Safety and efficacy of apatinib in patients with advanced gastric or gastroesophageal junction adenocarcinoma after the failure of two or more lines of chemotherapy (AHEAD): a prospective, single-arm, multicenter, phase IV study.

BACKGROUND: Apatinib, a highly selective VEGFR2 inhibitor, significantly improved efficacy versus placebo as a third- and later-line treatment for advanced gastric cancer in phase 2 and 3 trials. This prospective, single-arm, multicenter phase IV AHEAD study was conducted to verify the safety and efficacy of apatinib in patients with advanced or metastatic gastric or gastroesophageal adenocarcinoma after at least two lines of systematic therapy in clinical practice settings. METHODS: Patients with advanced gastric cancer who had previously failed at least two lines of chemotherapy received oral apatinib until disease progression, death or unacceptable toxicity. The primary endpoint was safety. The secondary endpoints included objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). Adverse events were summarized by the incidence rate. Median OS and PFS were estimated using the Kaplan-Meier method. ORR, DCR, OS at 3 and 6 months, and PFS at 3 and 6 months were calculated, and their 95% CIs were estimated according to the Clopper-Pearson method. RESULTS: Between May 2015 and November 2019, a total of 2004 patients were enrolled, and 1999 patients who received at least one dose of apatinib were assessed for safety. In the safety population, 87.9% of patients experienced treatment-related adverse events (TRAEs), with the most common hypertension (45.2%), proteinuria (26.5%), and white blood cell count decreased (25.3%). Additionally, 51% of patients experienced grade ≥ 3 TRAEs. Fatal TRAEs occurred in 57 (2.9%) patients. No new safety concerns were reported. Among the 2004 patients included in the intention-to-treat population, the ORR was 4.4% (95% CI, 3.6-5.4%), and DCR was 35.8% (95% CI, 33.7-38.0%). The median PFS was 2.7 months (95% CI 2.2-2.8), and the median OS was 5.8 months (95% CI 5.4-6.1). CONCLUSIONS: The findings in the AHEAD study confirmed the acceptable and manageable safety profile and clinical benefit of apatinib in patients with advanced gastric cancer as a third- or later-line of treatment. TRIAL REGISTRATION: This study was registered with ClinicalTrials.gov NCT02426034. Registration date was April 24, 2015.

Neutrophil-to-Apolipoprotein A1 Ratio Predicted Overall Survival in Hepatocellular Carcinoma Receiving Transarterial Chemoembolization.

PURPOSE: The purpose of this study was to investigate the predictive capability of neutrophil-to-apolipoprotein A1 ratio (NAR) for predicting overall survival (OS) among patients with hepatocellular carcinoma (HCC) receiving transarterial chemoembolization (TACE). PATIENTS AND METHODS: We investigated the clinical features of 554 patients with HCC receiving TACE and assessed NAR's predictive value for OS with 222 patients (the discovery cohort) and 332 patients (the validation cohort). The association of NAR with circulation lectin-type oxidized low-density lipoprotein receptor-1-positive (LOX-1+ ) polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) was illustrated. RESULTS: Multivariate Cox regression revealed that lymphocyte count; Tumor, Node, Metastasis (TNM) stage; and NAR were independent prognostic factors in the discovery cohort. The validation cohort confirmed the independent prognostic value of TNM stage and NAR. Patients with low NAR (<2.7) displayed significantly increased OS in the discovery cohort (59.8 months vs. 21 months), the validation group (38.0 months vs. 23.6 months), and the total cohort (44.1 months vs. 22.0 months). A Cox proportional hazards model was used to combine Cancer of the Liver Italian Program (CLIP) score with discretized NAR. C-index illustrated that NAR-integrated CLIP score was the best model compared with NAR and CLIP score. Furthermore, NAR-CLIP presented superior predictive capacity for 10-, 20-, 30-, 40-, 50-, and 60-month survival compared with CLIP score by survival receiver-operator characteristic analysis in the discovery cohort, validation cohort, and total cohort. NAR was significantly associated with LOX-1+ PMN-MDSCs by linear regression. CONCLUSION: This study identified NAR as an independent predictor for OS among patients with HCC receiving TACE. NAR reflected circulation LOX-1+ PMN-MDSC level. IMPLICATIONS FOR PRACTICE: The present study identified neutrophil-to-apolipoprotein A1 ratio (NAR) as an independent predictor for overall survival among patients with hepatocellular carcinoma receiving transarterial chemoembolization. NAR reflected circulation level of lectin-type oxidized low-density lipoprotein receptor-1-positive polymorphonuclear myeloid-derived suppressor cells.

