Impact of Genomic Alterations on the Clinical Outcome of Patients With Hepatitis B-related Hepatocellular Carcinoma Receiving Curative Surgery: A Retrospective Cohort Study.

BACKGROUND/AIM: The aim of the current study was to obtain comprehensive genomic information on viral hepatitis B (HBV)-related hepatocellular carcinoma (HCC) and identify potential biomarkers of early recurrence in patients receiving curative surgery. PATIENTS AND METHODS: A total of 104 patients with HBV-related HCC receiving curative surgery at Kaohsiung Chang Gung Memorial Hospital between January 2017 and December 2020 were identified, including 52 patients each with and without recurrence. Next-generation sequencing was performed to investigate genomic alterations caused by surgical resection of specimens. The Kaplan-Meier method was used to estimate disease-free survival and overall survival. RESULTS: The landscape of gene mutations in HCC patients of our cohort showed a median number of single nucleotide variants of 250, a median number of insertions and deletions of 22, and a median number of protein-coding mutations of 185. The 10 most frequently mutated genes were TP53 (43%), TTN (39%), MUC16 (28%), PCLO (25%), OBSCN (22%), ADGRV1 (19%), ALB (18%), SYNE1 (18%), DNAH17 (17%), and RYR1 (17%). The tumour mutation burden was 4.8 mutations per megabase, and high microsatellite instability was reported in only three patients. In addition, the mutational signatures showed that aristolochic acid exposure was highly implicated in our HCC cohort. Five mutant genes, TBC1D4, ITGA4, RPS6KA3, VWA8, and FMN2, were more frequent in the recurrence group than that in the non-recurrence group. CONCLUSION: Our results present an in-depth genomic analysis of HBV-related HCC. The study findings provide an improved understanding of the related molecular mechanisms and identify potential biomarkers associated with early tumour recurrence after curative resection.

E-cadherin expression in the tumor microenvironment of advanced epidermal growth factor receptor-mutant lung adenocarcinoma and the association with prognosis.

BACKGROUND: The expression of programmed death-ligand 1 (PD-L1), tumor-infiltrating lymphocytes (TILs), E-cadherin, and vimentin in lung cancer tumor microenvironment is known to impact patient survival or response to therapy. The expression of these biomarkers may also differ between primary lung tumors and brain metastatic tumors. In this study, we investigated the interaction between these biomarkers in lung tumors with or without concomitant brain metastasis and the interaction with paired brain metastatic tumors. METHODS: The study included 48 patients with stage IV epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma. Sixteen of the forty-eight patients were diagnosed with brain metastasis, while the remaining thirty-two were not. All sixteen patients with brain metastasis had brain tumors. The expression of PD-L1, TILs (CD8+ T lymphocytes and FOXP3+ regulatory T lymphocytes), E-cadherin, and vimentin were evaluated using immunohistochemical (IHC) staining. RESULTS: Patients with brain metastasis exhibited a higher frequency of exon 19 deletion and uncommon EGFR mutations, a higher lung tumor vimentin score, worse progression-free survival (PFS), and overall survival (OS) than patients without brain metastasis. IHC staining showed no difference between paired lung and brain tumors. Patients with low PD-L1 expression had better PFS and OS. After multivariate analysis, higher body mass index, the presence of brain metastasis, bone metastasis, and uncommon EGFR mutations were correlated with worse PFS, while the presence of brain metastasis and high lung tumor E-cadherin score was associated with worse OS. CONCLUSIONS: In patients with stage IV EGFR-mutant lung adenocarcinoma, high E-cadherin expression in the lung tumor might be associated with worse OS. Vimentin expression in the lung tumor was positively related to the risk of brain metastasis.

Slow skeletal muscle troponin T acts as a potential prognostic biomarker and therapeutic target for hepatocellular carcinoma.

