Rickman D.S. de Reynies A. Balabaud C. Rebouissou S. Jeannot E. et al.Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.Hepatology. 2007; 45: 42-52Crossref PubMed Scopus (842) Google Scholar]. In this work, a transcriptome analysis with genotype and phenotype correlation has been performed in a surgical series of 120 HCCs and 3 hepatocellular adenomas. The transcriptome analysis identified 6 subgroups (G1–G6) of HCCs associated with clinical and genetic characteristics. HCC of G1 and G2 group were associated with HBV infection. G3 HCC mainly included p53 mutation but without HBV infection. G4 HCC was a heterogeneous group of HCCs.G5 and G6 were highly related to β-catenin activation. Interestingly, HCC groups G1 to G3 were associated with a high rate of chromosomal instability in contrast to G5 and G6 where no chromosome deletion, then no LOH was identified. Hence, claims that FAI, largely correlated with LOH, in these subgroups of HCC is questionable and emphasized the fact that the FAI ratio is only a rough measure of cumulative mutational damage. It should be emphasized that the correlation of FAI and viral hepatitis was not assessed in the paper by Schwartz and colleagues. Despite these limitations, FAI represents a promising new tool to predict recurrence after LT, even if it is only of benefit postoperatively in patients falling beyond the Milan criteria. In the post-transplant setting, FAI could provide useful information to adapt immunosuppressive therapy and adjuvant chemotherapy thus attenuating the risk of recurrence [[23]Zimmerman M.A. Trotter J.F. Wachs M. Bak T. Campsen J. Skibba A. et al.Sirolimus-based immunosuppression following liver transplantation for hepatocellular carcinoma.Liver Transpl. 2008; 14: 633-638Crossref PubMed Scopus (171) Google Scholar]. In the pre-transplant setting, the application of molecular markers needs further evaluation before being used as selection criteria for liver transplantation. The authors thank Dr. Emir Hoti for reading the manuscript.