Regina Cheuk-Lam Lo

Clinicopathological and prognostic significance of serum and tissue Dickkopf-1 levels in human hepatocellular carcinoma

Although Dickkopf‐1 (DKK1) is known to be a negative regulator of the Wnt/β‐catenin pathway, it has been recently found to be upregulated in cancers.

Regorafenib as Second-Line Systemic Therapy May Change the Treatment Strategy and Management Paradigm for Hepatocellular Carcinoma

At the European Society of Medical Oncology World Congress of Gastrointestinal Cancer held in Barcelona, Spain, on 30th June 2016, positive outcomes were reported by the Study of Regorafenib after Sorafenib in Patients with Hepatocellular Carcinoma (RESORCE) trial, which investigated the efficacy of regorafenib as second-line therapy after sorafenib failure [1]. In this clinical trial, the group who received regorafenib achieved a survival benefit of approximately 2.8 months compared to the placebo group. Overall survival (OS) was 10.6 months in the regorafenib arm compared with 7.8 months in the placebo arm, with a hazard ratio (HR) of 0.62 (95% confidence interval [CI]: 0.50–0.78; p<0.001). These are groundbreaking results. The positive outcome achieved by this second-line systemic therapy is a major development, especially after the numerous reports of failures in clinical studies of first-and second-line systemic therapeutic agents (table ​(table1).1). Regorafenib therapy is expected to significantly prolong life expectancy by approximately 2.8 months in patients with hepatocellular carcinoma (HCC) who develop progressive disease (PD) during sorafenib therapy. This development will certainly lead to drastic changes in the treatment strategy and management paradigm for HCC. Table 1 Phase III Clinical Trials of Japanese Participation for HCC Design of the RESORCE Trial The RESORCE trial enrolled 573 patients with advanced HCC corresponding to Barcelona Clinic Liver Cancer (BCLC) stage B or C who were unresponsive to sorafenib. The patients were divided into placebo and regorafenib arms at a 1:2 ratio for the daily administration of placebo and oral regorafenib (160 mg), respectively, for three weeks on and one week off (four weeks/cycle) (fig. ​(fig.1).1). Geographic region, performance status on the Eastern Cooperative Oncology Group scale, α-fetoprotein level (≥400 or <400 ng/mL), macrovascular invasion, and extrahepatic disease were used as allocation factors. This study excluded patients who were intolerant of sorafenib and who discontinued the treatment because of side effects. It enrolled only those patients who discontinued sorafenib because of evidence of PD on imaging studies. In addition, patients were included only if they had received ≥400 mg sorafenib for at least 20 of 28 days immediately prior to radiologically detected PD. In other words, this trial was designed (1) to ensure regorafenib tolerance among patients, and to reduce the occurrence of the drug-specific skin symptoms because the compound is structurally similar to sorafenib [2,3] (fig. ​(fig.2)2) and (2) to reduce the effect of post-trial treatment on OS in both the placebo and treatment arms by using a homogeneous group of patients who developed PD due to sorafenib failure. Fig. 1 Design of the RESORCE Trial. ECOG PS=Eastern Cooperative Oncology Group Performance Status; RECIST=Response Evaluation Criteria in Solid Tumors. Fig. 2 Chemical structure of Regorafenib is very similar to that of Sorafenib. In general, post-progression survival (PPS) is defined as the time interval between the diagnosis of PD after primary treatment and the patients death, and OS is the sum of PPS and progression-free survival (PFS). Therefore, even significant differences in PFS can be canceled out because PPS is prolonged. Indeed, OS showed a stronger correlation with PPS than with PFS in a clinical trial of sorafenib [4]. Because HCC responds extremely well to locoregional therapy, it is often used as post-trial treatment even in cases in which locoregional therapy is no longer applicable and molecular targeted agents are subsequently administered in accordance with the protocol, provided that the patients general condition is stable. This rarely happens with other types of cancer and is therefore essentially unique to HCC, owing to the availability of powerful locoregional therapies such as intra-arterial infusion chemotherapy [5, 6, 7], transcatheter arterial chemoembolization (TACE) [8, 9], and radiofrequency ablation [10, 11, 12]. These post-trial treatments are capable of canceling out any difference in the primary endpoint OS by prolonging PPS [13]. Indeed, previous clinical trials of second-line agents other than regorafenib have always included patients intolerant to sorafenib, which may have increased the influence of post-trial treatment and thus contributed to their negative outcomes. Patients unresponsive to sorafenib are those who develop PD during sorafenib therapy and are likely to have relatively poor hepatic function and overall general condition. By contrast, patients intolerant to sorafenib are those who discontinue the treatment because of side effects; these patients are in relatively stable conditions because of negligible amounts of internalized sorafenib, and a lack of HCC progression. Because of their clinical stability, patients intolerant to sorafenib are inevitably treated by locoregional therapy or various other post-trial treatments, including the re-administration of sorafenib, regardless of whether they received an actual second-line agent or placebo during the trial. With this in mind, clinical trials of second-line agents should enroll only patients who are unresponsive to sorafenib [14]. The RESORCE trial was the first clinical study to reflect this point in the trial design (fig. ​(fig.1).1). The benefit of excluding patients intolerant to sorafenib was demonstrated in the subanalysis of a previous phase II study of axitinib, which generated an excellent HR and a significant study outcome [15, 16]. The second noteworthy point in the design of the RESORCE trial is that the allocation factors of macrovascular invasion and extrahepatic disease were treated as independent stratification factors. In general, the designs of previous clinical trials of molecular targeted agents involved allocation factors specifying “vascular invasion and/or extrahepatic spread” or “neither.” However, because vascular invasion is an extremely poor prognostic factor for HCC, assigning vascular invasion to the same category as extrahepatic spread may have influenced the outcome of these clinical trials. For example, when the treatment group contains more patients with vascular invasion but the placebo group includes more patients with extrahepatic spread, such sampling bias will put the treatment group at a significant disadvantage. In fact, such allocation imbalance apparently contributed to a negative outcome in a clinical trial of brivanib as second-line therapy [17] (table ​(table22). Table 2 Imbalance between Brivanib and Placebo Arm in BRISK-PS Trial The design of the RESORCE trial is excellent because it reflects what was learned from the negative outcomes of past trials and the reasons for those outcomes.