Slow skeletal muscle troponin T (TNNT1) as a poor prognostic indicator is upregulated in colon and breast cancers. However, the role of TNNT1 in the disease prognosis and biological functions of hepatocellular carcinoma (HCC) is still unclear. The Cancer Genome Atlas (TCGA), real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to evaluate the TNNT1 expression of human HCC. The impact of TNNT1 levels on disease progression and survival outcome was studied using TCGA analysis. Moreover, the bioinformatics analysis and HCC cell culture were used to investigate the biological functions of TNNT1. Besides, the immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) were used to detect the extracellular TNNT1 of HCC cells and circulating TNNT1 of HCC patients, respectively. The effect of TNNT1 neutralization on oncogenic behaviors and signaling was further validated in the cultured hepatoma cells. In this study, tumoral and blood TNNT1 was upregulated in HCC patients based on the analyses using bioinformatics, fresh tissues, paraffin sections, and serum. From the multiple bioinformatics tools, the TNNT1 overexpression was associated with advanced stage, high grade, metastasis, vascular invasion, recurrence, and poor survival outcome in HCC patients. By the cell culture and TCGA analyses, TNNT1 expression and release were positively correlated with epithelial-mesenchymal transition (EMT) processes in HCC tissues and cells. Moreover, TNNT1 neutralization suppressed oncogenic behaviors and EMT in hepatoma cells. In conclusion, TNNT1 may serve as a non-invasive biomarker and drug target for HCC management. This research finding may provide a new insight for HCC diagnosis and treatment.

ONC201 Suppresses Neuroblastoma Growth by Interrupting Mitochondrial Function and Reactivating Nuclear ATRX Expression While Decreasing MYCN.

Neuroblastoma (NB) is characterized by several malignant phenotypes that are difficult to treat effectively without combination therapy. The therapeutic implication of mitochondrial ClpXP protease ClpP and ClpX has been verified in several malignancies, but is unknown in NB. Firstly, we observed a significant increase in ClpP and ClpX expression in immature and mature ganglion cells as compared to more malignant neuroblasts and less malignant Schwannian-stroma-dominant cell types in human neuroblastoma tissues. We used ONC201 targeting ClpXP to treat NB cells, and found a significant suppression of mitochondrial protease, i.e., ClpP and ClpX, expression and downregulation of mitochondrial respiratory chain subunits SDHB and NDUFS1. The latter was associated with a state of energy depletion, increased reactive oxygen species, and decreased mitochondrial membrane potential, consequently promoting apoptosis and suppressing cell growth of NB. Treatment of NB cells with ONC201 as well as the genetic attenuation of ClpP and ClpX through specific short interfering RNA (siRNA) resulted in the significant upregulation of the tumor suppressor alpha thalassemia/mental retardation X-linked (ATRX) and promotion of neurite outgrowth, implicating mitochondrial ClpXP proteases in MYCN-amplified NB cell differentiation. Furthermore, ONC201 treatment significantly decreased MYCN protein expression and suppressed tumor formation with the reactivation of ATRX expression in MYCN-amplified NB-cell-derived xenograft tumors. Taken together, ONC201 could be the potential agent to provide diversified therapeutic application in NB, particularly in NB with MYCN amplification.

Genetic Interference of FGFR3 Impedes Invasion of Upper Tract Urothelial Carcinoma Cells by Alleviating RAS/MAPK Signal Activity.

Upper tract urothelial cancer (UTUC) is a less common disease in Western countries but has a high level of prevalence in Asian populations. Compared to bladder cancer, unique etiologic and genomic factors are involved in UTUC. Fibroblast growth factor receptor 3 (FGFR3) up-regulation has been proposed as a promising target for bladder cancer therapy. In this study, we aimed to profile the expression of FGFR3 in Asian and Caucasian UTUC tissues and to evaluate the in vitro therapeutic efficacy of small interference RNA (siRNA)-mediated FGFR3 silencing in UTUC treatment. The FGFR3 expression levels in renal pelvis tissues and microarray sections from Asian and Caucasian patients with UTUC, respectively, were measured via immunohistochemistry. The BFTC-909 and UM-UC-14 UTUC cell lines were used to examine the effects of FGFR3 silencing on proliferation, migration, epithelial-mesenchymal transition (EMT) marker expression, and signaling machinery. FGFR3 expression increased as the TNM stage increased in both Asian and Caucasian UTUC tumors, and no statistical difference was identified between the two groups. In vitro studies demonstrated that FGFR3 siRNA delivery significantly inhibited proliferation and migration and suppressed the expression of EMT markers and transcription factors in UTUC cells. Mechanistically, FGFR3 silencing alleviated the constitutive expression of RAS and the phosphorylation of MAPK signaling mediators, including ERK1/2 and JNK1/2. FGFR3 silencing elicited an apoptosis-inducing effect similar to that of FGFR inhibition. Conclusion: siRNA-targeted FGFR3 expression may impede the expansion and invasion of UTUC cells by alleviating the RAS/MAPK signaling pathway. The genetic interference of FGFR3 expression via siRNA in UTUC cells may constitute a useful therapeutic strategy.

