Accepted Manuscript Clinical trial watch Reports from the International Liver Cancer Association (ILCA) Congress 2014 Jean-Charles Nault PII: DOI: Reference:

S0168-8278(14)00859-9 http://dx.doi.org/10.1016/j.jhep.2014.11.015 JHEPAT 5435

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Journal of Hepatology

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2 October 2014 8 November 2014 12 November 2014

Please cite this article as: Nault, J-C., Reports from the International Liver Cancer Association (ILCA) Congress 2014, Journal of Hepatology (2014), doi: http://dx.doi.org/10.1016/j.jhep.2014.11.015

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Reports from the International Liver Cancer Association (ILCA) Congress 2014

Jean-Charles Nault1,2,3

1. Inserm, UMR-1162, Génomique fonctionnelle des Tumeurs solides, IUH, Paris, F-75010 France 2. Université Paris Descartes, Labex Immuno-Oncology, Sorbonne Paris Cité, Faculté de Médecine, Paris, France 3. Service d'Hépatologie, Hôpital Jean Verdier, AP-HP, Bondy, and Université Paris 13, Bobigny, France

Corresponding author: Jean-Charles Nault, INSERM UMR-1162 Université Paris Descartes, 27 rue Juliette Dodu, Paris 75010, France. Email : [email protected] Phone number : 33 1 53 72 51 94 Fax number : 33 1 53 72 51 92 Author Contribution: Conceiving and writing the manuscript (JCN) Conflicts of interest: none

Summary The International Liver Cancer Association (ILCA) Congress took place in Kyoto, Japan, from 4 to 7 September 2014, and ranged from basic to clinical studies in the area of primary liver cancer, including hepatocellular carcinoma (HCC), but also cholangiocarcinoma. In the field of basic and translational research, several studies attempted to refine our knowledge of biological events involved in liver carcinogenesis and sought to identify new therapeutic targets for improving clinical care in the future. In the present work, a subjective selection of studies among the large number of abstract available in the ILCA meeting is presented and places into context.

From identification of potential therapeutic targets … Sorafenib remains the sole approved treatment for patients with advanced HCC[1, 2]. However, the efficacy of sorafenib is limited, with an increase in survival of 2.8 months compared to a placebo and median time to radiological progression of 5.5 months. Consequently, identification of the most likely responders to sorafenib and, conversely, of the mechanisms of resistance to sorafenib, is a priority. This will help in selecting patients who will benefit from sorafenib, and in proposing combined treatments to increase its efficacy and bypass resistance.

In his lecture, Lars Zender reviewed it results recently published in Nature Medicine[3]. He used RNA interference (RNAi) combined with hydrodynamic vain tail injection in a mouse model harboring HCC in order to discover the molecular determinants of resistance to sorafenib. He identified mitogen-activated protein kinase 14 (MAPK14) (coding for p38α) as a key actor in sorafenib resistance in his mouse model. Downregulation of MAPK14 increased tumor shrinkage induced by sorafenib and consequently increased survival. In addition, phospho-Atf2 (p-Atf2), a well-known downstream target positively regulated by MAPK14, was assessed by immunohistochemistry in tumor biopsies of patients treated with sorafenib. In addition, the authors showed that overexpression of p-Atf2 was associated with poor overall survival in patients treated by sorafenib[3]. However, we could suggest that the use of p-Atf2 as a biomarker predictive of resistance to sorafenib needs to be validated in a prospective cohort of patients. In addition, this type of study opens up new avenues in treatment with second-generation MAP2K14 inhibitors (such as skepinone-L and PH-797804) to improve the efficacy of sorafenib. Ideally, this combination should be tested in patients selected according to the p-Atf2 level of their tumors. In parallel, however, we need to go beyond biotherapy like sorafenib for all comers, and propose different ways of managing patients with advanced HCC. Over the last few years, using next-generation sequencing, several teams have described the genetic landscape of HCC and have increased our knowledge of driver genes involved in liver carcinogenesis[4-6]. This initial step is mandatory in order to identify the main therapeutic targets of future clinical trials. However, this strategy continues to be restrained by the limited number of targeted therapies available and, consequently, the absence of biotherapy adapted to each therapeutic target

