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Are there opportunities for chemotherapy in the treatment of hepatocellular cancer?

  • Uzma Asghar
    Affiliations
    Department of Oncology, UCL Medical School, Royal Free Campus, London, UK
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  • Tim Meyer
    Correspondence
    Corresponding author. Address: UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6BT, UK. Tel.: +44 0207 679 6731; fax: +44 0203 108 2025.
    Affiliations
    Department of Oncology, UCL Medical School, Royal Free Campus, London, UK

    UCL Cancer Institute, London, UK
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Open AccessPublished:October 04, 2011DOI:https://doi.org/10.1016/j.jhep.2011.07.031

      Summary

      Hepatocellular cancer is a significant global health problem yet the prognosis for the majority of patients has not changed significantly over the past few decades. For patients with advanced disease, sorafenib is currently the standard of care providing a survival advantage of 2–3 months in selected patients. Cytotoxic chemotherapy has been used for over 30 years but definite evidence that it prolongs survival has been lacking. Resistance remains a significant barrier for both targeted and cytotoxic agents and an understanding of the underlying mechanisms is critical if outcomes are to be improved. Here, we summarise the past and current data that constitute the evidence base for chemotherapy in HCC, review the causes of chemoresistance and suggest strategies to overcome these barriers.

      Keywords

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      • Reply to: “Sorafenib prolongs survival, but what happens to the symptoms?”
        Journal of HepatologyVol. 57Issue 1
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          The main focus of our paper concerns the potential role of cytotoxic chemotherapy, which needs to be considered in the context of other standards of care [1]. As stated, the current standard of care for advanced HCC is sorafenib. An exhaustive review of the SHARP [2] and AP [3] trial data was not within the scope of our review since these have been covered extensively in previous publications. We do, however, highlight both the survival benefit and the toxicity associated with sorafenib. Survival remains the primary end point for most phase III trials and is both robust and, as Dr.
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      • Sorafenib prolongs survival, but what happens to the symptoms?
        Journal of HepatologyVol. 57Issue 1
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          A recent review published in the Journal of Hepatology discusses the opportunities for chemotherapy in hepatocellular carcinoma (HCC) [1]. In this review, the authors conclude that “Sorafenib is currently regarded as the standard of care in selected patients with advanced HCC based on two large randomised placebo controlled trials” [2,3]. Furthermore, the introduction suggests its use as a palliative therapeutic. But what actually defines a palliative treatment? A decent definition of palliative chemotherapy is given by V.R.
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      Introduction

      Hepatocellular cancer (HCC) is the third most common cause of cancer death worldwide with incidence matching mortality reflecting the poor prognosis of this disease. Potentially curative interventions such as transplantation, resection, and thermal ablation are applicable for the 30% whose tumours or liver function meet defined criteria [
      • Llovet J.M.
      • Burroughs A.
      • Bruix J.
      Hepatocellular carcinoma.
      ]. Even among those treated with curative intent, relapse rates are up to 50%. For patients who have large or multifocal disease but with good liver function, performance status, and a patent main portal vein, trans-arterial chemoembolization has been shown to improve survival compared to best supportive care [
      • Llovet J.M.
      • Bruix J.
      Systematic review of randomized trials for unresectable hepatocellular carcinoma: chemoembolization improves survival.
      ]. Of the remaining 50% patients, around 10–20% will have such advanced tumours or liver disease that only supportive care is appropriate leaving 20–30% potentially suitable for palliative systemic therapy [
      • Llovet J.M.
      • Di Bisceglie A.M.
      • Bruix J.
      • Kramer B.S.
      • Lencioni R.
      • Zhu A.X.
      • et al.
      Design and endpoints of clinical trials in hepatocellular carcinoma.
      ]. For those that are potential candidates for systemic therapy, the expected median survival without treatment ranges from 4 to 8 months based on outcomes from best supportive care (BSC) or placebo arms of controlled trials in this patient group [
      • Llovet J.M.
      • Bustamante J.
      • Castells A.
      • Vilana R.
      • Ayuso M.C.
      • Sala M.
      • et al.
      Natural history of untreated nonsurgical hepatocellular carcinoma: rationale for the design and evaluation of therapeutic trials.
      ,
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ,
      • Barbare J.C.
      • Bouche O.
      • Bonnetain F.
      • Raoul J.L.
      • Rougier P.
      • Abergel A.
      • et al.
      Randomized controlled trial of tamoxifen in advanced hepatocellular carcinoma.
      ]. Until recently there was no proven effective systemic therapy. However, the demonstration that the multi-targeted kinase inhibitor sorafenib results in a 44–47% improvement in median overall survival has dismissed the prevailing nihilism that HCC is an unresponsive tumour and has established a new standard of care [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ,
      • Cheng A.L.
      • Kang Y.K.
      • Chen Z.
      • Tsao C.J.
      • Qin S.
      • Kim J.S.
      • et al.
      Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial.
      ]. Sorafenib is the standard against which future systemic therapies need to be measured and is currently the control arm for several ongoing randomised phase III trials.
      Chemotherapy has been widely regarded as ineffective because of toxicity and poor response, however, recent trials, with appropriate patient selection, have provided some evidence that chemotherapy may have a role. Furthermore, a greater understanding of the molecular mechanisms of tumour resistance, and how to overcome them, present an opportunity to explore the combination of chemotherapy with targeted agents, or to select patient groups that are more likely to respond. Here, we summarise the past and current data that constitute the evidence base for chemotherapy in HCC, review the causes of chemoresistance and suggest strategies to improve outcomes.