The Ring Study: an international comparison of PD-L1 diagnostic assays and their interpretation in non-small cell lung cancer, head and neck squamous cell cancer and urothelial cancer.

PD-L1 immunohistochemistry has been approved as a diagnostic assay for immunotherapy. However, an international comparison across multiple cancers is lacking. This study aimed to assess the performance of PD-L1 diagnostic assays in non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC) and urothelial cancer (UC). The excisional specimens of NSCLC, HNSCC and UC were assayed by Ventana SP263 and scored at three sites in each country, including Australia, Brazil, Korea, Mexico, Russia and Taiwan. All slides were rotated to two other sites for interobserver scoring. The same cohort of NSCLC was assessed with Dako 22C3 pharmDx PD-L1 for comparison. The PD-L1 immunopositivity was scored according to the approved PD-L1 scoring algorithms which were the percentage of PD-L1-expressing tumour cell (TC) and tumour proportion score (TPS) by Ventana SP263 and Dako 22C3 staining, respectively. In NSCLC, the comparison demonstrated the comparability of the SP263 and 22C3 assays (cut-off of 1%, κ=0.71; 25%, κ=0.75; 50%, κ=0.81). The interobserver comparisons showed moderate to almost perfect agreement for SP263 in TC staining at 25% cut-off (NSCLC, κ=0.72 to 0.86; HNSCC, κ=0.60 to 0.82; UC, κ=0.68 to 0.91) and at 50% cut-off for NSCLC (κ=0.64 to 0.90). Regarding the immune cell (IC) scoring in UC, there was a lower correlation (concordance correlation coefficient=0.10 to 0.68) and poor to substantial agreements at the 1%, 5%, 10% and 25% cut-offs (κ= -0.04 to 0.76). The interchangeability of SP263 and 22C3 in NSCLC might be acceptable, especially at the 50% cut-off. In HNSCC, the performance of SP263 is comparable across five countries. In UC, there was low concordance of IC staining, which may affect treatment decisions. Overall, the study showed the reliability and reproducibility of SP263 in NSCLC, HNSCC and UC.

Leukocyte cell-derived chemotaxin 2 regulates epithelial-mesenchymal transition and cancer stemness in hepatocellular carcinoma.

Leukocyte cell-derived chemotaxin 2 (LECT2) acts as a tumor suppressor in hepatocellular carcinoma (HCC). However, the antineoplastic mechanism of LECT2, especially its influence on hepatic cancer stem cells (CSCs), remains largely unknown. In The Cancer Genome Atlas cohort, LECT2 mRNA expression was shown to be associated with stage, grade, recurrence, and overall survival in human HCC patients, and LECT2 expression was downregulated in hepatoma tissues compared with the adjacent nontumoral liver. Here, we show by immunofluorescence and immunoblot analyses that LECT2 was expressed at lower levels in tumors and in poorly differentiated HCC cell lines. Using functional assays, we also found LECT2 was capable of suppressing oncogenic behaviors such as cell proliferation, anchorage-independent growth, migration, invasiveness, and epithelial-mesenchymal transition in hepatoma cells. Moreover, we show exogenous LECT2 treatment inhibited CSC functions such as tumor sphere formation and drug efflux. Simultaneously, hepatic CSC marker expression was also downregulated, including expression of CD133 and CD44. This was supported by infection with adenovirus encoding LECT2 (Ad-LECT2) in HCC cells. Furthermore, in animal experiments, Ad-LECT2 gene therapy showed potent efficacy in treating HCC. We demonstrate LECT2 overexpression significantly promoted cell apoptosis and reduced neovascularization/CSC expansion in rat hepatoma tissues. Mechanistically, we showed using immunoblot and immunofluorescence analyses that LECT2 inhibited ß-catenin signaling via the suppression of the hepatocyte growth factor/c-MET axis to diminish CSC properties in HCC cells. In summary, we reveal novel functions of LECT2 in the suppression of hepatic CSCs, suggesting a potential alternative strategy for HCC therapy.

