Summary Background The ability of circulating microRNAs (miRNAs) to detect preclinical hepatocellular carcinoma has not yet been reported. We aimed to identify and assess a serum miRNA combination ...that could detect the presence of clinical and preclinical hepatocellular carcinoma in at-risk patients. Methods We did a three-stage study that included healthy controls, inactive HBsAg carriers, individuals with chronic hepatitis B, individuals with hepatitis B-induced liver cirrhosis, and patients with diagnosed hepatocellular carcinoma from four hospitals in China. We used array analysis and quantitative PCR to identify 19 candidate serum miRNAs that were increased in six patients with hepatocellular carcinoma compared with eight control patients with chronic hepatitis B. Using a training cohort of patients with hepatocellular carcinoma and controls, we built a serum miRNA classifier to detect hepatocellular carcinoma. We then validated the classifiers' ability in two independent cohorts of patients and controls. We also established the classifiers' ability to predict preclinical hepatocellular carcinoma in a nested case-control study with sera prospectively collected from patients with hepatocellular carcinoma before clinical diagnosis and from matched individuals with hepatitis B who did not develop cancer from the same surveillance programme. We used the sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) to evaluate diagnostic performance, and compared the miRNA classifier with α-fetoprotein at a cutoff of 20 ng/mL (AFP20). Findings Between Aug 1, 2009, and Aug 31, 2013, we recruited 257 participants to the training cohort, and 352 and 139 participants to the two independent validation cohorts. In the third validation cohort, 27 patients with hepatocellular carcinoma and 135 matched controls were included in the nested case-control study, which ran from Aug 1, 2009, to Aug 31, 2014. We identified a miRNA classifier (Cmi ) containing seven differentially expressed miRNAs (miR-29a, miR-29c, miR-133a, miR-143, miR-145, miR-192, and miR-505) that could detect hepatocellular carcinoma. Cmi showed higher accuracy than AFP20 to distinguish individuals with hepatocellular carcinoma from controls in the validation cohorts, but not in the training cohort (AUC 0·826 95% CI 0·771–0·880 vs 0·814 0·756–0·872, p=0·72 in the training cohort; 0·817 0·769–0·865 vs 0·709 0·653–0·765, p=0·00076 in validation cohort 1; and 0·884 0·818–0·951 vs 0·796 0·706–0·886, p=0·042 for validation cohort 2). In all four cohorts, Cmi had higher sensitivity (range 70·4–85·7%) than did AFP20 (40·7–69·4%) to detect hepatocellular carcinoma at the time of diagnosis, whereas its specificity (80·0–91·1%) was similar to that of AFP20 (84·9–100%). In the nested case-control study, sensitivity of Cmi to detect hepatocellular carcinoma was 29·6% (eight of 27 cases) 12 months before clinical diagnosis, 48·1% (n=13) 9 months before clinical diagnosis, 48·1% (n=13) 6 months before clinical diagnosis, and 55·6% (n=15) 3 months before clinical diagnosis, whereas sensitivity of AFP20 was only 7·4% (n=2), 11·1% (n=3), 18·5% (n=5), and 22·2% (n=6) at the corresponding timepoints (p=0·036, p=0·0030, p=0·021, p=0·012, respectively). Cmi had a larger AUC than did AFP20 to identify small-size (AUC 0·833 0·782–0·883 vs 0·727 0·664–0·792, p=0·0018) and early-stage (AUC 0·824 0·781–0·868 vs 0·754 0·702–0·806, p=0·015) hepatocellular carcinoma and could also detect α-fetoprotein-negative (AUC 0·825 0·779–0·871) hepatocellular carcinoma. Interpretation Cmi is a potential biomarker for hepatocellular carcinoma, and can identify small-size, early-stage, and α-fetoprotein-negative hepatocellular carcinoma in patients at risk. The miRNA classifier could be valuable to detect preclinical hepatocellular carcinoma, providing patients with a chance of curative resection and longer survival. Funding National Key Basic Research Program, National Science and Technology Major Project, National Natural Science Foundation of China.