      Systemic chemotherapy – historical perspective

      Systemic chemotherapy has been used for the treatment of advanced HCC for over 30 years but definitive evidence that cytotoxic drugs improve survival compared to BSC has not been provided [
      • Burroughs A.
      • Hochhauser D.
      • Meyer T.
      Systemic treatment and liver transplantation for hepatocellular carcinoma: two ends of the therapeutic spectrum.
      ]. The report by Olweny [
      • Olweny C.L.
      • Toya T.
      • Katongole-Mbidde E.
      • Mugerwa J.
      • Kyalwazi S.K.
      • Cohen H.
      Treatment of hepatocellular carcinoma with adriamycin. Preliminary communication.
      ] in 1975 of a 79% response rate for doxorubicin in a small single arm phase II trial was followed by a series of further uncontrolled phase II trials and resulted in doxorubicin being regarded by many as the standard chemotherapy drug for this disease. The reported response rates in the subsequent studies did not confirm the original study and have varied between 1% and 35%. Meanwhile a range of other drugs and combinations have also been assessed [
      • Burroughs A.
      • Hochhauser D.
      • Meyer T.
      Systemic treatment and liver transplantation for hepatocellular carcinoma: two ends of the therapeutic spectrum.
      ,
      • Simonetti R.G.
      • Liberati A.
      • Angiolini C.
      • Pagliaro L.
      Treatment of hepatocellular carcinoma: a systematic review of randomized controlled trials.
      ]. The PIAF regimen (cisplatin, interferon, doxorubicin and fluorouracil) was initially reported to have a response rate of 26% in a phase II trial of fifty patients, however, it was associated with significant toxicity including 2 treatment related deaths [
      • Leung T.W.
      • Patt Y.Z.
      • Lau W.Y.
      • Ho S.K.
      • Yu S.C.
      • Chan A.T.
      • et al.
      Complete pathological remission is possible with systemic combination chemotherapy for inoperable hepatocellular carcinoma.
      ]. Importantly, this trial did confirm the chemosensitivity of HCC demonstrating a complete pathological response in four out of nine patients who went on to surgical resection.
      Remarkably, there are only two small randomised trials that have compared chemotherapy with BSC. One study of 106 patients compared doxorubicin with BSC reporting a median survival of 2.7 vs. 1.9 months respectively and a 25% mortality rate associated with doxorubicin [
      • Lai C.L.
      • Wu P.C.
      • Chan G.C.
      • Lok A.S.
      • Lin H.J.
      Doxorubicin versus no antitumor therapy in inoperable hepatocellular carcinoma. A prospective randomized trial.
      ]. Although this trial was statistically significant, the survival in both groups was well below that expected and cannot be regarded as definitive evidence of efficacy. Moreover, the treatment related mortality was in excess of that seen in subsequent doxorubicin trials where treatment related mortality was 3% or less [
      • Yeo W.
      • Mok T.S.
      • Zee B.
      • Leung T.W.
      • Lai P.B.
      • Lau W.Y.
      • et al.
      A randomized phase III study of doxorubicin versus cisplatin/interferon alpha-2b/doxorubicin/fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma.
      ]. A second study compared enteric coated tegafur/uracil to best supportive care in a randomised trial of 56 patients with HCC [
      • Ishikawa T.
      • Ichida T.
      • Sugitani S.
      • Tsuboi Y.
      • Genda T.
      • Sugahara S.
      • et al.
      Improved survival with oral administration of enteric-coated tegafur/uracil for advanced stage IV-A hepatocellular carcinoma.
      ]. Overall survival in the treated group was 12.1 months while that in the BSC group was only 6.2 months. However, almost a third of the BSC group were not included in the final analysis leading to potential bias in an already small and underpowered study. Consequently, previous systematic reviews have concluded that chemotherapy has no role in the treatment of HCC [
      • Llovet J.M.
      • Bruix J.
      Systematic review of randomized trials for unresectable hepatocellular carcinoma: chemoembolization improves survival.
      ,
      • Simonetti R.G.
      • Liberati A.
      • Angiolini C.
      • Pagliaro L.
      Treatment of hepatocellular carcinoma: a systematic review of randomized controlled trials.
      ].

      Randomised trials

      In the past few years, there have been four large randomised trials reported, two of which provide some evidence that chemotherapy may improve survival. All have used doxorubicin rather than BSC as the control arm (Table 1). In a trial performed in Hong Kong, doxorubicin was compared with PIAF (n = 188) and, while the superior response rate of PIAF was confirmed; 10.5% vs. 20.9%, the difference in survival; 6.8 vs. 8.7 months, was not significant [
      • Yeo W.
      • Mok T.S.
      • Zee B.
      • Leung T.W.
      • Lai P.B.
      • Lau W.Y.
      • et al.
      A randomized phase III study of doxorubicin versus cisplatin/interferon alpha-2b/doxorubicin/fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma.
      ]. The β-tubulin binding drug T138067 which inhibits microtubule formation was studied in a large global trial (n = 339) and found to have an almost identical median survival to doxorubicin (5.7 vs. 5.6 months respectively) [
      • Posey J.
      • Johnson P.
      • Mok T.
      • Hirmand M.
      • Dahlberg S.
      • Kwei L.
      • et al.
      Results of a phase 2/3 open-label, randomized trial of T138067 versus doxorubicin (DOX) in chemotherapy-naïve, unresectable hepatocellular carcinoma (HCC).
      ]. This trial remains in abstract form only despite having been presented in 2005. The thymidylate synthase inhibitor nolatrexed was compared to doxorubicin (n = 445) in a trial conducted in Western patients and found to a have significantly worse survival than doxorubicin; 5.1 vs. 7.4 months respectively (HR 0.753 p = 0.0068) [
      • Gish R.G.
      • Porta C.
      • Lazar L.
      • Ruff P.
      • Feld R.
      • Croitoru A.
      • et al.
      Phase III randomized controlled trial comparing the survival of patients with unresectable hepatocellular carcinoma treated with nolatrexed or doxorubicin.
      ]. Nolatrexed was also associated with more toxicity with a greater number of patients withdrawn due to adverse events, patient choice or death. Finally, the combination of oxaliplatin and fluorouracil (FOLFOX 4) has been compared with doxorubicin in a large population of Asian patients (n = 371) [
      • Qin S.
      • Bai Y.
      • Ye S.
      • Fan J.
      • Lim H.
      • Cho J.Y.
      • et al.
      Phase III study of oxaliplatin plus 5-fluorouracil/leucovorin (FOLFOX4) versus doxorubicin as palliative systemic chemotherapy in advanced HCC in Asian patients.
      ]. Although presented and published in abstract form in 2010, the full publication is not available. The median survival in the doxorubicin arm was 5.0 months and 6.4 months for FOLFOX 4. At the final analysis after 249 events, the result failed to achieve significance (HR 0.797 [0.625, 1.017] p = 0.0695). However, on longer follow-up after 305 events, the difference just achieved significance (p = 0.0425). It is notable that this trial used doxorubicin at a lower dose of 50 mg/m2 in contrast to the three preceding trials that all used 60 mg/m2.
      Table 1Randomised controlled trials of doxorubicin for HCC.
      1Dox at 60 mg/m2.
      2Dox at 50 mg/m2.
      RR, partial response rate according to WHO or RECIST.
      What can be concluded from these trials is that real response rate to doxorubicin is between 2% and 10% and that both doxorubicin and the FOLFOX 4 regimen may provide some survival benefit in comparison to control treatment.

      How does chemotherapy compare with sorafenib?