DLK2 Acts as a Potential Prognostic Biomarker for Clear Cell Renal Cell Carcinoma Based on Bioinformatics Analysis.

Clear cell renal cell carcinoma (ccRCC) is the most common RCC subtype with a high mortality. It has been reported that delta-like 1 homologue (DLK1) participates in the tumor microenvironmental remodeling of ccRCC, but the relationship between delta-like 2 homologue (DLK2, a DLK1 homologue) and ccRCC is still unclear. Thus, this study aims to investigate the role of DLK2 in the biological function and disease prognosis of ccRCC using bioinformatics analysis. The TNMplot database showed that DLK2 was upregulated in ccRCC tissues. From the UALCAN analysis, the overexpression of DLK2 was associated with advanced stage and high grade in ccRCC. Moreover, the Kaplan-Meier plotter (KM Plotter) database showed that DLK2 upregulation was associated with poor survival outcome in ccRCC. By the LinkedOmics analysis, DLK2 signaling may participated in the modulation of ccRCC extracellular matrix (ECM), cell metabolism, ribosome biogenesis, TGF-ß signaling and Notch pathway. Besides, Tumor Immune Estimation Resource (TIMER) analysis showed that the macrophage and CD8+ T cell infiltrations were associated with good prognosis in ccRCC patients. Finally, DLK2 overexpression was associated with the reduced macrophage recruitments and the M1-M2 polarization of macrophage in ccRCC tissues. Together, DLK2 may acts as a novel biomarker, even therapeutic target in ccRCC. However, this study lacks experimental validation, and further studies are required to support this viewpoint.

Significant association between serum Wisteria floribunda agglutinin-positive Mac-2-binding protein and prognosis of hepatocellular carcinoma after surgical treatment.

Serum Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+-M2BP) is a novel marker for evaluating fibrosis and predicting the development of hepatocellular carcinoma (HCC). However, the role of WFA+-M2BP in the prognosis of HCC patients after curative surgery remains unknown. In this study, we aimed to evaluate the prognostic role of serum WFA+-M2BP in HCC patients after curative resection and liver transplantation. We enrolled 460 HCC patients (357 resection and 103 transplantation) to analyze the risk factors for HCC recurrence and patient's survival. We employed time-to-event models using univariate and multivariable Cox proportional hazards regression analyses and calculated the hazard ratios (HRs) and adjusted HRs with their corresponding 95% confidence intervals (CIs). The levels of WFA+-M2BP were 0.19-14.51 COI (median 1.08) in patients of hepatectomy and 0.47-19.90 COI (median 6.0) in transplant patients. The levels of WFA+-M2BP in liver transplant patients is much higher than that of hepatectomy patients. Overall, liver fibrotic stage was positively correlated to WFA+-M2BP levels (P<0.0001). This study demonstrated that elevated WFA+-M2BP level (COI ≥0.75) was associated with a higher HCC recurrence rate in the resection group (P<0.001). Survival analysis showed that an elevated WFA+-M2BP level (COI ≥1.43) is associated with a higher mortality risk after surgical resection (P=0.0088) in the univariate analysis only. In liver transplant patients, WFA+-M2BP level (COI ≥3.81) did not predict HCC recurrence at all, but was associated poor survival after transplantation, with a borderline significance (P=0.0943). Serum WFA+-M2BP is a reliable marker for liver fibrosis in the present study. It is also reliable marker to predict prognosis of HCC after surgical resection. However, the prognostic role of WFA+-M2BP in HCC related transplants is equivocal, which is different from that of surgical resection.

Skin wound healing assessment via an optimized wound array model in miniature pigs.

An appropriate animal wound model is urgently needed to assess wound dressings, cell therapies, and pharmaceutical agents. Minipig was selected owing to similarities with humans in body size, weight, and physiological status. Different wound sizes (0.07-100 cm2) were created at varying distances but fail to adequately distinguish the efficacy of various interventions. We aimed to resolve potential drawbacks by developing a systematic wound healing system. No significant variations in dorsal wound closure and contraction were observed within the thoracolumbar region between boundaries of both armpits and the paravertebral region above rib tips; therefore, Lanyu pigs appear suitable for constructing a reliable dorsal wound array. Blood flow signals interfered with inter-wound distances ˂ 4 cm; a distance > 4 cm is therefore recommended. Wound sizes ≥ 4 cm × 4 cm allowed optimal differentiation of interventions. Partial- (0.23 cm) and full-thickness (0.6 cm) wounds showed complete re-epithelialization on days 13 and 18 and strongest blood flow signals at days 4 and 11, respectively. Given histological and tensile strength assessments, tissue healing resembling normal skin was observed at least after 6 months. We established some golden standards for minimum wound size and distance between adjacent wounds for effectively differentiating interventions in considering 3R principles.