Background/Aims To characterize IL-17-producing cells, a newly defined T helper cell subset with potent pro-inflammatory properties, in hepatocellular carcinoma (HCC) and to determine their ...prognostic values. Methods One hundred and seventy-eight HCC patients were enrolled randomly. Distribution and phenotypic features of IL-17-producing cells were determined by flow cytometry and/or immunohistochemistry. Results Compared with corresponding non-tumor regions, the levels of Th17 cells were significantly increased in tumors of HCC patients ( P < 0.001). Most intratumoral Th17 cells exhibited an effector memory phenotype with increased expression of CCR4 and CCR6. Intratumoral IL-17-producing cell density was associated with overall survival (OS, P = 0.001) and disease-free survival (DFS, P = 0.001) in HCC patients. Multivariate Cox analysis revealed that intratumoral IL-17-producing cell density was an independent prognostic factor for OS (HR = 2.351, P = 0.009) and DFS (HR = 2.256, P = 0.002). Moreover, the levels of intratumoral Th17 cells were positively correlated with microvessel density in tumors ( r = 0.616, P = 0.001). Conclusion Accumulation of intratumoral IL-17-producing cells may promote tumor progression through fostering angiogenesis, and intratumoral IL-17-producing cell could serve as a potential prognostic marker and a novel therapeutic target for HCC.
Although cancer patients exhibit a generalized immunosuppressive status, substantial evidence indicates that the inflammatory reaction at a tumor site can promote tumor growth and progression. ...Hepatocellular carcinoma (HCC) is usually derived from inflamed cirrhotic liver with extensive leukocyte infiltration. We recently found that proinflammatory T helper (Th)17 cells are accumulated in HCC tissue, where they promote disease progression by fostering angiogenesis. Here we show that interleukin (IL)‐17‐producing cells were enriched predominantly in peritumoral stroma of HCC tissues, and their levels were well correlated with monocyte/macrophage density in the same area. Most peritumoral CD68+ cells exhibited an activated phenotype. Accordingly, tumor‐activated monocytes were significantly superior to the suppressive tumor macrophages in inducing expansion of Th17 cells from circulating memory T cells in vitro with phenotypic features similar to those isolated from HCCs. Moreover, we found that tumor‐activated monocytes secreted a set of key proinflammatory cytokines that triggered proliferation of functional Th17 cells. Inhibition of monocytes/macrophages inflammation in liver markedly reduced the level of tumor‐infiltrating Th17 cells and tumor growth in vivo. Conclusion: The proinflammatory Th17 cells are generated and regulated by a fine‐tuned collaborative action between different types of immune cells in distinct HCC microenvironments, and allows the inflammatory response of activated monocytes to be rerouted in a tumor‐promoting direction. Selectively modulating the “context” of inflammatory response in tumors might provide a novel strategy for anticancer therapy. (HEPATOLOGY 2009.)
Advanced hepatitis B virus (HBV)‐related hepatocellular carcinoma HCC with poor prognosis is often associated with chronic inflammation, immune tolerance, and marked heterogeneity. The interleukin‐6 ...(IL‐6)/JAK/STAT3 signal pathways play multiple regulatory roles in modulating inflammation and immunity in cancers. Polarization of myeloid‐derived suppressor cells (MDSCs) is involved in HBV‐related immunosuppression and CD8+ T‐cell activation through ERK/IL‐6/STAT3. Icaritin is a small molecule that has displayed anticancer activities through IL‐6/JAK/STAT3 pathways in tumor cells and immune cells including CD8+ T cells, MDSCs, neutrophils, and macrophages. This study aimed to confirm icaritin immunomodulation in advanced HBV‐related HCC patients with poor prognosis. Immunomodulation of MDSCs was evaluated in BALB/c mice in vivo. Immunomodulation of serum cytokines and a panel of immune checkpoint proteins were assessed in HBV‐related, histologically confirmed HCC patients. Poor prognostic characteristics included HBV infection, bulky tumors, Child‐Pugh B classification, and metastasis. Clinical end‐points included safety, tumor response, and overall survival (OS). Icaritin treatment‐induced dynamics of serum cytokines IL‐6, IL‐8, IL‐10, and tumor necrosis factor‐α, and soluble immune checkpoint proteins TIM3, LAG3, CD28, CD80, and CTLA‐4 were assessed. No grade III/IV treatment‐related adverse events were observed. Time‐to‐progression was significantly associated with the prognostic factors. Improved survival was observed in the advanced HCC patients with dynamic changes of cytokines, immune checkpoint proteins, and immune cells. Median OS (329‐565 days) was significantly correlated with baseline hepatitis B surface antigen positivity, cytokines, tumor neoantigens, and Stenotrophomonas maltophilia infection. Composite biomarker scores of high‐level α‐fetoprotein and T helper type I (Th1)/Th2 cytokines associated with favorable survival warrant further clinical development of icaritin as an alternative immune‐modulatory regimen to treat advanced HCC patients with poor prognosis.