      Sorafenib is an oral multi-targeted kinase inhibitor which blocks tumour growth and angiogenesis by inhibition of Raf, RET, FLT3 c-Kit, VEGFR-1,-2 and -3, and PDGF-α and -β. Two placebo controlled randomised phase III trials have now been reported, both of which have confirmed a significant survival advantage in favour of sorafenib. The first, performed in a predominantly Western population demonstrated a response rate of only 2% but a 44% improvement in median overall survival from 7.9 to 10.7 months (HR 0.69, p <0.001) [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ]. The second trial, conducted in an Asian population, again demonstrated a low response rate at 3.3% but confirmed a 47% improvement in overall survival from 4.2 to 6.5 months (HR 0·68, p = 0·014] [
      • Cheng A.L.
      • Kang Y.K.
      • Chen Z.
      • Tsao C.J.
      • Qin S.
      • Kim J.S.
      • et al.
      Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial.
      ]. The lower absolute difference in survival between the two trials was attributed to more advanced disease in the Asian patients as indicated by number of disease sites and worse performance status.
      With the establishment of sorafenib as the standard of care, it is no longer possible to conduct a trial comparing chemotherapy with BSC and to our knowledge there are no trials underway to directly compare sorafenib with chemotherapy. The best that can be done is to compare efficacy and toxicity across trials in similar patient populations as defined by key prognostic criteria. Performance status, Child–Pugh score, and tumour burden have been proposed as stratification factors in trials of BCLC stage C [
      • Llovet J.M.
      • Bru C.
      • Bruix J.
      Prognosis of hepatocellular carcinoma: the BCLC staging classification.
      ] patients [
      • Llovet J.M.
      • Di Bisceglie A.M.
      • Bruix J.
      • Kramer B.S.
      • Lencioni R.
      • Zhu A.X.
      • et al.
      Design and endpoints of clinical trials in hepatocellular carcinoma.
      ] and a recent analysis of predictors of one-year survival in patients with BCLC stage B and C disease identified six factors associated with increased survival: publication before 2000, low prevalence of alcoholic liver disease, high prevalence of HCV-related disease, high percentage of ECOG PS = 0, low percentage of patients with ascites, and high percentage of Okuda stage I disease [
      • Cabibbo G.
      • Enea M.
      • Attanasio M.
      • Bruix J.
      • Craxi A.
      • Camma C.
      A meta-analysis of survival rates of untreated patients in randomized clinical trials of hepatocellular carcinoma.
      ]. In comparing across trials, these factors need to be considered and are summarised in Table 2.
      Table 2Comparison of patient characteristics and outcome for patients treated with chemotherapy and sorafenib in randomised trials.
      An important aspect of both sorafenib trials was the selection of only Child–Pugh A patients so that the impact of coexistent liver disease on the survival endpoint was minimised. The only randomised trial in the Western population using chemotherapy was that performed by Gish et al. [
      • Gish R.G.
      • Porta C.
      • Lazar L.
      • Ruff P.
      • Feld R.
      • Croitoru A.
      • et al.
      Phase III randomized controlled trial comparing the survival of patients with unresectable hepatocellular carcinoma treated with nolatrexed or doxorubicin.
      ] in which only 73% were Child–Pugh A compared to 95% in the SHARP trial [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ]. Thus, we do not have a matched Western patient group treated with chemotherapy or sorafenib and are unable to make a meaningful comparison. However, for the Asia-Pacific populations, the key patient characteristics were well matched in terms of age, aetiology, Child–Pugh score and performance status. Survival across the three trials is more or less identical (Table 2). Toxicity is also an important consideration in comparing treatment modalities. For sorafenib treated patients the dominant side effects are diarrhoea and hand foot skin reaction (HFSR), occurring at grade 3 or 4 in 8–11% but no treatment related deaths were reported in either the SHARP trial or the Asia-Pacific study [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ,
      • Cheng A.L.
      • Kang Y.K.
      • Chen Z.
      • Tsao C.J.
      • Qin S.
      • Kim J.S.
      • et al.
      Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial.
      ]. For the Western population of patients treated with doxorubicin, the dominant symptomatic grade 3 and 4 toxicity was fatigue occurring in 10.8% of patients with no treated related deaths reported [
      • Gish R.G.
      • Porta C.
      • Lazar L.
      • Ruff P.
      • Feld R.
      • Croitoru A.
      • et al.
      Phase III randomized controlled trial comparing the survival of patients with unresectable hepatocellular carcinoma treated with nolatrexed or doxorubicin.
      ], while in the Asian population febrile neutropenia occurred in 17% and there was a 3% treatment related mortality [
      • Yeo W.
      • Mok T.S.
      • Zee B.
      • Leung T.W.
      • Lai P.B.
      • Lau W.Y.
      • et al.
      A randomized phase III study of doxorubicin versus cisplatin/interferon alpha-2b/doxorubicin/fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma.
      ]. Full toxicity data relating to FOLFOX for HCC remains to be published [
      • Qin S.
      • Bai Y.
      • Ye S.
      • Fan J.
      • Lim H.
      • Cho J.Y.
      • et al.
      Phase III study of oxaliplatin plus 5-fluorouracil/leucovorin (FOLFOX4) versus doxorubicin as palliative systemic chemotherapy in advanced HCC in Asian patients.
      ].