Synergistic effects of α-Mangostin and sorafenib in hepatocellular carcinoma: New insights into α-mangostin cytotoxicity.

Sorafenib remains the standard first-line treatment for advanced hepatocellular carcinoma (HCC), although other clinical trials are currently underway for treatments that show better curative effects. However, some patients are not sensitive to sorafenib. α-Mangostin, extracted from the pericarp of the mangosteen, which is widely used as a traditional medicine, has anticancer and anti-proliferative properties in various types of cancers, including HCC. In the present study, we found that combining sorafenib and α-Mangostin could be synergistically toxic to HCC both in vitro and in vivo. We then demonstrated that the combination of sorafenib and α-Mangostin enhances the inhibition of cell proliferation in HCC cell lines. Combination therapy leads directly to apoptosis. In xenograft mouse models, the in vivo safety and effectivity was confirmed by a reduction in tumor size after combination treatment. RNA sequencing and protein testing showed that the expression of LRRC8A and RNF181 genes and mTOR and MAPK pathways may be associated with the synergistic effect of the two drugs. In conclusion, our results highlight the synergistic effect of the combination of sorafenib and α-Mangostin, which indicates a potential treatment for advanced HCC for patients that are not sensitive to sorafenib therapy.

Endoplasmic reticulum stress induced LOX-1+ CD15+ polymorphonuclear myeloid-derived suppressor cells in hepatocellular carcinoma.

A recent study indicated that Lectin-type oxidized LDL receptor-1 (LOX-1) was a distinct surface marker for human polymorphisms myeloid-derived suppressor cells (PMN-MDSC). The present study was aimed to investigate the existence LOX-1 PMN-MDSC in hepatocellular carcinoma (HCC) patients. One hundred and twenty-seven HCC patients, 10 patients with mild active chronic hepatitis B, 10 liver cirrhosis due to hepatitis B, 10 liver dysplastic node with hepatitis B and 50 health control were included. LOX-1+  CD15+ PMN-MDSC were significantly elevated in HCC patients compared with healthy control and patients with benign diseases. LOX-1+  CD15+ PMN-MDSC in circulation were positively associated with those in HCC tissues. LOX-1+  CD15+ PMN-MDSCs significantly reduced proliferation and IFN-γ production of T cells with a dosage dependent manner with LOX-1-  CD15+ PMNs reached negative results. The suppression on T cell proliferation and IFN-γ production was reversed by ROS inhibitor and Arginase inhibitor. ROS level and activity of arginase of LOX-1 + CD15+ PMN were higher in LOX-1+  CD15+ PMN-MDSCs than LOX-1-  CD15+ PMNs, as well as the expression of the NADPH oxidase NOX2 and arginase I. RNA sequence revealed that LOX-1+ CD15+ PMN-MDSCs displayed significantly higher expression of spliced X-box -binding protein 1 (sXBP1), an endoplasmic reticulum (ER) stress marker. ER stress inducer induced LOX-1 expression and suppressive function for CD15+ PMN from health donor. For HCC patients, LOX-1+  CD15+ PMN-MDSCs were positively related to overall survival. Above all, LOX-1+  CD15+ PMN-MDSC were elevated in HCC patients and suppressed T cell proliferation through ROS/Arg I pathway induced by ER stress. They presented positive association with the prognosis of HCC patients.

microRNA-dependent gene regulatory networks in maize leaf senescence.