Low energy shock wave therapy attenuates mitochondrial dysfunction and improves bladder function in HCl induced cystitis in rats.

BACKGROUND: We examine the effects of low energy shock wave (LESW) on bladder and mitochondrial function in a rat model of HCl induced cystitis, and the influence of dynamic bladder filling volume on LESW responses. Dysregulation of mitochondria function may impact the urothelial barrier and contribute to bladder dysfunction in patients with Interstitial cystitis/bladder pain syndrome (IC/BPS). METHODS: Female Sprague-Dawley rats underwent urethral catheterization and intravesical instillation of 0.2 ml of 0.4 N HCl (N = 32) or 0.2 ml saline (N = 8) kept for 90 s. After HCl instillation, the bladder received LESW treatment while filled with 0 ml, 0.2 ml or 0.4 ml saline or no LESW treatment. Continuous cystometry (CMG) was performed on day 8. The bladder was harvested after CMG for histology and Western blotting. RESULTS: HCl provoked bladder overactivity, bladder wall inflammation marked by infiltration of mast cells, increased bax/bcl2 ratio consistent with increased TUNEL staining and increased release of mitochondrial-integrity markers (cleaved caspase 3 and Cytochrome c). LESW treatment suppressed HCl provoked bladder overactivity in association with lower inflammatory reaction, mast cells infiltration, and a lower bax/bcl2 ratio also reflected by reduced TUNEL staining and mitochondrial-integrity markers irrespective of the volume of saline in bladder at the time of LESW. CONCLUSIONS: These findings support that antiinflammatory effect of LESW in chemical cystitis is associated with the reversal of the molecular-cellular perturbations in mitochondrial dependent intrinsic apoptotic pathway.

Comprehensive Clinicopathologic Analysis for Mismatch Repair Protein Expression in Unselected Endometrial Carcinoma Patients With an Emphasis on the Role of MLH1 Deficiency.

Screening for mismatch repair (MMR) deficiency in unselected patients with endometrial carcinoma (EC) and the clinicopathologic descriptions of ECs with MMR deficiency have been well demonstrated in Western populations, but studies on Asian populations are relatively scarce. In this study, we described the clinicopathologic features of ECs according to MMR status in unselected Taiwanese patients. We also conducted subgroup analysis of MMR-deficient (dMMR) cases according to the presence or absence of MLH1. Patients diagnosed with ECs between January 2017 and February 2020 at our institution were included. Immunohistochemistry analysis of MLH1, PMS2, MSH2, and MSH6 proteins on endometrial primary tumors and clinicopathologic variables were assessed retrospectively. A total of 231 EC patients were enrolled, of whom 50 (21.6%) had dMMR tumors. Of these 50 cases, 39 had tumors that lacked MLH1 expression and 11 were positive for MLH1. The overall dMMR group was significantly related to older age, parity, and high histologic grade compared with the MMR-proficient (pMMR) group. ECs with MLH1 deficiency were obviously associated with several poor pathologic features, including high histologic grade, lymph node metastasis, and lymphovascular space invasion. Moreover, we first reported that parity and the late age at menopause are strongly correlated with MLH1-related dMMR EC group compared with pMMR group. In conclusion, triaging EC patients into pMMR, MLH1-related dMMR and non-MLH1-related dMMR groups by immunohistochemistry analysis may help clinicians to predict disease behavior and guide further management. The strong association between parity and MLH1-related dMMR ECs warrants further investigation on the underlying mechanism.

Comprehensive analysis of PD-L1 in non-small cell lung cancer with emphasis on survival benefit, impact of driver mutation and histological types, and archival tissue.