      Combining chemotherapy with sorafenib and other targeted agents

      In common with other cancers, a number of key signalling pathways are dysregulated in HCC providing an opportunity for pharmacological intervention with targeted agents [
      • Llovet J.M.
      • Bruix J.
      Molecular targeted therapies in hepatocellular carcinoma.
      ] (Fig. 1). Combining targeted therapy with chemotherapy improved survival in several tumour types and similar combinations have been explored in HCC (Table 3). The combination of doxorubicin and sorafenib has been explored in a phase I dose escalation trial which demonstrated that both drugs could be given at full dose without additional toxicity despite the slight increase in doxorubicin exposure [
      • Richly H.
      • Henning B.F.
      • Kupsch P.
      • Passarge K.
      • Grubert M.
      • Hilger R.A.
      • et al.
      Results of a Phase I trial of sorafenib (BAY 43–9006) in combination with doxorubicin in patients with refractory solid tumors.
      ]. Eighteen patients with advanced HCC were enrolled in a follow-on study confirming a modest increase in the AUC (21%) and Cmax (33%) for doxorubicin. The most frequent non-haematological toxicity was diarrhoea and dose reductions or discontinuation of sorafenib was required in 33% cases due to emergent toxicity, most commonly HFSR [
      • Richly H.
      • Schultheis B.
      • Adamietz I.A.
      • Kupsch P.
      • Grubert M.
      • Hilger R.A.
      • et al.
      Combination of sorafenib and doxorubicin in patients with advanced hepatocellular carcinoma: results from a phase I extension trial.
      ]. Recently the results of a randomised double blind placebo controlled phase II (n = 96) trial comparing the combination of doxorubicin and sorafenib vs. doxorubicin alone have been published [
      • Abou-Alfa G.K.
      • Johnson P.
      • Knox J.J.
      • Capanu M.
      • Davidenko I.
      • Lacava J.
      • et al.
      Doxorubicin plus sorafenib vs. doxorubicin alone in patients with advanced hepatocellular carcinoma: a randomized trial.
      ]. The primary endpoint was time to progression which was superior in the combination group; median 6.4 vs. 2.8 months (p= 0.02), and overall survival, a secondary endpoint, was improved from a median of 6.5 to 13.7 months (p= 0.006). Adverse events were consistent with the known toxicity profile of sorafenib with HFSR in 6.4% and diarrhoea in 10.6% of those receiving the combination. Haematological toxicity was not increased by the addition of sorafenib but all grade systolic dysfunction and hypertension was more common with the combination; 19% vs. 2% and 17% vs. 0% respectively. While the difference in survival appears impressive, this trial cannot determine what, if any, additional contribution was made by doxorubicin in the combination arm. With sorafenib now established as the standard of care, a trial comparing sorafenib vs. sorafenib combined with doxorubicin is required and is currently underway.
      Figure thumbnail gr1
      Fig. 1Activated signal transduction pathways in HCC and associated molecularly targeted agents. PDGFR, platelet derived growth factor receptor; EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; VEGFR, vascular endothelial growth factor receptor.
      Table 3Trials in which chemotherapy has been combined with targeted agents.
      PFS, Progression free survival; OS, Overall survival.
      Bevacizumab, the anti-VEGF monoclonal antibody, has proved effective in combination with chemotherapy for breast [
      • Miller K.D.
      • Chap L.I.
      • Holmes F.A.
      • Cobleigh M.A.
      • Marcom P.K.
      • Fehrenbacher L.
      • et al.
      Randomized phase III trial of capecitabine compared with bevacizumab plus capecitabine in patients with previously treated metastatic breast cancer.
      ] and colon cancer [
      • Giantonio B.J.
      • Catalano P.J.
      • Meropol N.J.
      • O’Dwyer P.J.
      • Mitchell E.P.
      • Alberts S.R.
      • et al.
      Bevacizumab in combination with oxaliplatin, fluorouracil, and leucovorin (FOLFOX4) for previously treated metastatic colorectal cancer: results from the Eastern Cooperative Oncology Group Study E3200.
      ,
      • Saltz L.B.
      • Clarke S.
      • Diaz-Rubio E.
      • Scheithauer W.
      • Figer A.
      • Wong R.
      • et al.
      Bevacizumab in combination with oxaliplatin-based chemotherapy as first-line therapy in metastatic colorectal cancer: a randomized phase III study.
      ,
      • Hurwitz H.
      • Fehrenbacher L.
      • Novotny W.
      • Cartwright T.
      • Hainsworth J.
      • Heim W.
      • et al.
      Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer.
      ] despite its limited single agent activity. In HCC there have only been single arm phase II trials exploring a number of different chemotherapy regimens with bevacizumab [
      • Zhu A.X.
      • Blaszkowsky L.S.
      • Ryan D.P.
      • Clark J.W.
      • Muzikansky A.
      • Horgan K.
      • et al.
      Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma.
      ,
      • Sun W.
      • Sohal D.
      • Haller D.G.
      • Mykulowycz K.
      • Rosen M.
      • Soulen M.C.
      • et al.
      Phase 2 trial of bevacizumab, capecitabine, and oxaliplatin in treatment of advanced hepatocellular carcinoma.
      ,
      • Hsu C.H.
      • Yang T.S.
      • Hsu C.
      • Toh H.C.
      • Epstein R.J.
      • Hsiao L.T.
      • et al.
      Efficacy and tolerability of bevacizumab plus capecitabine as first-line therapy in patients with advanced hepatocellular carcinoma.
      ] (Table 3). Results are in line with those of chemotherapy or bevacizumab alone [
      • Boige V.
      • Raoul J.L.
      • Pignon J.P.
      • Bouche O.
      • Blanc J.F.
      • Dahan L.
      • et al.
      Multicentre phase II trial of capecitabine plus oxaliplatin (XELOX) in patients with advanced hepatocellular carcinoma: FFCD 03–03 trial.
      ,
      • Louafi S.
      • Boige V.
      • Ducreux M.
      • Bonyhay L.
      • Mansourbakht T.
      • de B.T.
      • et al.
      Gemcitabine plus oxaliplatin (GEMOX) in patients with advanced hepatocellular carcinoma (HCC): results of a phase II study.
      ,
      • Siegel A.B.
      • Cohen E.I.
      • Ocean A.
      • Lehrer D.
      • Goldenberg A.
      • Knox J.J.
      • et al.
      Phase II trial evaluating the clinical and biologic effects of bevacizumab in unresectable hepatocellular carcinoma.
      ] but no randomised trials are available. The anti-EGFR antibodies cetuximab and panitumumab, have also been effectively combined with chemotherapy in colorectal cancer [
      • Van C.E.
      • Kohne C.H.
      • Hitre E.
      • Zaluski J.
      • Chang Chien C.R.
      • Makhson A.
      • et al.
      Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer.
      ,
      • Douillard J.Y.
      • Siena S.
      • Cassidy J.
      • Tabernero J.
      • Burkes R.
      • Barugel M.
      • et al.
      Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study.
      ] where response has been linked to KRAS mutational status. In HCC there is only one reported phase II trial in which anti-EGFR therapy, using cetuximab, has been combined with chemotherapy [
      • Asnacios A.
      • Fartoux L.
      • Romano O.
      • Tesmoingt C.
      • Louafi S.S.
      • Mansoubakht T.
      • et al.
      Gemcitabine plus oxaliplatin (GEMOX) combined with cetuximab in patients with progressive advanced stage hepatocellular carcinoma: results of a multicenter phase 2 study.
      ] (Table 3) and further studies are needed.

      Overcoming chemoresistance in HCC

      In general, a variety of mechanisms contribute to chemoresistance including tumour hypoxia, expression of DNA repair enzymes and abrogation of apoptotic effector pathways by altered expression of genes such as TP53. For doxorubicin, one of the most important causes of resistance is increased expression of the drug transporter family known as the ATP-binding cassette (ABC) transporters. The most common member of this family is the ABCB1 (also known as MDR1; multidrug resistance gene 1) which encodes for P-glycoprotein. Overexpression of P-glycoprotein is associated with lower accumulation of doxorubicin in HCC cells and with worse prognosis in patients. Recent progresses in our understanding of mechanisms of resistance in HCC present opportunities to improve the response to cytotoxic agents. Potential strategies are discussed below and summarised in Table 4.
      Table 4Overview of the mechanisms of resistance to chemotherapy in hepatocellular carcinoma (HCC).