BACKGROUND: Maize grain yield depends mainly on the photosynthetic efficiency of functional leaves, which is controlled by an array of gene networks and other factors, including environmental conditions. MicroRNAs (miRNAs) are small RNA molecules that play important roles in plant developmental regulation. A few senescence-associated miRNAs (SA-miRNAs) have been identified as important participants in regulating leaf senescence by modulating the expression levels of their target genes. RESULTS: To elucidate miRNA roles in leaf senescence and their underlying molecular mechanisms in maize, a stay-green line, Yu87-1, and an early leaf senescence line, Early leaf senescence-1 (ELS-1), were selected as experimental materials for the differential expression of candidate miRNAs. Four small RNA libraries were constructed from ear leaves at 20 and 30 days after pollination and sequenced by Illumina deep sequencing technology. Altogether, 81 miRNAs were detected in both lines. Of these, 16 miRNAs of nine families were differentially expressed between ELS-1 andYu87-1. The phenotypic and chlorophyll content analyses of both lines identified these 16 differentially expressed miRNAs as candidate SA-miRNAs. CONCLUSIONS: In this study, 16 candidate SA-miRNAs of ELS-1 were identified through small RNA deep sequencing technology. Degradome sequencing results indicated that these candidate SA-miRNAs may regulate leaf senescence through their target genes, mainly transcription factors, and potentially control chlorophyll degradation pathways. The results highlight the regulatory roles of miRNAs during leaf senescence in maize.

Genetic dissection of the maize kernel development process via conditional QTL mapping for three developing kernel-related traits in an immortalized F2 population.

Kernel development is an important dynamic trait that determines the final grain yield in maize. To dissect the genetic basis of maize kernel development process, a conditional quantitative trait locus (QTL) analysis was conducted using an immortalized F2 (IF2) population comprising 243 single crosses at two locations over 2 years. Volume (KV) and density (KD) of dried developing kernels, together with kernel weight (KW) at different developmental stages, were used to describe dynamic changes during kernel development. Phenotypic analysis revealed that final KW and KD were determined at DAP22 and KV at DAP29. Unconditional QTL mapping for KW, KV and KD uncovered 97 QTLs at different kernel development stages, of which qKW6b, qKW7a, qKW7b, qKW10b, qKW10c, qKV10a, qKV10b and qKV7 were identified under multiple kernel developmental stages and environments. Among the 26 QTLs detected by conditional QTL mapping, conqKW7a, conqKV7a, conqKV10a, conqKD2, conqKD7 and conqKD8a were conserved between the two mapping methodologies. Furthermore, most of these QTLs were consistent with QTLs and genes for kernel development/grain filling reported in previous studies. These QTLs probably contain major genes associated with the kernel development process, and can be used to improve grain yield and quality through marker-assisted selection.

Comparison of five models for end-stage liver disease in predicting the survival rate of patients with advanced hepatocellular carcinoma.

Prognosis of patients with advanced hepatocellular carcinoma (HCC) is under expectation. Life expectancy more than 3 months is one inclusion criteria for molecular targeted drugs in clinical trials. The main purpose of this research is to compare Model for End-Stage Liver Disease (MELD) and four MELD-based prognostic models in predicting the survival rate of advanced HCC patients. One hundred eighty-three patients with advanced HCC who were not amendable to standard anti-tumor therapy were retrospectively analyzed. Data were collected to classify patients according to MELD, Model for End-Stage Liver Disease with the incorporation of serum sodium (MELD-NA), Model for End-Stage Liver Disease to ascites and sodium (MELD-AS), integrated Model for End-Stage Liver Disease (iMELD), and Model for End-Stage Liver Disease to sodium (MESO) scores at diagnosis. 1-, 3-, and 6-month survivals were the end points used in the analysis. When predicting 1-month survival, MELD-AS, MELD, and MESO were the top 3 ranking staging systems. When predicting 3-month survival, area under the receiver operating characteristic curve (AUC) of MELD-AS is significantly higher than that of the other models (P < 0.05). When predicting 6-month survival, AUCs of MELD-AS and MELD-NA are significantly higher than those of the other models (P < 0.05). Cutoff point of MELD-AS is 23.11 with 40.5 % sensitivity and 93.8 % specificity at 1 month, 9.5 with 76.9 % sensitivity and 59.5 % specificity at 3 months, and 18.5 with 27.0 % sensitivity and 89.1 % specificity at 6 months. MELD-based scores of death group are significantly higher than those of survivors within 1 and 3 months (P < 0.001). Independent prognostic factors identified by multivariate analysis included persistent ascites, serum sodium, and thrombosis. MELD-AS is the best model in the prediction of short and intermediate survival among the five models for end-stage liver disease analyzed for Chinese advanced HCC patients.