identified by whole-exome and whole-genome sequencing. Substantial effort is being made to develop new compounds targeting the genetic drivers of liver carcinogenesis. Along this line, somatic activating mutations of CTNNB1 (catenin (cadherin-associated protein), beta 1) coding for B-catenin occur in around 20 to 40% of HCC[7]. However, until recently, no safe compounds have shown any significant activity against the Wnt/B-catenin pathway[8]. Consequently, inhibitors of Wnt/b-catenin are not used in clinical practice. Budhu et al. (A High-Throughput Screen Identifies Wnt-Beta-Catenin Inhibitors For A Stem-Like Subtype Of Hepatocellular Carcinoma) used high through-put drug screening and identified two compounds (pimozide and fiduxison) that inhibited the Wnt/B-catenin pathway in hepatocellular cell lines. They showed that those compounds mainly affected HCC cell lines harboring stem cell markers and the Wnt-TGFβ (Transforming growth factor beta) pathway not due to CTNNB1-activating mutations. Interestingly, pimozide has been already tested in several clinical trials to treat psychiatric disorders with favorable safety signals [9]. However, the efficacy of such compounds against CTNNB1 mutations, the doses required to demonstrate an anti-tumor effect in humans and their safety profile in cirrhotic patients, remain to be determined. In addition, around 5 to 10% of HCC harbor focal amplification of the FGF19 locus that leads to constitutive activation of the FGF/FGFR (fibroblast growth factor/fibroblast growth factor receptor) pathway[10]. Hagel et al. (First Isoform Selective Inhibitor Of Fgfr4 For The Treatment Of Genomically Defined Patients With Hepatocellular Carcinoma) developed a specific inhibitor of FGFR4, named BLU9931, that decreased FGFR4 signalling and proliferation and induced apoptosis in HCC cell lines. The FGF19 transgenic mouse is a mouse model prone to developing

HCC. As previously described, when FGF19 transgenic mice were crossed with FGFR4 knockout mice, they failed to develop HCC[11]. This suggests that FGFR4 inhibition might be a clue to treating HCC with FGF19 amplification. Consequently, they hypothesize that this inhibitor could target HCC cell lines harboring FGF19 amplification; moreover, they showed that the BLU9931 inhibitor was able to decrease FGFR signaling in these HCC cell lines and induce tumor regression in a xenograft mouse model. Clearly, this study suggested that this selective FGFR4 inhibitor (BLU9931) is a good candidate for clinical trials in patients with HCC harboring the FGF19 amplification. A study by Quetglas et al. (Igf2 Is An Oncogenic Driver And Druggable Target In HCC) confirmed that a subset of HCC (around 20%) harbored activation of the IGF/IGFR1 (insulin growth factor/insulin growth factor receptor 1) pathway due to IGF2 (insulin growth factor 2) upregulation (as previously described in[12]). They showed that IGF2 upregulation was the consequence of decreased activity of the IGF2 adult promoter induced by its hypermethylation. In addition, they demonstrated an increase in IGF2 fetal promoter activity due to its hypomethylation. Overexpression of IGF2 or IGF1R combined with the myc/akt oncogene leads to HCC formation via hydrodynamic tail injection in mice. The authors used BI836845, a monoclonal antibody directed against the ligand IGF2, to decrease IGF2/IG1R pathway activation and, consequently, decrease tumor formation in this mouse model. However, toxicity as severe hypoglycemia has limited the use of other IGF inhibitors in clinical trials[13]. The authors concluded that if this monoclonal antibody targeting IGF2 showed no limiting toxicity, it could be tested for treating patients with HCC harboring IGF pathway activation.