Sox9 confers stemness properties in hepatocellular carcinoma through Frizzled-7 mediated Wnt/β-catenin signaling

Sox9, an SRY-related HMG box transcription factor, is a progenitor/precursor cell marker of the liver expressed during embryogenesis and following liver injury. In this study, we investigated the role of Sox9 and its molecular mechanism with reference to stemness properties in hepatocellular carcinoma (HCC). Here, we observed upregulation of Sox9 in human HCC tissues compared with the non-tumorous liver counterparts (p < 0.001). Upregulation of Sox9 transcript level was associated with poorer tumor cell differentiation (p = 0.003), venous invasion (p = 0.026), advanced tumor stage (p = 0.044) and shorter overall survival (p = 0.042). Transcript levels of Sox9 and CD24 were positively correlated. Silencing of Sox9 in HCC cells inhibited in vitro cell proliferation and tumorsphere formation, sensitized HCC cells to chemotherapeutic agents, and suppressed in vivo tumorigenicity. In addition, knockdown of Sox9 suppressed HCC cell migration, invasion, and in vivo lung metastasis. Further studies showed that Sox9 endowed stemness features through activation of Wnt/β-catenin signaling, which was confirmed by the partial rescue effect on tumorigenicity and self-renewal upon transfection of active β-catenin in Sox9 knockdown cells. By ChIP and luciferase promoter assays, Frizzled-7 was identified to be the direct transcriptional target of Sox9. In conclusion, Sox9 confers stemness properties of HCC through Frizzled-7 mediated Wnt/β-catenin pathway.