Alanine aminotransferase to hemoglobin ratio is an indicator for disease progression for hepatocellular carcinoma patients receiving transcatheter arterial chemoembolization.

The prognosis of hepatocellular carcinoma (HCC) patients receiving transcatheter arterial chemoembolization (TACE) is far from being identified. The present study aimed to assess the role of blood cell counts, routine liver function tests, and alanine aminotransferase to hemoglobin ratio (AHR) in predicting the progression-free survival (PFS) of these patients. A total of 243 HCC patients receiving TACE were analyzed retrospectively. Cancer of the Liver Italian Program (CLIP) score system was indentified to be the best score system for this patient subgroup according to the Akaike information criterion (AIC) index and linear trend χ (2). Then, prognostic value of parameters was determined by integration into the CLIP score system. As a result, AHR was confirmed to be an independent predictor for the PFS of HCC patients receiving TACE (p = 0.001) with the other parameters failing to reach statistical significance. Moreover, AHR improved the performance of CLIP by adjusting into it, thus improving its discriminatory ability. AHR defined ≤0.4583 as low level and >0.4583 as high level. And, patients were also dichotomized into two groups accordingly. HCC patients receiving TACE with low AHR presented higher 1 year DCR (41.9 vs 18.1 %) compared with patients with high AHR levels. Furthermore, AHR level was associated with prognostic factors such as lower ALP, total bilirubin, and portal vein thrombosis. In summary, the present study firstly indentified AHR as an independent prognostic factor in HCC patients receiving TACE. The subgroup of HCC patients with lower AHR presented preferable disease control and were the idealistic candidates for TACE.

Platelet-to-lymphocyte ratio acts as a prognostic factor for patients with advanced hepatocellular carcinoma.

The platelet count, as an inflammation marker, is involved in the progress of tumor invasion. However, the prognostic value of platelet counts and the platelet-to-lymphocyte ratio (PLR) has not been investigated in patients with advanced hepatocellular carcinoma (HCC). This study aimed to determine the prognostic value of platelet counts and PLR in HCC patients. A total of 243 ethnic Chinese advanced HCC patients from two major hospitals, not receiving systemic sorafenib, were analyzed retrospectively. The prognostic value of differential blood cell counts and PLR for overall survival (OS) was determined by integrating the Cancer of the Liver Italian Program (CLIP) score system and model for end-stage liver disease by using a stepwise model of multivariate Cox regression. The Kaplan-Meier method and receiver operating characteristic (ROC) curves were utilized accordingly. PLR was confirmed to be an independent predictor for OS (p < 0.01), while the remaining parameters had no predictive value. Then, advanced HCC patients were dichotomized into two groups based on the PLR value (≤111.23 or >111.23), according to ROC analysis. Patients with a high PLR had a lower 3-month survival rate (37.6 vs. 57.6%) compared with patients with a low PLR. PLR was associated with aggressive malignant behavior, characterized by distant metastasis and portal vein thrombosis. Additionally, PLR was not associated with the CLIP score and Child-Pugh grade. PLR was identified as an independent prognostic factor for advanced HCC patients not receiving systemic sorafenib; the predictive ability of PLR partially relies on its association with the aggressive nature of HCC.

Autophagic LC3B overexpression correlates with malignant progression and predicts a poor prognosis in hepatocellular carcinoma.

