{'fraction_non_alphanumeric': 0.03281027104136947, 'fraction_numerical': 0.020978434169673574, 'mean_word_length': 5.10684089162183, 'pattern_counts': {'":': 0, '<': 3, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Second-line treatment options in hepatocellular carcinoma

For many years, sorafenib has been the only approved systemic treatment for advanced hepatocellular carcinoma (HCC). For over a decade, randomized controlled trials exploring the efficacy of new drugs both in first- and second-line treatment have failed to prove any survival benefit. However, in the past few years, several advances have been made especially in pretreated patients; phase III trials of regorafenib, cabozantinib, and ramucirumab in patients with elevated α-fetoprotein have demonstrated efficacy in patients progressing after or intolerant to sorafenib. In addition, early phase I and II trials have shown promising results of immunotherapy alone or in combination with tyrosine-kinase inhibitors or monoclonal antibodies in the same setting of patients. In this review, we will discuss the evidence on second-line options for HCC, focusing on the latest results that are currently refining the treatment scenario.


Introduction
Hepatocellular carcinoma (HCC) is the most frequent primary liver tumor, and it is a worldwide leading cause of morbidity and mortality. According to recent statistics, about 40,000 HCC new cases and 30,000 deaths are expected in the United States alone in 2018. 1 Chronic liver inflammation is a procarcinogenic condition that is associated to the majority of known HCC risk factors: hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol abuse/ exposure to toxic agents, nonalcoholic fatty liver disease (NAFLD), and autoimmune hepatitis. In 30% of cases, no risk factor is identified and HCC is defined as cryptogenetic; however, it is important to point out that many of these risk factors are preventable (such as HBV infection prevented by active vaccination) or can be handled with habit modifications (alcohol abuse and nonalcoholic steatohepatitis [NASH] that is a precursor of NAFLD) or with effective treatment (directacting antiviral agents for HCV infection). 2 Although this should hopefully lead to a drastic change in the epidemiology of HCC in the next decades, HCC currently remains a major health problem worldwide. 3 Sorafenib, a multi-target tyrosine-kinase inhibitor (TKI), was the first drug to demonstrate efficacy in advanced HCC with well-preserved liver function (Child-Pugh A). Ten years ago, in the SHARP randomized trial and afterward in the Asia-Pacific randomized trial, the comparison of sorafenib to placebo led to a significant improvement in overall survival (OS) and time to progression (TTP). 4,5 The drug was consequently approved for the treatment of advanced disease, and has remained the gold standard for over a decade. After the negative results of several randomized controlled trials (RCTs), [6][7][8][9][10] in 2018 lenvatinib has proved to be noninferior to sorafenib in the first-line setting 11 and has subsequently received approval by Food and Drugs Administration (FDA) and European Medicines Agency (EMA). 12,13 Unfortunately, both adaptive resistance and intrinsic resistance represent crucial issues in the management of patients receiving first-line treatment of HCC. Disease progression or significant toxicity leading to first-line therapy discontinuation is a frequent event and further treatment options are eagerly needed. Disappointingly, the first attempt to provide a gold standard in the second-line treatment was unsuccessful.
We performed a structured search on the PubMed database and on the proceedings of the main oncology and hepatology conferences, identifying clinical trials of second-line therapy in HCC between 1 January 2008 and 15 February 2019. We also searched Clinicaltrials.gov with the terms 'hepatocellular carcinoma' and 'liver cancer' to identify ongoing post-sorafenib clinical trials. ISSN: 1740-4398 REVIEW -Second-line therapy in HCC drugsincontext.com Hence, in this review, we will discuss the evidence on secondline options for HCC, focusing on the latest results that are currently refining the treatment scenario.

Negative randomized control trials
Following the publication of the SHARP trial and based on promising phase II trials, several drugs have been tested in RCTs in patients experiencing failure of first-line sorafenib. In these trials, given the absence of an active standard treatment, TKIs or monoclonal antibodies (MoAbs) with different target profile were compared to placebo and best supportive care (BSC).