Advanced hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC) is often associated with chronic inflammation, liver injury, immune tolerance, and poor clinical outcomes. Due to drug toxicity and side‐effects, current targeted drugs including sorafenib are unfortunately not favorable treatment options, particularly for those advanced HCC patients with poor prognosis. Herbal‐derived natural small molecule icaritin showed anticancer immunomodulatory efficacy with superior safety and improved survival associated with coordinated immune dynamics. Composite biomarkers associated with favorable survival would warrant further clinical development of icaritin as an alternative “natural compound” immune therapy in advanced HBV‐related HCC patients with poor prognosis.
Little is known about the composition and generation of plasma cell subsets in patients with hepatocellular carcinoma (HCC) and how these associate with outcomes. We investigated whether, or how, ...plasma cells differentiate and function in patients with HCC and mice with liver tumors.
We analyzed subset composition and distribution of plasma cells in HCC samples from 342 patients who underwent curative resection at the Cancer Center of Sun Yat-sen University in China; samples of non-tumor liver tissue were used as controls. We associated plasma cell profiles with patient outcomes. Tissue-derived leukocytes were analyzed by flow cytometry and real-time polymerase chain reaction. The ability of macrophages to regulate plasma cell differentiation was determined in ex vivo cultures of cells from human HCC tissues. C57BL/6 and BALB/c mice were given injections of Hepa1-6 cells, which formed hepatomas, or H22 cells, which formed ascitic hepatomas. Gene expression patterns were analyzed in human HCC, mouse hepatoma, and non-tumor tissues by real-time polymerase chain reaction. Mice with hepatomas were given injections of GSK126 (an inhibitor of histone H3 lysine 27 methyltransferase EZH2) and 5-AZA-dC (an inhibitor of DNA methyltransferases); tumor tissues were analyzed by immunofluorescence and immunohistochemistry for the presence of immune cells and cytokines.
B cells isolated from HCCs had somatic hypermutations and class-switch recombinations to the IgG phenotype that were not observed in non-tumor tissues. Increased level of plasma cells correlated with poor outcomes of patients. Activated CD4+ T cells from HCCs stimulated C-X-C motif chemokine 10 (CXCL10) production by macrophages. CXCL10 bound CXC chemokine receptor 3 on B cells and signaled via extracellular signal–regulated kinase to cause them to become IgG-producing plasma cells. IgG activated Fc receptors on macrophages and induced them to produce interleukin 6, interleukin 10, and C-C motif chemokine ligand 20 (CCL20). In mice with hepatomas, depletion of B cells prevented generation of these macrophage, increased the anti-tumor T cell response, and reduced growth of hepatomas. However, these effects were lost after injection of CXC chemokine receptor 3–positive plasma cells. Human HCC and mouse hepatoma tissues had increased expression of DNA methyltransferase 1 and EZH2 compared with non-tumor tissues. Injection of mice with GSK126 and 5-AZA-dC induced expression of CXCL10 by tumor cells and caused plasma cell polarization, suppression of the anti-tumor T cell response, and hepatoma growth.
Human HCC tissues contain B cells with class-switch recombinations to the IgG phenotype. Activated CD4+ T cells from HCCs stimulate CXCL10 production by macrophages; CXCL10 binds CXC chemokine receptor 3 on B cells and causes them to become IgG-producing plasma cells. IgG activates Fc receptor in macrophages to produce cytokines that reduce the anti-tumor immune response. In mice with hepatomas, depletion of B cells prevented generation of these macrophages, increased the anti-tumor T cell response, and reduced growth of hepatomas. This pathway involves increased expression of DNA methyltransferase 1 and EZH2 by HCC and hepatoma cells.