Autophagy is a process that involves lysosomal degradations of cellular organelles and closely related to tumor occurrence and progression. However, its importance in hepatocellular carcinoma (HCC) was still controversial. Therefore, this study is aimed to address the clinicopathologic effect of microtubule-associated protein 1 light chain 3B (LC3B) and Beclin-1, as autophagic markers, in HCC patients. Tissue microarray-based immunohistochemistry was used to examine the expression of LC3B and another autophagy key regulator (Beclin-1) in 156 operable HCC patients. Kaplan-Meier analysis, chi-square test, and Spearman's correlation analysis were used to analyze correlation of LC3B and Beclin-1 and their influence on clinical characteristics and prognosis. We found that the expression level of LC3B was significantly associated with vascular invasion (P = 0.008), lymph node metastasis (P < 0.001), and Beclin-1 expression level (P < 0.001). However, LC3B was not related to other clinicopathological features, including hepatitis B virus infection, liver cirrhosis, tumor number, tumor size, pathology grade, and tumor-node-metastasis (TNM) stage. Besides, correlation between the expression of Beclin-1 and clinicopathological features were not identified. Survival analysis showed that patients with high LC3B expression had a poorer 5-year overall survival (OS) rate than those with low LC3B expression (high vs. low: 79.5 % vs. 20.5 %, P = 0.026). And high LC3B expression tended to be related with shorter progression-free survival (PFS) (P = 0.074), whereas the expression level of Beclin-1 did not show statistically significant association with OS or PFS. Further multivariate analysis revealed that lymph node metastasis (P = 0.047) and LC3B expression level (P = 0.047) were independent factors to predict the prognosis of OS in all patients. Our study demonstrated that high expression of LC3B, correlated with vascular invasion and lymph node metastasis, might be a novel prognostic biomarker and would be a potential therapy target for HCC, especially in operable patients.

Neutrophil-to-lymphocyte ratio acts as a prognostic factor for patients with advanced hepatocellular carcinoma.

Few studies investigated the prognosis of patients with advanced hepatocellular carcinoma (aHCC). This study was aimed to determine the prognostic value of differential blood cell counts including blood white cells, neutrophil, lymphocyte, neutrophil-lymphocyte ratio (NLR), and platelet in patients with aHCC. A total of 205 ethnic Chinese aHCC patients receiving non-systematic sorafenib were analyzed retrospectively. The prognostic value of differential blood cell counts and NLR for overall survival (OS) was determined by integration into Cancer of the Liver Italian Program (CLIP) score system using backward elimination model of multivariate Cox regression. As a result, NLR was confirmed to be an independent predictor for OS (p = 0.001) with the rest parameters presented negative results. Then, aHCC patients were dichotomized into two groups according to NLR values ≤ 2.43 or >2.43. Patients with low NLR presented lower CLIP score and higher 6-month survival rate (56.1 vs 25.9%) compared with patients with high NLR level. Besides, low NLR level was associated with favorable prognostic factors such as lower α-fetoprotein, alkaline phosphatase, and total bilirubin, as well as decreased incidence of ascites, portal vein thrombosis, and metastasis. Besides, low NLR level was associated less white cells and neutrophil granulocytes, as well as more lymphocyte. In summary, the present study firstly indentified NLR as an independent prognostic factor in aHCC patients receiving no systematic sorafenib.

Prognosis value of mitotic kinase Aurora-A for primary duodenal adenocarcinoma.

Others and we have demonstrated that hypoxia-inducible factor 1α (HIF-1α) and transcriptionally upregulated Aurora-A are required for disease progression in several tumors. We investigated the clinicopathological value of HIF-1α and Aurora-A in primary duodenal adenocarcinoma (PDA). Using immunohistochemistry, we evaluated Aurora-A and HIF-1α expression semiquantitatively in 140 PDA cases. There were 76 cases from one institute that formed the training set; 64 cases from another two institutes were used as the testing set to validate the prognostic value of Aurora-A and HIF-1α expression. Aurora-A expression was high or sufficient in the tumor zone, whereas expression was low in the adjacent normal epithelia. High Aurora-A expression, identified using the training set receiver operator characteristic (ROC) analysis-generated cutoff score, predicted poorer overall survival both in the testing set (18.0 vs. 45.1 %, P = 0.001) and training set (23.1 vs. 53.9 %, P = 0.011). Multivariate Cox regression confirmed that Aurora-A was an independent prognostic factor. Contrary to previous studies, we did not detect any correlation between Aurora-A and HIF-1α. Survival analysis showed that HIF-1α level was not correlated with patient outcome (P = 0.466). Activation of Aurora-A, an independent negative prognostic biomarker, might be used to identify particular PDA patients for more selective therapy.