Brivanib
Brivanib is an oral inhibitor of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptor that was tested in HCC patients both in first-line and in second-line therapy. 7

Everolimus
On the basis of preclinical evidence supporting the role of the mTOR pathway in hepatocarcinogenesis and of the clinical experience of the mTOR inhibitor everolimus in HCC, the phase III trial EVOLVE-1 was designed. 15 It enrolled 546 patients with advanced HCC whose disease progressed during or after sorafenib or who were sorafenib intolerant. The patients were randomized in a 2:1 ratio to receive everolimus 7.5 mg per day (n=362) or matched placebo (n=184

Tivantinib
In the METIV-HCC trial, tivantinib, a selective TKI targeting MET receptor, was tested in a biomarker-enriched population. 20 The HGF-MET pathway is involved in tumor development and metastasis and has a prognostic value. Moreover, MET is often overexpressed after sorafenib exposure. In a randomized phase II trial, interesting hints of efficacy of tivantinib were seen in MET-high tumors 21 ; therefore, only patients harboring this molecular characteristic (staining intensity score ≥2 in ≥50% of tumor cells) were enrolled in the subsequent randomized, double-blind, placebo-controlled trial. Unfortunately, efficacy outcomes were not significantly different between the groups, and the trial did not meet its primary endpoint (mOS was 8.4 months for tivantinib and 9.  10%) and discontinuation due to drugrelated adverse events (10 versus 4%); however, no difference in grade 5 adverse events was recorded. HR-QoL was assessed using FACT-G, FACT-Hep, EQ-5D, and EQ-VAS questionnaires; globally, the scores were similar between the groups except for the FACT-Hep total score that favored placebo (p<0.001). Nevertheless, the difference did not meet the established minimally important threshold of 8-9 points (129.31 points for regorafenib and 133.17 points for placebo). 25 The adverse events profile of regorafenib in this study is in line with the known drug safety profile from other indications, being hypertension (15%), HSFR (9%), and diarrhea (3%) the most common clinically relevant grade 3 or 4 events.
Based on the results of the RESORCE trial, regorafenib was the first drug to be approved by regulatory agencies as secondline treatment for HCC patients following first-line sorafenib. Subsequent analyses showed that, at least in this selected population of patients who well tolerated sorafenib and were subsequently eligible for a second-line treatment, the sequence sorafenib-regorafenib yields a mOS of 26 months versus 19.2 months of sorafenib-placebo, and that the survival benefit is independent of the pattern of the disease progression during prior sorafenib treatment and of their last sorafenib dose (800 or <800 mg/day). 26 The development of new distant metastases or vascular invasion was confirmed to be associated with worse survival irrespective of treatment. 27 Similar to previous observation with sorafenib, also for regorafenib treatment the early onset of dermatological toxicity correlates with better outcome. 28 As for molecular biomarkers, regorafenib benefit is independent of baseline AFP and cMET protein levels that are on the contrary negative prognostic factors. 29 Initial evidence suggests that some single-nucleotide polymorphisms (SNPs) assessed in the RESORCE population may have a prognostic and predictive impact: however, to date, no predictive molecular biomarker of regorafenib benefit has been established. 30

Cabozantinib
Cabozantinib is an oral multitarget TKI that was initially identified as a potent dual inhibitor of VEGFR-2 and c-MET. Its target profile also includes the inhibition of VEGFR-1 and 3, AXL, RET, FLT3, and TIE-2. It is important to point out that both MET and AXL overexpression has a negative prognostic impact and that MET might be induced by sorafenib treatment and can be a mechanism of resistance. 31,32 The first clinical experience tivantinib in HCC treatment was also confirmed in the Japanese population enrolled in the phase III trial JET-HCC. 22