BACKGROUND: The aim of the study was to assess programmed death-ligand-1 (PD-L1) expression in different histological types and gene mutation status of patients with non-small cell lung cancer (NSCLC). METHODS: A total of 4062 pathology-confirmed lung cancer patients were retrospectively screened at Kaohsiung Chang Gung Memorial Hospital from November 2010 to June 2017. There were 699 NSCLC patients with confirmed PD-L1 expression level retrospectively enrolled for analysis. RESULTS: There was a trend of higher PD-L1 expression in squamous cell carcinoma and adenosquamous cell carcinoma than in adenocarcinoma (p = 063). Significant higher PD-L1 expression in EGFR wild-type was noted (p < 0.001). No significant differences in PD-L1 expression were found between ALK wild- and mutant types, but there seem was a trend of high PD-L1 level noted in ALK mutation patients (p = 0.069). In EGFR mutation patients, a higher time to treatment failure (TTF) duration was observed in no PD-L1 expression (p = 0.011). Longer tumor tissue storage time correlated with lower PD-L1 expression in lung cancer (p < 0.001 for linear trend). CONCLUSIONS: There were a trend or significant differences in PD-L1 expression between different histological types in NSCLC, different EGFR and ALK status, and different tumor tissue storage time. A higher survival benefit was observed in no PD-L1 expression than with PD-L1 expression in adenocarcinoma, EGFR and ALK mutation patients. We recommend that PD-L1 assay should be performed as early as possible if tissue is available.

Galectin-1 Expression Is Associated with the Response and Survival Following Preoperative Chemoradiotherapy in Locally Advanced Esophageal Squamous Cell Carcinoma.

The galectin-1 has been found to be involved in poor outcomes after treatment of a variety of cancers. To the best of our knowledge, however, the significance of galectin-1 expression in the sensitivity to chemoradiotherapy (CCRT) of patients with locally advanced esophageal squamous cell carcinoma (ESCC) remains unclear. Expression levels of galectin-1 were evaluated by immunohistochemistry and correlated with the treatment outcome in 93 patients with locally advanced ESCC who received preoperative CCRT between 1999 and 2012. Galectin-1 expression was significantly associated with the pathological complete response (pCR). The pCR rates were 36.1% and 13.0% (p = 0.01) in patients with low and high galectin-1 expression, respectively. Univariate analyses revealed that galectin-1 overexpression, clinical 7th American Joint Committee on Cancer (AJCC) stage III and a positive surgical margin were significant factors of worse overall survival and disease-free survival. In multivariate analyses, galectin-1 overexpression and a positive surgical margin represented the independent adverse prognosticators. Therefore, galectin-1 expression both affects the pCR and survival in patients with locally advanced ESCC receiving preoperative CCRT. Our results suggest that galectin-1 may be a potentially therapeutic target for patients with ESCC treated with preoperative CCRT.

Exosome-derived microRNAs in oral squamous cell carcinomas impact disease prognosis.

OBJECTIVES: microRNA (miRNA) expression patterns have provided new insight as biomarkers of prognosis as well as novel therapeutic targets for several neoplasms. However, the role of exosomal miRNA in the prognosis of oral squamous cell carcinoma (OSCC) has not yet been completely clarified. Paired primary tumor and normal oral epithelial cells from OSCC patients were obtained, and the exosomal miRNA profiles between them were compared by miRNA microarray analysis. The miRNA levels in the serum exosomes of OSCC patients were verified by real-time quantitative reverse transcription PCR (qRT-PCR) analysis. Finally, the biological functions and the potential as a prognostic marker of the selected miRNA candidates were analyzed in the OSCC cells and patients, respectively. RESULTS: Exosomal miR-155 and miR-21 were significantly upregulated, and exosomal miR-126 was dramatically downregulated in the primary OSCC cells and the serum of OSCC patients. In the analysis of oncogenic behaviors, coculture with either miR-155-rich or miR-21-rich exosomes could promote cell proliferation and invasion accompanied with downregulation of PTEN and Bcl-6 tumor suppressors. Moreover, treatment with miR-126-rich exosomes inhibited oncogenic behaviors and oncogene EGFL7 expression in OSCC cells. Finally, exosomal miR-126 was reduced in the serum of the late-staged OSCC patients, and downregulation of blood exosomal miR-126 was associated with poor survival in OSCC patients. CONCLUSION: Exosomal miR-155 and miR-21 are oncogenic miRNAs which suppress PTEN and Bcl-6 expression, and exosomal miR-126 acts as a tumor suppressor which downregulates EGFL7 in OSCC. Furthermore, blood exosomal miRNAs may serve as biomarkers for the diagnosis and prognosis of OSCC.