      Targeting G2 in TP53 mutated tumours

      An important mechanism of action for many anti-cancer agents, including platinum based drugs, is the induction of DNA damage. This causes the stabilisation of p53 leading to cell cycle arrest and DNA repair or apoptosis depending on the extent of the damage. TP53 mutations occur in around 50% of human carcinomas and are an important cause of drug resistance. In HCC, the worldwide prevalence of TP53 mutations has been estimated at around 28% [
      • Buendia M.A.
      Genetics of hepatocellular carcinoma.
      ] and 37% in the Chinese population [
      • Woo H.G.
      • Wang X.W.
      • Budhu A.
      • Kim Y.H.
      • Kwon S.M.
      • Tang Z.Y.
      • et al.
      Association of TP53 mutations with stem cell-like gene expression and survival of patients with hepatocellular carcinoma.
      ]. The highest rates are seen in aflatoxin exposed populations in which over 50% have a specific mutation at codon 249 [
      • Bressac B.
      • Kew M.
      • Wands J.
      • Ozturk M.
      Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa.
      ]. In addition, the X gene of HBV (HBx), which integrates into the host genome, is frequently mutated, yet mutant HBx proteins retain their ability to bind to p53 and attenuate p53-mediated DNA repair and apoptosis [
      • Hussain S.P.
      • Schwank J.
      • Staib F.
      • Wang X.W.
      • Harris C.C.
      TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer.
      ].
      p53 is required for regulation of the G1 checkpoint, but even in the absence of functional p53, cancers retain the G2 checkpoint allowing cell cycle arrest and repair of drug-induced DNA damage. Targeting the G2 checkpoint in TP53 mutated tumours therefore allows cells carrying DNA lesions into mitosis, prompting mitotic catastrophe and cell death. Potentially, this strategy provides a means of selectively sensitising tumour cells to chemotherapy-induced DNA damage since normal tissues retain intact functional p53 (Fig 2). Thus, the combination of Chk1 inhibition and DNA damage could provide a means of targeting cells in which G1 is already defective. AZD7762 is a potent ATP-competitive checkpoint kinase inhibitor that has been evaluated in preclinical models in combination with the antimetabolite gemcitabine, and the topoisomerase-1 inhibitor irinotecan. Both drugs induce G2 arrest, which is abrogated by the addition of AZD7762 and in xenografts, the combination of cytotoxics was more effective than either drug used separately [
      • Zabludoff S.D.
      • Deng C.
      • Grondine M.R.
      • Sheehy A.M.
      • Ashwell S.
      • Caleb B.L.
      • et al.
      AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies.
      ]. Phase 1 trials of AZD7762 in combination with gemcitabine have been completed but not yet reported. Another key regulator of the G2 checkpoint is cycline dependent kinase-1 (CDC2), which is inactivated by phosphorylation mediated by the tyrosine kinase, Wee-1 (Fig. 2). Inhibition of Wee-1 by the small molecule MK-1775 has recently been shown to abrogate the G2 DNA damage checkpoint and induce apoptosis in response to the cytotoxic drugs gemcitabine, carboplatin and cisplatin, but is inactive as a single agent [
      • Hirai H.
      • Iwasawa Y.
      • Okada M.
      • Arai T.
      • Nishibata T.
      • Kobayashi M.
      • et al.
      Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents.
      ]. The effect was selective for p53-deficient cell lines and caused little toxicity in vivo. Of interest, the phosphorylation of CDC2Y15 can be measured in skin hair follicles providing a clinically applicable pharmacodynamic biomarker. Phase I dose escalation trials are currently underway on MK-1775 as both a single agent or in combination with gemcitabine, cisplatin and carboplatin in solid cancers.
      Figure thumbnail gr2
      Fig. 2Pharmacological mechanisms to abrogate the G2 cell cycle checkpoint by inhibition of Wee-1 kinase and Chk 1. (A) Cell cycle: p53 is the key regulator of G1 checkpoint while p53 deficient tumours are dependent on G2 checkpoint. (B) The G2 checkpoint is regulated by the protein kinase Chk1, which is activated in response to DNA damage. Chk1 phosphorylates the protein phosphatase Cdc25, preventing Cdc25 from dephosphorylating and activating Cdc2. Inhibition of Chk1, therefore, promotes entry into mitosis. Similarly inhibition of Wee-1 also prevents mitotic delay by inhibiting phosphorylation of Cdc2. Green arrow indicates activation. Red line indicates inhibition.

      Targeting the PI3K/Akt/mTOR pathway

      The phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is an important growth factor signalling pathway that mediates several cellular processes including proliferation, survival and motility. Increased activation of this pathway is common in cancer, driving tumorigenesis and conferring resistance to chemotherapy. For example Wang et al. have shown that the oncogenic transcription factor octamer 4 (Oct4) is upregulated in chemotherapy resistant HCC cell lines due to demethylation of the Oct4 gene [
      • Wang X.Q.
      • Ongkeko W.M.
      • Chen L.
      • Yang Z.F.
      • Lu P.
      • Chen K.K.
      • et al.
      Octamer 4 (Oct4) mediates chemotherapeutic drug resistance in liver cancer cells through a potential Oct4-AKT-ATP-binding cassette G2 pathway.
      ]. Overexpression is associated with increased expression of stem cell markers including CD133, CD90 and CD19, tumourigenicity and the activation of Akt. In addition, there was upregulation of the drug efflux pump ABCG2 which could be inhibited by the PI3K inhibitor LY294002, suggesting the presence of an Oct4-AKT-ABCG2 pathway in resistant HCC. Further evidence that Akt is important for chemoresistance in HCC stem cells comes from cell sorting experiments in which the CD133+ subgroup (stem cell phenotype) was found to have greater resistance to doxorubicin, and 5FU compared with CD133− cells [
      • Ma S.
      • Lee T.K.
      • Zheng B.J.
      • Chan K.W.
      • Guan X.Y.
      CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway.
      ]. The mechanism of resistance was reduced apoptosis associated with activation of Akt and Bcl2 survival pathways with inactivation of proapoptotic BAD. In this model, the preferential survival of CD133+ cells in the presence of chemotherapy could also be abolished by inhibition of Akt. CD133+ are known to account for 1.3–13.6% of cells in the bulk of primary HCC cells yet may be the critical target since they have property of self replication.
      There are number of inhibitors of mTOR available in the clinic, including sirolimus, everolimus, temsirolimus and AP23573. As single agents, these drugs are cytostatic and have little effect on apoptosis yet, in combination with cytoxic drugs such as cisplatin, have been shown to enhance apoptosis [
      • Beuvink I.
      • Boulay A.
      • Fumagalli S.
      • Zilbermann F.
      • Ruetz S.
      • O’Reilly T.
      • et al.
      The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damaged induced apoptosis through inhibition of p21 translation.
      ,
      • Tam K.H.
      • Yang Z.F.
      • Lau C.K.
      • Lam C.T.
      • Pang R.W.
      • Poon R.T.
      Inhibition of mTOR enhances chemosensitivity in hepatocellular carcinoma.
      ]. The effect is p53-dependent and not observed in TP53 −/− or TP53 mutant cell lines and appears to be mediated by an effect on p21 (Fig 3). Thus, there is a clear rationale for combining DNA damaging agents with mTOR inhibitors in p53 wild type tumours.
      Figure thumbnail gr3
      Fig. 3DNA damage induced by cisplatin activates p53 which increases levels of p21 leading to cell cycle arrest allowing potential for DNA repair. In the presence of the mTOR inhibitor everolimus, the rise in p21 is inhibited favouring an apoptotic response to p53 activation mediated by the pro-apoptotic molecules Puma, Noxa and Bax.
      Even in TP53 wild type tumours, the approach may be valid since in vivo studies suggest combination therapy is effective through inhibition of angiogenesis. Piguet et al. have shown that combination of doxorubicin and sirolimus is more effective than either drug alone in a TP53 mutant HCC rat model [
      • Piguet A.C.
      • Semela D.
      • Keogh A.
      • Wilkens L.
      • Stroka D.
      • Stoupis C.
      • et al.
      Inhibition of mTOR in combination with doxorubicin in an experimental model of hepatocellular carcinoma.
      ]. The reduced growth was associated with reduced angiogenesis and lowered levels of p21 in the endothelial cells which were TP53 wild type.
      Another approach to increasing chemosensitivity by modulation of the mTOR pathway is to mimic endogenous regulators. MicroRNAs (miRNAs) are small RNA sequences that bind to target RNA sequences thereby regulating post-transcriptional expression. MicroRNA-199a-3p (miR-199a-3p) has been shown to bind directly to mTOR mRNA and decrease mTOR protein expression with a consequent restoration of sensitivity to doxorubicin in HCC cell lines presenting an opportunity to develop analogues that can mimic this activity [
      • Fornari F.
      • Milazzo M.
      • Chieco P.
      • Negrini M.
      • Calin G.A.
      • Grazi G.L.
      • et al.
      MiR-199a–3p regulates mTOR and c-Met to influence the doxorubicin sensitivity of human hepatocarcinoma cells.
      ].
      Clinical trials combining mTOR inhibitors with both chemotherapy and radiotherapy are ongoing.