Regorafenib
Regorafenib is an oral multitarget inhibitor that is already approved for refractory colorectal cancer and gastrointestinal stromal tumors (GISTs). Although it is structurally similar to sorafenib, regorafenib shows a more potent pharmacological activity and a distinct molecular target profile, blocking the activity of several protein kinases involved in the regulation of tumor angiogenesis (VEGFR1, VEGFR2, VEGFR3, and TIE2), oncogenesis (KIT, RET, RAF1, BRAF, and BRAF V600E ), and the tumor microenvironment (PDGFR and FGFR). In a single-arm phase II trial, 36 patients with Barcelona Clinic Liver Cancer (BCLC) staging classification B or C HCC that would not benefit from treatments of established efficacy were enrolled. 23 Notably, patients who experienced discontinuation of previous sorafenib due to poor drug tolerability were excluded. The primary endpoint of the study was to assess the safety of regorafenib as second-line treatment in HCC patients who had disease progression after sorafenib; secondary endpoints included efficacy (including TTP and OS) and pharmacokinetics.
In half of the cases, adverse events were the reasons for treatment discontinuation, but only in 19% it was deemed to be related to regorafenib. All the 36 patients experienced at least a treatment-related adverse event, and dose modifications were frequent (97%); nevertheless, the safety profile in this setting of patients was in line with the known safety from other indications, and it was globally deemed manageable, being diarrhea, fatigue, and hand-foot skin reaction (HFSR) the most frequent adverse events. Notably, in the efficacy analysis, mTTP was 4.3 months (95% CI: 2.9-13.1) and mOS was 13.8 months (95% CI: 9.3-18.3) with a DCR of 72%. On these bases, the drug was moved to a phase III RCT, the RESORCE study, 24 that was planned as a double-blind, placebo-controlled, international trial. Randomization was stratified by geographical region (Asia versus rest of world), macrovascular invasion (yes versus no), extrahepatic disease (yes versus no), AFP concentration (<400 versus ≥400 ng/mL), and Eastern Cooperative Oncology Group (ECOG) performance status (PS) (0 versus 1). As for the phase II study, eligible patients must have a BCLC stage B or C not amenable to locoregional treatments, a Child-Pugh A, and they had to be previously treated with sorafenib in first-line treatment. To select a study population that could more likely tolerate regorafenib, patients needed to be sorafenib-tolerant, meaning that they must have received ≥400 mg of sorafenib daily for at least 20 of the 28 days preceding discontinuation. As a consequence, all the patients enrolled in the RESORCE trial had discontinued sorafenib because of disease progression. The drug was administered at the recommended dosing schedule of 160 mg once daily in repeating cycles of 3 weeks on treatment followed by 1 week off treatment and the treatment needed to be initiated within 10 weeks of sorafenib discontinuation. After a median follow-up of 7 months, the ISSN: 1740-4398 REVIEW -Second-line therapy in HCC drugsincontext.com of cabozantinib in HCC involved 41 patients (19 sorafenib naïve, 22 sorafenib pretreated) enrolled in 1 of the 9 cohorts of a phase II randomized discontinuation trial. 33,34 Patients received a 12-week lead-in treatment with 100 mg per day of cabozantinib. After restaging, patients with stable disease (SD) were randomized to cabozantinib or placebo, patients with a partial response (PR) could continue open-label cabozantinib treatment, and patients with progressive disease (PD) at or before week 12 permanently discontinued treatment. The primary endpoint of the lead-in phase was ORR at week 12, and the primary endpoint of the randomized phase was PFS. Randomization was halted before the preplanned accrual was reached due to initial efficacy results. In the HCC cohort, cabozantinib demonstrated a week-12-ORR of 5% and a week-12-DCR of 66%. Among the patients who had SD at week 12 (n=22), 12 patients were randomized to placebo and 10 to cabozantinib. With the limitation of this small sample size, no significant difference in PFS was observed between the two groups. In the overall population of 41 treated patients, mPFS from the start of the study was 5.2 months and mOS 11.5 months. The most frequent adverse events (diarrhea, weight loss, and HFSR) were mainly mild to moderate.
Based on these preliminary signals of clinical activity, a phase III, randomized, double-blind, controlled trial comparing cabozantinib to placebo in patients with HCC who had received prior sorafenib therapy was conducted (the CELESTIAL trial). 35

Ramucirumab
We have already discussed the initial clinical development of the MoAb ramucirumab in HCC. Following the results of the REACH trial 16 in the subgroup of patients with baseline AFP concentration of 400 ng/mL or greater, the REACH-2 trial was designed to assess the benefit of ramucirumab specifically in this subgroup of patients. The results of this trial were presented at the 2018 ASCO Meeting. 41  The results of phase III trials in second-line treatment for HCC are summarized in Table 1.