Down‐regulation of TIMP2 by HIF‐1α/miR‐210/HIF‐3α regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma

Cancer metastasis is a multistep process that involves a series of tumor‐stromal interaction, including extracellular matrix (ECM) remodeling, which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases (TIMP) 2 in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly down‐regulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.8%; 23 of 55) in human HCCs and was significantly associated with liver invasion and poorer survival outcomes of HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced cell invasive ability and ECM degradation associated with formation of invadopodia‐like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Using an orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line, MHCC‐97L, significantly reduced HCC progression as well as pulmonary metastasis. Mechanistically, TIMP2 suppression, in a hypoxic environment, was induced through a regulatory feedback circuit consisting of hypoxia‐inducible factor (HIF) 1 alpha, microRNA‐210 (miR‐210), and HIF‐3α. Conclusion: TIMP2 is frequently down‐regulated in human HCCs and its down‐regulation is associated with aggressive tumor behavior and poorer patient outcome. Its suppression is under the regulation of a novel feedback circuit consisting of HIF‐1α/miR‐210/HIF‐3α. TIMP2 is an important regulator of ECM degradation and HCC metastasis. (Hepatology 2016;64:473‐487)

Pilot study of high‐intensity focused ultrasound ablation as a bridging therapy for hepatocellular carcinoma patients wait‐listed for liver transplantation

The objective of this study was to investigate the outcomes of high‐intensity focused ultrasound (HIFU) ablation as a bridging therapy for patients with hepatocellular carcinoma (HCC) who had been wait‐listed for deceased donor liver transplantation (DDLT). Adult patients with unresectable and unablatable HCCs within the University of California San Francisco criteria who had been wait‐listed for DDLT were screened for their suitability for HIFU ablation as a bridging therapy if they were not suitable for transarterial chemoembolization (TACE). Treatment outcomes for patients receiving HIFU ablation, TACE, and best medical treatment (BMT) were compared. Fifty‐one patients were included in the analysis. Before the introduction of HIFU ablation, only 39.2% of the patients had received bridging therapy (TACE only, n = 20). With HIFU ablation in use, the rate increased dramatically to 80.4% (TACE + HIFU, n = 41). The overall dropout rate was 51% (n = 26). Patients in the BMT group had a significantly higher dropout rate (P = 0.03) and significantly poorer liver function as reflected by higher Model for End‐Stage Liver Disease scores and higher Child‐Pugh grading. Clinically relevant ascites was found in 5 patients in the HIFU group and 2 patients in the BMT group, but none was found in the TACE group (P = 0.01 and P = 0.03, respectively). The TACE and HIFU groups had comparable percentages of tumor necrosis in excised livers (P = 0.35), and both were significantly higher than that in the BMT group (P = 0.01 and P = 0.02, respectively). In conclusion, HIFU ablation was safe even for HCC patients with Child‐Pugh C disease. Its adoption increased the percentage of patients receiving bridging therapy from 39.2% to 80.4%. A randomized controlled trial for further validation of its efficacy is warranted. Liver Transpl 20:912–921, 2014.

Characterization of interleukin-1β in Helicobacter pylori-induced gastric inflammation and DNA methylation in interleukin-1 receptor type 1 knockout (IL-1R1-/-) mice

Helicobacter pylori infection induced interleukin-1β (IL-1β) production and is associated with aberrant DNA methylation and gastric diseases. Here, we investigated the role of IL-1β in H. pylori-induced gastric inflammation and DNA methylation using IL-1 receptor type 1 knockout (IL-1R1(-/-)) mice, and compared the therapeutic efficacy of antimicrobial therapy with IL-1 receptor antagonist (IL-1ra). IL-1R1(-/-) and wild-type (WT) mice were infected with H. pylori for 16, 24 and 32 weeks. Infected WT mice at 24 weeks were given either antimicrobial therapy or IL-1ra. Comparing to the IL-1R1(-/-) mice, infected WT mice with functional IL-1β signaling had higher gastritis scores, higher IL-1β and iNOS mRNA expression, higher nitric oxide (NO) production and increased frequency of E-cadherin (E-cad) methylation at all the time points analyzed. IL-1β release was significantly elevated in infected WT mice than normal controls at 16 weeks post-infection (p<0.005). Treatment of infected mice with antimicrobial therapy and IL-1ra significantly reduced the degree of gastritis (p<0.005; p<0.05, respectively), iNOS expression (p<0.0001; p<0.01, respectively) and NO production (both p<0.001) compared with untreated controls. Mice receiving antimicrobial therapy had significantly lower IL-1β expression than untreated controls (p<0.0001). Both treatments reduced the incidence of E-cad methylation in infected mice compared with controls, however, no statistical significance was observed. There was no significant alteration of total DNA methyltransferase (DNMT) activity. These results demonstrated that IL-1β played a crucial role in H. pylori-induced gastric inflammation and DNA methylation. H. pylori eradication and IL-1ra administration could ameliorate inflammatory stress.