      Overcoming hypoxia associated chemoresistance

      Hypoxia has been proposed a significant cause of chemoresistance and is particularly relevant in the treatment of HCC in which chemotherapy is administered with the concurrent induction of acute hypoxia during transarterial chemoembolisation (TACE). The cellular response to hypoxia is mediated by hypoxia-inducible (HIF-1), a heterodimer composed of HIF-1α and HIF-1β. Under normoxic conditions HIF-1α is rapidly hydroxylated, ubiquinated and degraded by the proteosome. In hypoxic conditions, hydroxylation is inhibited allowing HIF-1α levels to rise and dimerise with HIF-1β. The dimer binds to the hypoxia response elements, transactivating a number of genes responsible for angiogenesis, erythropoiesis and glycolysis. Recent evidence also suggests that PDGF is a target gene for HIF-1 and that an autocrine loop involving Akt/HIF and PDGF is responsible for hypoxia-induced resistance to cisplatin (Fig. 4) [
      • Lau C.K.
      • Yang Z.F.
      • Ho D.W.
      • Ng M.N.
      • Yeoh G.C.
      • Poon R.T.
      • et al.
      An Akt/hypoxia-inducible factor-1alpha/platelet-derived growth factor-BB autocrine loop mediates hypoxia-induced chemoresistance in liver cancer cells and tumorigenic hepatic progenitor cells.
      ]. Hence, the combination of chemotherapy with inhibitors of PDGF signalling or the AKT pathway provides a potential means of overcoming hypoxia-induced resistance and there is a clear rationale for combining sorafenib, which blocks PDGF signalling, with TACE or systemic chemotherapy.
      Figure thumbnail gr4
      Fig. 4Hypoxia causes an increase in the transcription factor hypoxia inducible factor HIF-1α which transactivates a number of hypoxia responsive genes including platelet derived growth factor (PDGFβ) and vascular endothelial growth factor (VEGF). In an autocrine feedback loop, PDGF binds to its receptor activating the phosphatidylinositol 3-kinase (PI3K)/Akt pathway which in turn increases HIF-1α. The activation of Akt promotes drug resistance via activation of glycogen synthase kinase-3β (GSK-3β). The feedback loop can be inhibited by the PI3K inhibitor LY294002, the HIF-1α inhibitor YC1 or the PDGF receptor inhibitor AG1296
      [
      • Lau C.K.
      • Yang Z.F.
      • Ho D.W.
      • Ng M.N.
      • Yeoh G.C.
      • Poon R.T.
      • et al.
      An Akt/hypoxia-inducible factor-1alpha/platelet-derived growth factor-BB autocrine loop mediates hypoxia-induced chemoresistance in liver cancer cells and tumorigenic hepatic progenitor cells.
      ]
      .