Beyond the problem of identification of new drugs targeting key oncogenic pathways involved in liver carcinogenesis, we are faced with a new level of complexity due to the intratumoral and intertumoral heterogeneity of liver cancers. While intratumoral heterogeneity has rarely been described in HCC, intertumoral heterogeneity is a well-known phenomenon. With cirrhosis, multiple tumors may arise at the same time in a given patient and can be due to metastasis of one HCC or to multifocal carcinogenesis with growth of independent tumor clones. A previous study[14] had compared the primary tumors and the corresponding tumor portal thrombosis at the vinicity of the primary tumors using whole-exome sequencing and found a high rate of concordance at the genomic level. However, this study was performed on a limited number of samples and didn’t compare different tumors localized at different location in the same liver (multiple HCC). The study of Sia et al. (Molecular Heterogeneity Of Multinodular Hepatocellular Carcinoma), using CGH SNP array (comparative genomic hybridization-single nucleotide polymorphism) and microarray analysis, showed that 38% of multiple HCC treated by liver transplantation are clonal, indicating a metastatic process. In contrast, 62% of HCC are independent tumors, suggesting a multifocal process in carcinogenesis on cirrhosis. In addition, the authors showed that HCC related to HCV (hepatitis C virus) infection were more frequently clonal suggesting a metastatic process compared to HCC related to HBV (hepatitis B virus) infection that derived more frequently from a multifocal process of carcinogenesis. It could be a clue to explain the different prognosis sometimes observed between HCV related HCC and those due to chronic HBV infection[15]. The elucidation of tumor heterogeneity is crucial for understanding the mechanisms of primary and secondary resistance as targeted therapies directed against driver genes become available.

In cholangiocarcinoma, the spectrum of somatic mutations greatly differs from that observed in HCC[16]. Recurrent mutations in IDH1, IDH2, BAP1, ARID1A, T53, PBMR1, KRAS and SMAD4 have been reported in cholangiocarcinoma, although their frequencies vary according to histological type and etiology (intrahepatic cholangiocarcinoma, extrahepatic cholangiocarcinoma, gallbladder cancer, liver fluke-related cholangiocarcinoma and other cholangiocarcinomas)[17-19]. In addition, cholangiocarcinoma harbors a wide range of FGFR fusions (FGFR2-TACC3, FGFR2-AHCYL1, FGFR2-BICC1) that constitute potential therapeutic targets[20-22]. At this ILCA meeting, Ton et al. (Decoding Tumor Heterogeneity Informs Targeted Therapies In Intrahepatic Cholangiocarcinoma) presented an integrative analysis of intrahepatic cholangiocarcinoma and identified activation of NOTCH signaling as a prognostic biomarker. They found that cholangiocarcinoma harboring activation of NOTCH signaling had a poor survival. Consequently, this tumor subtype with a poor prognosis will be an ideal candidate to test targeted therapies. They showed that inhibition of NOTCH signaling using a gamma secretase inhibitor led to decreased cell proliferation and decreased tumor formation in a xenograft model. Gamma secretase inhibitors are highly effective in treating cholangiocarcinoma cell lines with activation of the NOTCH pathway. Consequently, targeting NOTCH signaling using gamma secretase inhibitors could be an option for treating cholangiocarcinoma in the future. However, we need to deal with the genetic landscape of cholangiocarcinoma, including other genetic alterations that can be targeted by biotherapy, such as the IDH1/2 (Isocitrate dehydrogenase 1 / 2) mutation or FGFR fusion. Currently, clinical trials are-on going testing IDH1/IDH2[23, 24] and FGFR4 inhibitors[25] in human cancers and

these targeted therapies will be ideally used in biomarker driven clinical trials to treat unresecable or metastatic cholangiocarcinoma[16].

… to real-life and clinical trials A recent multicentric prospective cohort study (Trinchet et al. Incidence And Characteristics Of Primary Liver Cancer In Hbv- Or Hcv-Related Compensated Cirrhosis. A Multicenter Prospective Cohort Study In 1672 Patients Anrs Co12 Cirvir) that included 1,672 patients with HBV- or HCVrelated compensated cirrhosis regularly followed up using ultrasonography showed that the cumulative incidence of HCC was 13.4% at 5 years; 81% were diagnosed within Milan criteria and 65% received curative treatment. In that cohort, 136 patients died and 52% of deaths were liver-related, mainly due to liver failure and HCC. That study suggested that ultrasonography in compensated cirrhosis can help to diagnose cancer at curative stages even though, in 19% of cases, HCC are still diagnosed outside of Milan criteria. This subset of patients with a diagnosis of HCC falling outside of Milan criteria represents a sector for improvement in clinical care in the future. Along the same lines, in support of HCC screening in cirrhotic patients, a Japanese multicentric randomized controlled trial comprising 658 patients, and directed by Kudo (B-Mode Ultrasonography Versus Contrast-Enhanced Ultrasonography For Surveillance Of Hepatocellular Carcinoma: A Prospective Multicenter Randomized Controlled Trial), compared HCC screening via classical ultrasonography every 3-4 months with contrast-enhanced sonography (313 patients using classical ultrasonography and 309 using contrast-enhanced sonography). They used sonazoid as a contrast agent taken up by Kupffer cells. The low-cost of sonazoid is also an