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With poor prognosis and limited treatment options for advanced hepatocellular carcinoma (HCC), development of novel therapeutic agents is urgently needed. This single-arm phase I study sought to ...assess the safety and preliminary efficacy of icaritin in human as a potential oral immunotherapy in addition to the immune-checkpoint inhibitors.
Eligible advanced HCC patients with Child-Pugh Class A or B were administered with a fixed oral dose of icaritin at either 600 or 800 mg b.i.d. The primary endpoint was safety, and the secondary endpoints included time-to-progression (TTP), overall survival (OS) and the clinical benefit rate (CBR). Icaritin treatment induced immune biomarkers and immune-modulating activities in myeloid cells were also explored.
No drug-related adverse events ≥ Grade 3 were observed in all 20 enrolled HCC patients. Among the 15 evaluable patients, 7 (46.7%) achieved clinical benefit, representing one partial response (PR, 6.7%) and 6 stable disease (SD, 40%). The median TTP was 141 days (range: 20-343 days), and the median OS was 192 days (range: 33-1036 days). Durable survival was observed in PR/SD patients with a median OS of 488 days (range: 72-773). TTP was significantly associated with the dynamic changes of peripheral neutrophils (p = 0.0067) and lymphocytes (p = 0.0337). Icaritin treatment induced changes in immune biomarkers-and immune-suppressive myeloid cells were observed.
Icaritin demonstrated safety profiles and preliminary durable survival benefits in advanced HCC patients, which were correlated with its immune-modulation activities and immune biomarkers. These results suggested the potential of icaritin as a novel oral immunotherapy for advanced HCC in addition to antibody-based PD-1/PD-L1 blockade therapies.
Clinicaltrial.gov identifier. NCT02496949 (retrospectively registered, July 14, 2015).
Macrophages （Mφ） are prominent components of solid tumors and exhibit distinct phenotypes in different microenvironments. We have recently found that tumors can alter the normal developmental process ...of Mφ to trigger transient activation of monocytes, but the underlying regulatory mechanisms are incompletely understood. Here, we showed that the protein expression of transcription factor C/EBPβ was markedly elevated in tumor-associated Mφ both in vitro and human tumors in situ. The expression of C/EBPβ protein correlated with cytokine production in tumor-activated monocytes. Moreover, we found that C/EBPβ expression was regulated at the post-transcriptional level and correlated with sustained reduction of microRNA-155 （miR-155） in tumor-activated monocytes. Bioinformatic analysis revealed that C/EBPβ is a potential target of miR-155 and luciferase assay confirmed that C/EBPβ translation is suppressed by miR-155 through interaction with the 3＇UTR of C/EBPβ mRNA. Further analysis showed that induction of miR-155 suppressed C/EBPβ protein expression as well as cytokine production in tumor-activated monocytes, an effect which could be mimicked by silencing of C/EBPβ. These results indicate that tumor environment causes a sustained reduction of miR-155 in monocytes/Mφ, which in turn regulates the functional activities of monocytes/Mφ by releasing the translational inhibition of transcription factor C/EBPβ.
Viruses are constantly subject to natural selection to enrich beneficial mutations and weed out deleterious ones. However, it remains unresolved as to how the phenotypic gains or losses brought about ...by these mutations cause the viral genomes carrying the very mutations to become more or less numerous. Previous investigations by us and others suggest that viruses with plus strand (+) RNA genomes may compel such selection by bottlenecking the replicating genome copies in each cell to low single digits. Nevertheless, it is unclear if similarly stringent reproductive bottlenecks also occur in cells invaded by DNA viruses. Here we investigated whether tomato yellow leaf curl virus (TYLCV), a small virus with a single-stranded DNA genome, underwent population bottlenecking in cells of its host plants. We engineered a TYLCV genome to produce two replicons that express green fluorescent protein and mCherry, respectively, in a replication-dependent manner. We found that among the cells entered by both replicons, less than 65% replicated both, whereas at least 35% replicated either of them alone. Further probability computation concluded that replication in an average cell was unlikely to have been initiated with more than three replicon genome copies. Furthermore, sequential inoculations unveiled strong mutual exclusions of these two replicons at the intracellular level. In conclusion, the intracellular population of the small DNA virus TYLCV is actively bottlenecked, and such bottlenecking may be a virus-encoded, evolutionarily conserved trait that assures timely selection of new mutations emerging through error-prone replication.
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