      Targeting angiogenesis with chemotherapy – metronomic chemotherapy

      The fact that proliferating endothelial cells are genetically stable and less prone to develop drug resistance is one of the key justifications for targeting angiogenesis for cancer therapy. The concept of chemotherapy as an anti-angiogenic therapy was first proposed by Browder et al. [
      • Browder T.
      • Butterfield C.E.
      • Kraling B.M.
      • Shi B.
      • Marshall B.
      • O’Reilly M.S.
      • et al.
      Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer.
      ] as a means of overcoming tumour chemoresistance. Conventional chemotherapy is given at high doses according to intermittent schedules that allow normal tissue, including proliferating endothelium and bone marrow cells, to recover. By administering continuous, low dose cyclophosphamide, drug resistant tumours were more effectively treated as compared with conventional doses and schedules. Endothelial cell apoptosis was shown to precede tumour cell apoptosis suggesting the tumour growth was inhibited by the antiangiogenic effect of cyclophosphamide [
      • Browder T.
      • Butterfield C.E.
      • Kraling B.M.
      • Shi B.
      • Marshall B.
      • O’Reilly M.S.
      • et al.
      Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer.
      ]. Combining so called ‘metronomic chemotherapy’ with other antiangiogenic approaches, such as inhibition of VEGFR-2, has proved even more effective in preclinical experiments [
      • Klement G.
      • Huang P.
      • Mayer B.
      • Green S.K.
      • Man S.
      • Bohlen P.
      • et al.
      Differences in therapeutic indexes of combination metronomic chemotherapy and an anti-VEGFR-2 antibody in multidrug-resistant human breast cancer xenografts.
      ,
      • Klement G.
      • Baruchel S.
      • Rak J.
      • Man S.
      • Clark K.
      • Hicklin D.J.
      • et al.
      Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity.
      ,
      • Kerbel R.S.
      Improving conventional or low dose metronomic chemotherapy with targeted antiangiogenic drugs.
      ]. Clinical trials of metronomic chemotherapy [
      • Pasquier E.
      • Kavallaris M.
      • Andre N.
      Metronomic chemotherapy: new rationale for new directions.
      ] have been reported in a number of different tumour types including breast [
      • Colleoni M.
      • Orlando L.
      • Sanna G.
      • Rocca A.
      • Maisonneuve P.
      • Peruzzotti G.
      • et al.
      Metronomic low-dose oral cyclophosphamide and methotrexate plus or minus thalidomide in metastatic breast cancer: antitumor activity and biological effects.
      ,
      • Bottini A.
      • Generali D.
      • Brizzi M.P.
      • Fox S.B.
      • Bersiga A.
      • Bonardi S.
      • et al.
      Randomized phase II trial of letrozole and letrozole plus low-dose metronomic oral cyclophosphamide as primary systemic treatment in elderly breast cancer patients.
      ], ovarian [
      • Sanchez-Munoz A.
      • Mendiola C.
      • Perez-Ruiz E.
      • Rodriguez-Sanchez C.A.
      • Jurado J.M.
      • Alonso-Carrion L.
      • et al.
      Bevacizumab plus low-dose metronomic oral cyclophosphamide in heavily pretreated patients with recurrent ovarian cancer.
      ], glioblastoma [
      • Kong D.S.
      • Lee J.I.
      • Kim J.H.
      • Kim S.T.
      • Kim W.S.
      • Suh Y.L.
      • et al.
      Phase II trial of low-dose continuous (metronomic) treatment of temozolomide for recurrent glioblastoma.
      ] and non-Hodgkin’s lymphoma [
      • Buckstein R.
      • Kerbel R.S.
      • Shaked Y.
      • Nayar R.
      • Foden C.
      • Turner R.
      • et al.
      High-Dose celecoxib and metronomic “low-dose” cyclophosphamide is an effective and safe therapy in patients with relapsed and refractory aggressive histology non-Hodgkin’s lymphoma.
      ].
      Recently, there has been growing interest in exploring the potential benefits of metronomic chemotherapy in advanced HCC given its relative chemoresistance.
      In a orthotopic model of HCC, metronomic chemotherapy with cyclophosphamide, tagefur-uracil (UFT), doxorubicin or a combination thereof was found to be ineffective but, with the addition of VEGR-2 inhibition with the antibody DC101, was more effective than chemotherapy or DC101 alone [
      • Tang T.C.
      • Man S.
      • Lee C.R.
      • Xu P.
      • Kerbel R.S.
      Impact of metronomic UFT/cyclophosphamide chemotherapy and antiangiogenic drug assessed in a new preclinical model of locally advanced orthotopic hepatocellular carcinoma.
      ]. Furthermore, in a model of acquired resistance to sorafenib, the combination of UFT and low dose sorafenib delayed the emergence of resistance and reduced toxicity compared to a higher dose of sorafenib alone [
      • Tang T.C.
      • Man S.
      • Xu P.
      • Francia G.
      • Hashimoto K.
      • Emmenegger U.
      • et al.
      Development of a resistance-like phenotype to sorafenib by human hepatocellular carcinoma cells is reversible and can be delayed by metronomic UFT chemotherapy.
      ].
      The combination of sorafenib and metronomic UFT has been taken forward in a phase II trial of 53 patients from Taiwan [
      • Hsu C.H.
      • Shen Y.C.
      • Lin Z.Z.
      • Chen P.J.
      • Shao Y.Y.
      • Ding Y.H.
      • et al.
      Phase II study of combining sorafenib with metronomic tegafur/uracil for advanced hepatocellular carcinoma.
      ]. Patients were unsuitable for locoregional therapy, had Child–Pugh A liver disease and ECOG performance scores 0–2. The majority were hepatitis B surface antigen positive and had extrahepatic disease. Patients received sorafenib 400 mg BD and UFT continuously as first line therapy for advanced HCC and continued until disease progression or unacceptable toxicity. At a median follow up of 16.1 months, the median PFS was 3.7 months (95% CI: 1.9–5.5) and median OS 7.4 months (95% CI 3.4–11.4). According to the RECIST criteria, ORR was 8% (CR = 0; PR = 4) with 26 patients achieving disease stabilisation. The authors concluded that metronomic UFT can be safely combined with sorafenib and appears to improve efficacy of sorafenib in advanced HCC with minimal haematological toxicities. Predominant side effects were similar to sorafenib monotherapy with HFSR, diarrhoea and fatigue. These results should be interpreted in the context of survival figures from the Asia-Pacific study (China, Korea and Taiwan) in which single agent sorafenib was associated with a median survival of 6.6 months [
      • Cheng A.L.
      • Kang Y.K.
      • Chen Z.
      • Tsao C.J.
      • Qin S.
      • Kim J.S.
      • et al.
      Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial.
      ], however, randomised trials are required to demonstrate superiority.

      Molecular prediction of response to chemotherapy

      Tumours are increasingly subjected to molecular profiling in order to select the optimum therapy. In some cases, specific mutations or alterations in gene expression are known to predict drug sensitivity while in others, gene signatures have been associated with better outcomes.

      MGMT

      The sensitivity of tumours to certain alkylating agents has been linked with the expression levels of the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT). Temozolomide, streptozocin and decarbazine all induce DNA methylation at the O6 position of guanine which is not repaired in tumours where epigenetic silencing of the MGMT gene promoter CpG island prevents MGMT expression. Hence, in glioblastoma, response to temozolomide has been associated with MGMT expression in several studies [
      • Brandes A.A.
      • Tosoni A.
      • Cavallo G.
      • Reni M.
      • Franceschi E.
      • Bonaldi L.
      • et al.
      Correlations between O6-methylguanine DNA methyltransferase promoter methylation status, 1p and 19q deletions, and response to temozolomide in anaplastic and recurrent oligodendroglioma: a prospective GICNO study.
      ,
      • Hegi M.E.
      • Diserens A.C.
      • Gorlia T.
      • Hamou M.F.
      • de T.N.
      • Weller M.
      • et al.
      MGMT gene silencing and benefit from temozolomide in glioblastoma.
      ]. In HCC, methylation of the MGMT promoter has been variably reported and may be related to aetiology. In a series from Taiwan, hypermethylation was found in 39% HCC and there appeared to be a relationship between hypermethylation and aflatoxin B1 DNA adducts [
      • Zhang Y.J.
      • Chen Y.
      • Ahsan H.
      • Lunn R.M.
      • Lee P.H.
      • Chen C.J.
      • et al.
      Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma.
      ]. However, other studies, including those from Japan, have found relatively low levels of MGMT hypermethylation in comparison to other target genes [
      • Formeister E.J.
      • Tsuchiya M.
      • Fujii H.
      • Shpyleva S.
      • Pogribny I.P.
      • Rusyn I.
      Comparative analysis of promoter methylation and gene expression endpoints between tumorous and non-tumorous tissues from HCV-positive patients with hepatocellular carcinoma.
      ,
      • Nomoto S.
      • Kinoshita T.
      • Kato K.
      • Otani S.
      • Kasuya H.
      • Takeda S.
      • et al.
      Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma.
      ].