advantage is this setting. The rate of HCC identified did not significantly differ between the two groups, nor did the number of patients eligible for curative treatment. Nonetheless, this study attained it primary endpoint, since the mean size at tumor detection was 13 mm with contrastenhanced ultrasonography and 16 mm with classical ultrasonography (P=0.011). However, the Kupffer-specific contrast agent sonozoid is not available in western countries, and the time between two screenings (3-4 months) is not recommended in western countries, where screening using ultrasonography every 6 months is the gold standard[26]. In addition, we do not know whether detection of tumors 3 mm smaller (13 mm versus 16 mm) will increase the rate of curative treatment or improve survival. For HCC falling within Milan criteria (classified BCLC 0 or A using the Barcelona Liver Cancer Classification), liver transplantation is among the leading curative treatments[27]. It is meant to cure both the cancer and the underlying liver disease. However, liver transplantation for HCC is impaired by dropout due to tumor progression while on the waiting list, and by HCC recurrence after transplantation[27]. A double-blind randomized trial conducted in Germany (Hoffmann K, et al. Prospective, Randomized, Double-Blind, Multicenter, Phase 3 Clinical Study On Tace Combined With Sorafenib Vs. Tace Plus Placebo In Patients With Hcc Before Liver Transplantation – Heilivca Trial) compared 26 patients treated by TACE (Trans-arterial chemoembolization) and placebo to 24 patients treated by TACE and sorafenib listed for liver transplantation. The primary endpoint was time to progression; no difference was observed between the TACE+ placebo group (125 days) and the TACE+sorafenib group (171 days). The rate of severe adverse events was higher in the sorafenib group (50%) than in the placebo group (16%). The overall response rate did not differ between the sorafenib group (20.8%) and the

placebo group (26.9%). In contrast, the median operating time was increased in the sorafenib group (6.7 hours) versus the placebo group (5.4 hours). Consequently, the authors suggested that this study did not provide a clear positive signal for running a larger, multicentric phase 3 randomized trial to test sorafenib as a neoadjuvant treatment before liver transplantation. Moreover, the rate of liver transplantation seems to be different between the TACE+placebo arm (46.2%) and the TACE+sorafenib arm (22.7%) and the number of patients included is small (n=50) suggesting that the results of such study should be taken with caution Bruix et al. (STORM: A Phase III Randomized, Double-Blind, Placebo-Controlled Trial of Adjuvant Sorafenib after Resection or Ablation to Prevent Recurrence of Hepatocellular Carcinoma) presented the results (previously reported at the American Society of Clinical Oncology meeting 2014) of the largest phase 3 randomized trial ever conducted in the setting of adjuvant treatment after resection or radiofrequency ablation. 558 patients were randomized into the placebo arm and 556 into the sorafenib arm, for a total of 4 years of therapy after curative treatment (resection or radiofrequency ablation). That study did not meet its primary endpoint, as recurrence-free survival (RFS) was not statistically different between the sorafenib (median RFS=33.4 months) and placebo group (median RFS=33.8 months) (HR=0.940 [0.780-1.134] P=0.26). In addition, there was no significant difference in overall survival between the two groups of treatment. Treatment discontinuation due to adverse events was higher in the sorafenib group (23.9%) than in the placebo group (7.3%). Moreover, the median duration of treatment was shorter in the sorafenib arm (12.5 months) compared to the placebo arm (22.2 months) and was also shorter than the 4 years of treatments initially planned by the protocol. It could be one of the explanations of the absence of benefit of sorafenib in the adjuvant setting.