Immunotherapy and future perspectives
Immunotherapy has been the breakthrough in cancer treatment in the last few years, and it has completely revolutionized the cure strategy, with continuous advances and evidence of efficacy in several types of cancer. The possibility of unleashing T cells activation against cancer cells produces durable responses at the cost of limited toxicity. Although the knowledge of predictive factors is far from being optimal, efficacy of immunotherapy is often correlated to specific features that are independent of the site of primary tumor, such as PD-1/PD-L1 expression, tumor mutational burden (TMB), or presence of microsatellite-instable phenotype (MSI-H). [44][45][46] In HCC etiology, the role of chronic liver injury is prevalent and it is associated with inflammation and establishment of a suppressed immune environment. In addition, high expression of PD-L1 in tumors correlates with a poorer prognosis in patients with HBV-related HCC, and upregulation of PD-1 on CD8(+) T cells could predict a poorer disease progression and postoperative recurrence. 47 . Survival outcomes were encouraging with a 6-and 9-month survival of 83 and 74%, respectively, and an mTTP of 4 months. In recent updates of the study, 18-month OS rates were 57% in sorafenib-naïve patients and 44% in sorafenib-experienced patients, mOS was 28.6 months and 15.6 months, respectively, and survival was correlated with the degree of nivolumab antitumor activity. 51,52 As for safety, the most common adverse events of any grade were fatigue (23%), pruritus (21%), and rash (15%) and grade 3/4 AST and alanine aminotransferase (ALT) increase occurred in 4 and 2%, respectively. Symptomatic treatmentrelated adverse events were comparable in patients with and without HCV or HBV infection. As a secondary endpoint, PD-L1 expression levels were retrospectively assessed as a potential biomarker for nivolumab therapy.  54 After a median follow-up of 12.3 months, an ORR was recorded in 18 (17%) of the patients (CR 1% and PR 16%). Moreover, 44% of patients had SD as best response on treatment. Similar to nivolumab, also for pembrolizumab responses generally occurred at the first radiological assessment and were durable. The mTTP was 4.9 months, while mOS was 12.9 months. The most frequent serious adverse events were increased AST concentration in four (4%) participants, increased ALT concentration in two (2%), and adrenal insufficiency in two (2%) participants. The association with tumor proportion score was not significant, consistently with Checkmate 040 conclusions. However, to explore potential predictive biomarkers, investigators used a combined positive score (a measure of PD-L1-positive immune and tumor cell number) that was found to be associated with response to pembrolizumab. 55 Based on the KEYNOTE-224 results, the FDA granted accelerated approval for the use of pembrolizumab in patients previously treated with sorafenib. Merck has recently published a press release about the final analysis of the phase III trial KEYNOTE-240 (NCT02702401) evaluating pembrolizumab versus placebo as second-line treatment in Western population; although the co-primary endpoints OS and PFS were improved, these differences did not meet the statistical significance. 56 Ongoing and future trials Final considerations about the role of anti-PD-1 monotherapy in HCC will be drawn as soon as other RCTs will be completed; the results of the CheckMate-459 and the BGB-A317-301 trials (nivolumab or tislelizumab compared with sorafenib in the firstline setting, NCT02576509, NCT03412773) and of the KEYNOTE 394 (pembrolizumab versus placebo as second-line setting in Asian population, NCT03062358) are eagerly awaited.
In addition, other strategies are being pursued to maximize the potential of immunotherapy; in particular, some trials are exploring the combination of anti-PD-1/PD-L1 with TKIs, anti-VEGF, or anti-CTLA-4. Some results from phase I trials are already available, and they show a potential benefit compared to anti-PD-1 monotherapy. In a group of 18 subjects (6 previously treated and 12 treatment-naive), lenvatinib and pembrolizumab yielded high rates of ORR (46% PR) and a 92% DCR. 57 The combination of two checkpoint inhibitors, the anti-PD-L1 durvalumab and the anti-CTLA-4 tremelimumab given at the dose of 20 and 1 mg/kg I.V., respectively, for four doses followed by 20 mg/kg durvalumab alone every 4 weeks, was tested in a safety run-in cohort with 40 patients. No unexpected safety signals were seen in this unresectable HCC population that included 30% of sorafenib-naïve patients. Clinical activity was observed predominantly in uninfected patients (DCR 70 versus 44.4 and 45.5% of HCV and HBV infected). 58 Taking the advantage of the immunomodulatory effects of anti-VEGF therapy, the combination of atezolizumab 1200 mg and bevacizumab 15 mg/kg every three weeks is being explored. Among the 73 patients enrolled in a phase Ib trial, ORR was 32% with responses observed in all clinical significant subgroups and adverse events were consistent with the established safety profile of each agent. 59 Atezolizumab is also being tested in combination with the anti-glypican-3 codrituzumab. In the preliminary analyses on 18 patients recently reported, the combinations obtained 1 PR and 10 SD, ISSN: 1740-4398 REVIEW -Second-line therapy in HCC drugsincontext.com  was negative for the primary endpoint; in this case, indeed, the predictive biomarker, AFP elevation, is a clinical and not a molecular biomarker.
It has already been pointed out that the effect of antiangiogenic drugs has reached an efficacy ceiling with a mOS of first-line treatments constantly below 1 year. 64 Although much progress has been made, especially in the second-line treatment after sorafenib, it is mandatory for clinical research to focus on optimizing a treatment algorithm, rather than introducing into the market 'me-too' drugs. In the absence of a direct comparison between second-line options, some aspects of the RCTs that we have discussed may be used to help clinicians; for example, regorafenib should not be offered to patients who are sorafenib intolerant, whereas cabozantinib may be used also in third-line treatment. Ramucirumab differs from the other treatment options for the route of administration (I.V.) and for the presence of a predictive biomarker (AFP≥400 ng/mL).
In this context, immunotherapy has drawn considerable interest for its potential under many issues. First, its mechanism of action is completely different from the other approved drugs. Second, although a minority of patients could experience severe and even potentially life-threatening toxicities, immunotherapy has a more favorable profile that makes the combination with other compounds easier and that could be exploited in patients with impaired liver function. If initial results are confirmed in phase III trials and immunotherapy will become a standard of care in first-or second-line treatment for HCC, current evidence regarding second-line therapy after sorafenib exposure will be difficultly applicable. In addition, immunotherapy may produce early and durable responses that are already being investigated in other disease settings, such as neoadjuvant treatment or combination with local treatments increasing tumor antigens exposure. The recent announcement of the negative results of the KEYNOTE-240 trial might hamper the enthusiasm for immunotherapy, but it will be important to analyze the whole data to draw definitive conclusions.
Hopefully, with the advances in understanding tumor microenvironment and immune reactivity, 49 we will be able to select patients for their potential response to these new drugs.
some of which are durable. 60 The ongoing immunotherapy studies, both for advanced and for early-intermediate stage disease, are reported in Table 2.