      ERCC1

      Another important mechanism of DNA repair is through the nucleotide excision repair (NER) pathway which is responsible for the repair of interstrand and intrastrand crosslinks formed by cisplatin. The excision repair cross-complementation group 1 (ERCC1) is responsible for excising DNA adducts and has an important role in NER pathway. Increased levels of ERCC1 have been associated with resistance to platinum-based chemotherapy in colon, gastric, lung and ovarian cancer. In HCC, ERCC1 expression has been reported in to be increased in comparison to normal liver tissues [
      • Ueda S.
      • Shirabe K.
      • Morita K.
      • Umeda K.
      • Kayashima H.
      • Uchiyama H.
      • et al.
      Evaluation of ERCC1 expression for cisplatin sensitivity in human hepatocellular carcinoma.
      ,
      • Fautrel A.
      • Andrieux L.
      • Musso O.
      • Boudjema K.
      • Guillouzo A.
      • Langouet S.
      Overexpression of the two nucleotide excision repair genes ERCC1 and XPC in human hepatocellular carcinoma.
      ] and, in vitro, cisplatin sensitivity was increased using ERCC1 siRNA to inhibit ERCC1 expression. Interestingly, the expression of ERCC1 in hepatocytes has been shown to increase in response to epidermal growth factor (EGF). While basal levels could be inhibited by blocking the phosphoinositide-3-kinase (PI3K) pathway, the response to EGR was inhibited by blocking the mitogen-activated protein kinase (MAPK) pathway [
      • Andrieux L.O.
      • Fautrel A.
      • Bessard A.
      • Guillouzo A.
      • Baffet G.
      • Langouet S.
      GATA-1 is essential in EGF-mediated induction of nucleotide excision repair activity and ERCC1 expression through ERK2 in human hepatoma cells.
      ]. This provides a potential rationale for combining EGFR or MAPK inhibition with platinum chemotherapy in HCC. So far, the only reported trial combined the anti-EGFR antibody cetuximab with the combination of gemcitabine and oxaliplatin in a single arm phase II study [
      • Asnacios A.
      • Fartoux L.
      • Romano O.
      • Tesmoingt C.
      • Louafi S.S.
      • Mansoubakht T.
      • et al.
      Gemcitabine plus oxaliplatin (GEMOX) combined with cetuximab in patients with progressive advanced stage hepatocellular carcinoma: results of a multicenter phase 2 study.
      ]. The response rate was 20% with a median PFS of 4.7 months and OS of 9.5 months which was similar to that reported previously for chemotherapy alone [
      • Louafi S.
      • Boige V.
      • Ducreux M.
      • Bonyhay L.
      • Mansourbakht T.
      • de B.T.
      • et al.
      Gemcitabine plus oxaliplatin (GEMOX) in patients with advanced hepatocellular carcinoma (HCC): results of a phase II study.
      ].

      AEG1

      The recently identified gene, astrocyte elevated gene-1 (AEG-1), has also been shown to play a role in chemoresistance. This gene was first identified as a HIV and TNF-α inducible gene in primary foetal astrocytes but was shown to promote migration and invasion activating both the Wnt/β-catenin and NF-κB pathway [
      • Yoo B.K.
      • Emdad L.
      • Su Z.Z.
      • Villanueva A.
      • Chiang D.Y.
      • Mukhopadhyay N.D.
      • et al.
      Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression.
      ]. AEG-1 is expressed in over 90% of HCC samples and overexpression was found to convert non-tumourigenic HCC cell lines into aggressive tumours [
      • Yoo B.K.
      • Emdad L.
      • Su Z.Z.
      • Villanueva A.
      • Chiang D.Y.
      • Mukhopadhyay N.D.
      • et al.
      Astrocyte elevated gene-1 regulates hepatocellular carcinoma development and progression.
      ]. Recently AEG-1 was also shown to play an important role in chemoresistance by two separate mechanisms. Firstly, resistance to 5-fluorouracil (5FU) is promoted by the increased expression of the transcription factor LSF (late SV40 factor) which upregulates thymidylate synthase (TS), the target enzyme for 5FU, and also enhances the expression of dihydropyrimidine dehydrogenase (DPD), which catalyses the rate limiting step in the catabolism of 5FU. In xenografts, the combination of 5FU and AEG-1 siRNA was superior to either intervention alone in inhibiting growth [
      • Yoo B.K.
      • Gredler R.
      • Vozhilla N.
      • Su Z.Z.
      • Chen D.
      • Forcier T.
      • et al.
      Identification of genes conferring resistance to 5-fluorouracil.
      ]. Secondly, AEG-1 confers resistance to doxorubicin by increasing the expression of the multidrug resistance gene 1 (MDR1) by increasing its translation and inhibiting its proteosome mediated degradation. Again the combination of AEG siRNA and doxorubicin was found to be superior to either agent alone in preclinical models [
      • Yoo B.K.
      • Chen D.
      • Su Z.Z.
      • Gredler R.
      • Yoo J.
      • Shah K.
      • et al.
      Molecular mechanism of chemoresistance by astrocyte elevated gene-1.
      ].

      Conclusions

      Sorafenib is currently regarded as the standard of care in selected patients with advanced HCC based on two large randomised placebo controlled trials. Meanwhile, recent large randomised trials suggest that cytotoxic chemotherapy also has a modest impact on survival in this patient group. However, the efficacy of chemotherapy is compromised by resistance due to activation of signalling pathways, hypoxia and genetic aberrations. In the last ten years our understanding of the molecular basis of resistance has improved, providing an opportunity to explore novel schedules and rational combinations of targeted agents with chemotherapy. Targeting mTOR provides a promising means of abrogating the drug resistance arising from activation of the PI3K/Akt pathway in p53 wild type tumours while targeting the G2 checkpoint may overcome resistance in p53 mutated tumours. Sorafenib inhibits signalling through both the Raf/Mek/Erk pathway and may so reduce the expression of mdr1 and prevent activation of the hypoxia induced Akt/HIF1/PDGF autocrine loop. There are already data on the safety and efficacy of sorafenib in combination with doxorubicin and results of the comparison of this combination with sorafenib alone are awaited.
      Single arm phase II trials are difficult to interpret in this patient population and randomised phase II trials with time to progression as a primary endpoint should be performed using novel combinations compared with standard therapy in order to establish a justification for proceeding to phase III where survival remains the key endpoint [
      • Llovet J.M.
      • Di Bisceglie A.M.
      • Bruix J.
      • Kramer B.S.
      • Lencioni R.
      • Zhu A.X.
      • et al.
      Design and endpoints of clinical trials in hepatocellular carcinoma.
      ]. Patient selection remains of key importance. In general, only patients with good performance status and well preserved liver function should be treated, in whom HCC is the life limiting disease and increased toxicity due to liver impairment is minimised. Molecular pathology may further help define subpopulations that should be treated with targeted agents in combination with chemotherapy.

      Conflict of interest

      The authors declared that do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Acknowledgements

      This work was supported by the UCLH/UCL Department of Health’s NIHR Biomedical Research Centres funding scheme .

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