When screening fails or recurrence occurs after curative treatment, chemo-embolization and thus sorafenib are the main palliative treatments available in BCLC B and C patients, respectively[26]. However, no systemic agent is approved in second line after sorafenib failure, and several phase 2 clinical trials are ongoing worldwide. Kudo et al. (Randomized Phase Ii Trial Of Intravenous Ro5137382/Gc33 At 1600 Mg Every Other Week And Placebo In Previously Treated Patients With Unresectable Advanced Hepatocellular Carcinoma) presented results of a randomized phase 2 trial that compared, at a 2:1 ratio, a humanized monoclonal antibody directed against glypican 3 (121 patients) and a placebo (64 patients) in second line after failure of sorafenib. Glypican-3, a heparan sulfate proteoglycan, is an immunohistochemical marker for distinguishing early HCC from pre-neoplastic lesions[28]. That study failed to show a significant difference in the primary endpoint, progression-free survival, between the placebo arm (1.5 months) and the glypican 3 antibody (2.6 months) (P=0.87). Patients treated by the glypican 3 antibody more frequently exhibited fever, nausea and headache compared to patients treated by a placebo. No significant difference in progression-free survival was observed, even after stratification using glypican 3 expression, in tumors assessed using immunohistochemistry. Subsequent subgroup analyses suggested that patients with higher exposure to the drug or with high CD16 expression on circulating NK (Natural killer) cells may be more sensitive to glypican 3 inhibition. However, this post-hoc subgroup analysis requires further investigation, as the study failed to attain it primary endpoint. Finally, in non-resectable or metastatic cholangiocarcinoma, an international multicentric randomized phase 3 trial published in the New England Journal Of Medicine showed that a combination of gemcitabine and cisplatin increased overall survival (11.7 months) compared to

gemcitabine alone (8.1 months)[29]. At present, the combination of gemcitabine with a platinum salt is the sole approved chemotherapy available as a first line, and no systemic treatment has been approved as a second line. The result of a French multicentric single-arm study of sunitinib, a tyrosine kinase inhibitor targeting VEGFR (Vascular endothelial groth factor receptor), PDGFR (Platelet-derived growth factor receptor), cKIT and RET, after failure of the combination gemcitabine+platinum salt, was presented (Neuzillet C, et al. Safety, Translational and Preliminary Efficacy Results of Sunitinib in Patients with Advanced Intrahepatic Cholangiocarcinoma (Sun-Ck): A Gercor-Irc Multicenter Phase II Study). 51 patients were treated by sunitinib at a median dose of 37.5 mg/day. Median overall survival was 9.6 months (5.9-13.1) and median progression-free survival was 5.2 months (3.1-6.8). The rate of complete response was 0%, partial response 14.7% and stable disease 70.6%, for a disease control rate of 85.3%. Drug toxicity was manageable, with mainly asthenia, hand-foot syndrome, mucositis, diarrhea, anemia and thrombopenia. The authors concluded that overall survival observed in this phase 2 mono-arm study (9.6 months) exceeded the median overall survival reported in other studies in a second line[30]. For example, the combination of 5-fluouracil and irinotecan (FOLFIRI) in second line provided a median overall survival of 6.2 months[31] and the combination of gemcitabine and cisplatine in second line after progression under gemcitabine alone was associated with a median overall survival of 6.7 months[32]. Despite the fact that the absence of a placebo arm precluded drawing a firm conclusion in that study, the authors concluded that signals indicating the efficacy of sunitinib suggest the need for further clinical trials using sunitinib as a second-line treatment for non-resectable and metastatic cholangiocarcinomas. Conclusion

A recent European survey on cancer showed that HCC is one the most deadly cancers, similarly to pancreatic cancer and mesothelioma, with less than 15% survival at 5 years[33]. In the ILCA meeting, several sessions underlined the fact that identification of patients at risk of HCC, namely cirrhotic patients, is crucial for improving survival. Since HCC is one of the most frequent causes of death related to cirrhosis, screening via ultrasonography is aimed at diagnosing HCC at an early stage and proposing curative treatment. Continuous efforts are needed to reach this goal in most cirrhotic patients. Moreover, failure of the STORM trial to demonstrate the efficacy of sorafenib in an adjuvant setting follows upon the recent failure of several multicentric phase 3 randomized trials on advanced HCC, and indicates that we must urgently rethink the manner in which large clinical trials should be carried out[34]. Furthermore, continuous efforts are required in the field of HCC and cholangiocarcinoma in both basic and clinical studies. A preconference workshop focused on future HCC clinical trial design. During this workshop, several presentations have summarized the reasons why phase 3 randomized trials failed and discussed how the future clinical trials should be conducted. Clinical trials adapted to tumor biology, the better selection of patients in first and second line and the uses of appropriate endpoints have been highlighted as the most promising strategies. Knowledge obtained from basic studies must be used to test new drugs and therapeutic approaches in a carefully designed manner, so as to overcome recent setbacks in clinical trials.