Discussion
Advanced HCC still is a medical unmet need; for over a decade, sorafenib had been the only approved treatment in this setting, with several drugs failing to show a benefit both in first-and second-line setting in RCTs. 61 Trials conducted in the last decade have been generally characterized by an underestimation of driving mechanisms of HCC pathogenesis and progression; thus, many studies enrolled a heterogeneous population regardless of potential biomarkers of response. Similarly, promising drugs were moved from phase II to phase III on the basis of very small, single-arm trials and relying on surrogate endpoints such as ORR, PFS, and TTP that are not predictors of OS in HCC. In patients with compromised baseline liver function due to chronic liver disease, the toxicity of certain drugs might strongly impact on treatment compliance and efficacy. In addition, it has to be pointed out that trials generally enroll patients with Child-Pugh A liver function that are not representative of all HCC patients. As a consequence, scientific evidence of safety for Child-Pugh B patients that could be treated in clinical practice is always limited to observational or retrospective series. In a recently presented real-life experience after first-line sorafenib, investigators remarked that only a small proportion of patients (13.1%) met the criteria used for enrollment in RESORCE, CELESTIAL, and REACH-2 trials. By using modified eligibility criteria (such as including patients with ECOG PS 2 or with Child-Pugh score B7), the percentage of eligible patients would increase to 31.7%, but at the cost of a worse survival outcome 62 (mOS 6.2 versus 9.4 months of strict eligibility criteria).
Although the molecular underpinnings of HCC are being unveiled, the majority of recurrent somatic genetic alterations are not directly druggable 63 ; unsurprisingly, the larger part of the recently approved drugs were tested in a 'all-comers' setting and block, among the others, the VEGF pathway. Ramucirumab is actually the only drug that has proven efficacy in a biomarker-selected population, thanks to an initial preplanned subgroup analysis from the REACH trial that