Microalgal extract from thermotolerant Coelastrella sp. F50 retards the liver tumor progression by targeting hepatic cancer stem cells.

Microalgae extracts have shown antitumor activities. However, the antitumor mechanism of them is not yet completely clear, especially the effect on cancer stem cells (CSCs). This study aimed to elucidate the antitumor activity and mechanism of microalgal extract from thermotolerant Coelastrella sp. F50 (F50) in hepatocellular carcinoma (HCC). Oncogenic behaviors were analyzed using cell proliferation, colony formation, invasion, sphere formation, and side population cells (SPCs) assays in HCC cells after F50 treatment. The molecular mechanism was further studied by quantitative real-time PCR, immunoblot, and immunofluorescence analyses. The chemopreventive efficacy of F50 was evaluated in rat orthotopic hepatoma, and the hepatic pathologies were investigated by immunohistochemical, immunoblot, and immunofluorescence analyses. F50 specifically suppressed hepatic CSCs (tumor spheres, drug efflux, CD133/ABCG2 CSCs markers) with no cytotoxicity in vitro. In the animal experiments, prophylactic F50 administration significantly attenuated tumor progression and improved liver function in HCC-bearing rats. In the mechanistic analysis, F50 potentially inhibited cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) axis in HCC cells and rat hepatoma, and exogenous PGE2 restored CSCs properties in F50-treated HCC cells. In summary, F50 extract inhibits hepatic CSCs by COX-2/PGE2 downregulation and may facilitate a novel phytotherapy for HCC prevention.

Clinical and novel application of FibroScan, FIB-4 and aspartate aminotransferase-to-platelet ratio index in liver fibrosis evaluation in patients with hepatocellular carcinoma and their roles in oesophageal variceal prediction.

BACKGROUND: Non-invasive techniques for liver fibrosis diagnosis are very important for clinician especially in high-risk patients for liver biopsy. We further explored the diagnostic accuracy of FibroScan, FIB-4 and aminotransferase-to-platelet ratio index (APRI) in identifying liver fibrosis and assess their predictive role for oesophageal varices in patients with hepatocellular carcinoma (HCC). METHODS: In total, 380 patients who underwent surgery for HCC were included based on retrospective study design. Liver fibrosis was pathologically diagnosed using the Ishak scoring system. Liver stiffness parameters were measured using FibroScan. APRI and FIB-4 were calculated. Among those, 121 patients who received oesophagogastroduodenoscopic examination underwent variceal evaluation. RESULTS: For liver cirrhosis diagnosis with FibroScan, the optimal cut-off values for the patients with HCC overall, left HCC and right HCC were 8.85, 11.75 and 8.70 kPa (the accuracy were 78.7%, 78.4% and 79.2%, respectively). They had high areas under the receiver operating characteristic curve of 0.84, 0.84 and 0.85. The combined FibroScan, APRI and FIB-4 had very high specificity (more than 92%) for cirrhosis diagnosis. The optimal cut-off liver stiffness values for the diagnosis of varices were all 11.2 kPa. For predicting varices, the optimal cut-off values of FIB-4 and APRI were 2.64 and 0.71, their accuracy were 64.3%-78.4%, 69.4% and 72.7%, respectively. CONCLUSIONS: FibroScan, FIB-4 and APRI have moderate accuracy for liver fibrosis diagnosis and oesophageal varices prediction in patients with hepatoma. This is a study of these non-invasive techniques applied in specific hepatoma patients and with inevitable limitations and need future more studies for validation.

Hepatoma-derived growth factor participates in concanavalin A-induced hepatitis.