Figure 1: from identification of therapeutic targets to clinical trials The mechanism of resistance to sorafenib due to activation of MAPK14/ATF2 pathway was recently published in Nature Medecinea. In HCC, activation of IGF/IGFR pathway could be targeted by IGF2 antibody (IM Quetglas et alb)[3], activation of Wnt/catenin pathway by Wnt/catenin inhibitor Pimozide and Fiduxison (Buddhu et al.c), amplification of FGF19 by FGFR4 inhibitor BLU9931 (Hagel M, et ald). Activation of NOTCH pathway in cholangiocarcinoma could be targeted by gamma secretase inhibitors (Ton A, et ale). Moreover, tumor heterogeneity will be an important determinant of resistance to targeted therapies (Sia D, et al.f). Prospective cohort of HBV or HCV related cirrhotic patients and HCC incidence (Trinchet JC, et al.g) and randomized control trial of ultrasonography and contrast-enhanced ultrasonography in HCC screening (Kudo M, et alh) highlighted the need to support screening policie of HCC on cirrhosis. The results of randomized trials of sorafenib as a neoadjuvant treatment on waiting list for liver transplantation (Hoffmann K, et al.i) or as an adjuvant treatment after resection or radiofrequency ablation (Bruix J, et al.j) have been reported during the ILCA meeting 2014. Moreover, the results of clinical trials testing an antibody directed against glypican 3 in second line for advanced HCC (Kudo M, et alk) and testing sunitinib in second line for non-resecable or metastatic cholangiocarcinoma have also been presented (Neuzillet C, et al.l). In this ILCA meeting, a pre-conference workshop has paved the way of the future clinical trials in the field of

HCC. The analysis of tumor biology in order to propose biomarkers driven clinical trial, the adequate selection of patients and the use of robust endpoints will help to reach this goal. Ns=non-significant, Ab= antibody, D=day, M=months, PFS=progression free survival, RFS= recurrence free survival, US=ultrasonography, CEUS=contrast-enhanced ultrasonography, CholangioK=cholangiocarcinoma, TACE=transarterial chemoembolization, RFA=radiofrequency ablation, RCT= randomized control trial, NRT-SA= non-randomized control trial, single arm.

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Basic studies Hepatocellular carcinoma MAP2K14a

IGF/IGFR activationb

CholangioK

Wnt/catenin FGF19 c activation amplificationd

Notch activatione

Phospho-Atf2 Resistance to Sorafenib

Ab anti IGF2 BI836845

Wnt/catenin inhibitor Pimozide and Fiduxison

FGFR4 inhibitor BLU9931

Gamma secretase inhibitor

Assessment of tumor heterogeneityf

Clinical studies CholangioK

Hepatocellular carcinoma Epidemiology

Cohort HBV/HCV cirrhosisg

Screening

Neoadjuvant

Adjuvant

TACE+sorafenib Sorafenib vs vs placebo after US vs CEUS for h HCC screening TACE+placebo resection before LTi or RFAj

Observational study

RCT

13% of HCC 13 mm in CEUS at 5 years vs 16 mm in US 81% in milan (P=0.011) criteria Same rate of HCC

RCT

PFS 125 D in placebo vs 171 D in sorafenib (P=ns)

RCT

RFS 33.8 M in placebo vs 33.4 M in sorafenib (P=0.22)

Advanced

Advanced

Ab anti glypican 3 in second linek

Sunitinib in 2nd linel One arm trial

RCT

PFS 1.5 M in placebo vs 2.6 M in Ab anti GPC3(P=0.87)

NRT-SA

Overall survival 9.6 months Disease control rate 85.3%

New design of clinical trial in hepatocellular carcinoma