Hepatic Progenitor Cells: Their Role and Functional Significance in the New Classification of Primary Liver Cancers

Hepatic progenitor cells (HPCs) are bipotential cells residing in normal liver. Their proliferation is observed in reactive conditions of the liver and in primary liver cancers. The observation that some hepatocellular carcinomas (HCCs) express a biliary-like immunophenotype has led to the identification of HPCs in HCC. Accumulating evidence suggests that HPCs play a role as the cell of origin in a variety of primary liver cancers. This has led to the development of revolutionary concepts in hepatocarcinogenesis. In this article, the role and significance of HPCs in HCC, including its classification, are summarized and discussed.Hepatic progenitor cells (HPCs) are bipotential cells residing in normal liver. Their proliferation is observed in reactive conditions of the liver and in primary liver cancers. The observation that some hepatocellular carcinomas (HCCs) express a biliary-like immunophenotype has led to the identification of HPCs in HCC. Accumulating evidence suggests that HPCs play a role as the cell of origin in a variety of primary liver cancers. This has led to the development of revolutionary concepts in hepatocarcinogenesis. In this article, the role and significance of HPCs in HCC, including its classification, are summarized and discussed.

Hypoxia induces myeloid‐derived suppressor cell recruitment to hepatocellular carcinoma through chemokine (C‐C motif) ligand 26

A population of stromal cells, myeloid‐derived suppressor cells (MDSCs), is present in tumors. Though studies have gradually revealed the protumorigenic functions of MDSCs, the molecular mechanisms guiding MDSC recruitment remain largely elusive. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of solid cancers, whose growth often exceeds the growth of functional blood vessels. Here, using hepatocellular carcinoma as the cancer model, we show that hypoxia is an important driver of MDSC recruitment. We observed that MDSCs preferentially infiltrate into hypoxic regions in human hepatocellular carcinoma tissues and that hypoxia‐induced MDSC infiltration is dependent on hypoxia‐inducible factors. We further found that hypoxia‐inducible factors activate the transcription of chemokine (C‐C motif) ligand 26 in cancer cells to recruit chemokine (C‐X3‐C motif) receptor 1‐expressing MDSCs to the primary tumor. Knockdown of chemokine (C‐C motif) ligand 26 in cancer cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of chemokine (C‐C motif) ligand 26 production in cancer cells by the hypoxia‐inducible factor inhibitor digoxin or blockade of chemokine (C‐X3‐C motif) receptor 1 in MDSCs by chemokine (C‐X3‐C motif) receptor 1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth. Conclusion: This study unprecedentedly reveals a novel molecular mechanism by which cancer cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against solid cancers. (Hepatology 2016;64:797‐813)

Hepatocellular Tumors: Immunohistochemical Analyses for Classification and Prognostication

Following the classification of hepatocellular nodules by the International Working Party in 1995 and further elaboration by the International Consensus Group for Hepatocellular Neoplasia in 2009, entities under the spectrum of hepatocellular nodules have been better characterized. Research work hence has been done to answer questions such as distinguishing high-grade dysplastic nodules from early hepatocellular carcinoma (HCC), delineating the tumor cell origin of HCC, identifying its prognostic markers, and subtyping hepatocellular adenomas. As a result, a copious amount of data at immunohistochemical and molecular levels has emerged. A panel of immunohistochemical markers including glypican-3, heat shock protein 70 and glutamine synthetase has been found to be of use in the diagnosis of small, well differentiated hepatocellular tumors and particularly of HCC. The use of liver fatty acid binding protein (L-FABP), β-catenin, glutamine synthetase, serum amyloid protein and C-reactive protein is found to be helpful in the subtyping of hepatocellular adenomas. The role of tissue biomarkers for prognostication in HCC and the use of biomarkers in subclassifying HCC based on tumor cell origin are also discussed.

Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.