Hepatitis is an important health problem worldwide. Novel molecular targets are in demand for detection and management of hepatitis. Hepatoma-derived growth factor (HDGF) has been delineated to participate in hepatic fibrosis and liver carcinogenesis. However, the relationship between hepatitis and HDGF remains unclear. This study aimed to elucidate the role of HDGF during hepatitis using concanavalin A (ConA)-induced hepatitis model. In cultured hepatocytes, ConA treatment-elicited HDGF upregulation at transcriptional level and promoted HDGF secretion while reducing intracellular HDGF protein level and cellular viability. Similarly, mice receiving ConA administration exhibited reduced hepatic HDGF expression and elevated circulating HDGF level, which was positively correlated with serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. By using HDGF knockout (KO) mice, it was found the ConA-evoked cell death was prominently alleviated in KO compared with control. Besides, it was delineated HDGF ablation conferred protection by suppressing the ConA-induced neutrophils recruitment in livers. Above all, the ConA-mediated activation of tumor necrosis factor-α (TNF-α)/interleukin-1ß (IL-1ß)/interleukin-6 (IL-6)/cyclooxygenase-2 (COX-2) inflammatory signaling was significantly abrogated in KO mice. Treatment with recombinant HDGF (rHDGF) dose-dependently stimulated the expression of TNF-α/IL-1ß/IL-6/COX-2 in hepatocytes, further supporting the pro-inflammatory function of HDGF. Finally, application of HDGF antibody not only attenuated the ConA-mediated inflammatory cascade in hepatocytes, but also ameliorated the ConA-induced hepatic necrosis and AST elevation in mice. In summary, HDGF participates in ConA-induced hepatitis via neutrophils recruitment and may constitute a therapeutic target for acute hepatitis.

Combined Chibby and ß-Catenin Predicts Clinical Outcomes in Patients with Hepatocellular Carcinoma.

Chibby is an antagonist of ß-catenin and is considered a potential tumor suppressor protein, but the role of Chibby in hepatocellular carcinoma (HCC) has not been characterized. The expression patterns of Chibby and ß-catenin in HCC specimens and paired adjacent noncancerous tissues were measured by Western blotting and immunohistochemistry. The correlations between Chibby expression and clinicopathological parameters were analyzed. Then the biological functions of Chibby were analyzed in vitro. The Chibby protein was significantly downexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and is a risk factor for HCC recurrence and shorter survival. Furthermore, we found that in HCC tissues the high expression of ß-catenin with low expression of Chibby in the nuclei was an independent predictor for disease-free survival (DFS) (p = 0.012) and overall survival (OS) (p = 0.005). Subsequent genetic manipulation in vitro studies revealed that Chibby knockdown induced the expression of ß-catenin and C-myc, cyclin D1 protein, which promoted cell proliferation and invasiveness. In contrast, overexpression of Chibby decreased ß-catenin expression and inhibited the cell proliferation and invasiveness. Our results suggest that low expression of Chibby was associated with advanced tumor-node-metastasis (TNM) stage and poor differentiation. Furthermore, the combination of Chibby and ß-catenin can predict poor prognosis in patients with HCC. Chibby inhibited HCC progression by blocking ß-catenin signaling in vitro. Chibby is a biomarker and may be a potential therapeutic target for HCC.

The clinical impact of angiotensin-(1-7)/mitochondrial assembly receptor axis in esophageal squamous cell carcinoma patients receiving curative esophagectomy.

BACKGROUND: Mitochondrial assembly receptor (MasR), a receptor of angiotensin-(1-7), plays an important role in the anti-cancer effect of the peptide hormone. The aim of the current study was to evaluate the crucial role of angiotensin-(1-7)/MasR axis in esophageal squamous cell carcinoma (ESCC) patients who received curative esophagectomy. METHODS: The immunohistochemistry of MasR in 90 ESCC patients, including 52 patients with MasR overexpression and 38 patients with low MasR expression, was examined and correlated with their treatment outcomes. Two ESCC cell lines, TE11 and KYSE270, were treated with angiotensin-(1-7) to explore the biological function of MasR. RESULTS: A higher percentage of patients in the low MasR expression group experienced tumor recurrence than those in the MasR overexpression group (76% versus 54%, P = 0.029). Patients below 60 years of age and having early T status and negative pathologic N status were found to have significantly better disease-free survival (DFS) and overall survival (OS). Additionally, patients with MasR overexpression had higher DFS (88.1 months versus 50.0 months, p = 0.023) and OS (129.4 months versus 67.5 months, p = 0.028) relative to those with low MasR expression, although there was no significant difference in multivariable analysis. In vitro, these cell lines were treated with angiotensin-(1-7) and the results demonstrated that angiotensin-(1-7) could inhibit the growth of ESCC tumor cells in a dose-dependent manner. CONCLUSION: Low expression of MasR may be associated with poor prognosis in ESCC patients receiving curative esophagectomy. Further cohort study with larger population, or a prospective study is warranted to validate this finding.