Entecavir vs lamivudine for prevention of hepatitis B virus reactivation among patients with untreated diffuse large B-cell lymphoma receiving R-CHOP chemotherapy: a randomized clinical trial.

IMPORTANCE: Hepatitis B virus (HBV) reactivation is a serious complication for patients with lymphoma treated with rituximab-containing chemotherapies, despite lamivudine prophylaxis treatment. An optimal prophylactic antiviral protocol has not been determined. OBJECTIVE: To compare the efficacy of entecavir and lamivudine in preventing HBV reactivation in patients seropositive for the hepatitis B surface antigen with untreated diffuse large B-cell lymphoma receiving chemotherapy treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). DESIGN, SETTING, AND PATIENTS: Randomized, open-label, phase 3 study conducted from February 2008 through December 2012 at 10 medical centers in China. This study was a substudy of a parent study designed to compare a 3-week with a 2-week R-CHOP chemotherapy regimen for untreated diffuse large B-cell lymphoma. Patients enrolled in the parent study who were seropositive for the hepatitis B surface antigen and had normal liver function, serum HBV DNA levels of less than 103 copies/mL, and no prior antiviral therapy were randomized to entecavir (n = 61) or lamivudine (n = 60). INTERVENTIONS: Daily entecavir (0.5 mg) or lamivudine (100 mg) beginning 1 week before the initiation of R-CHOP treatment to 6 months after completion of chemotherapy. MAIN OUTCOMES AND MEASURES: The primary efficacy end point was the incidence of HBV-related hepatitis. The secondary end points included rates of HBV reactivation, chemotherapy disruption due to hepatitis, and treatment-related adverse events. RESULTS: There were 121 patients randomly assigned to receive entecavir (n = 61) or lamivudine (n = 60). The date of last patient follow-up was May 25, 2013. The rates were significantly lower for the entecavir group vs the lamivudine group for HBV-related hepatitis (0% vs 13.3%, respectively; difference between groups, 13.3% [95% CI, 4.7% to 21.9%]; P = .003), HBV reactivation (6.6% vs 30%; difference, 23.4% [95% CI, 10.2% to 36.6%]; P = .001), and chemotherapy disruption (1.6% vs 18.3%; difference, 16.7% [95% CI, 6.4% to 27.0%]; P = .002). Of the 61 patients in the entecavir group, 15 (24.6%) experienced treatment-related adverse events. Of 60 patients in the lamivudine group, 18 (30%) experienced treatment-related adverse events (difference between entecavir and lamivudine groups, 5.4% [95% CI, -10.5% to 21.3%]; P = .50). CONCLUSIONS AND RELEVANCE: Among patients seropositive for the hepatitis B surface antigen with diffuse large B-cell lymphoma undergoing R-CHOP chemotherapy, the addition of entecavir compared with lamivudine resulted in a lower incidence of HBV-related hepatitis and HBV reactivation. If replicated, these findings support the use of entecavir in these patients. TRIAL REGISTRATIONS: clinicaltrials.gov Identifier: NCT01793844; Chinese Clinical Trial Registry Identifier: CTR-TRC-11001687.

An optimized antiviral modification strategy for prevention of hepatitis B reactivation in patients undergoing prophylactic lamivudine and chemotherapy: a pilot study.

In patients receiving prophylactic lamivudine (LAM) and chemotherapy, hepatitis B virus (HBV) reactivation cannot be eliminated without knowing the latent causes and optimal management. In our previous study, virus breakthrough and relapse were highly suspected as potential virologic causes for HBV reactivation. Therefore, we reviewed 24 previous studies and 447 patients who underwent chemotherapy and prophylactic LAM, with an incidence of 7.2 % HBV reactivation. Virus breakthrough and relapse were seldom investigated in these studies. In addition, 72 patients that underwent prophylactic LAM and chemotherapy at our centers were also analyzed. Among them, eight patients developed virus breakthrough, with another nine developing virus relapse after discontinuation of LAM. Eight patients received antiviral modification, which included administration of adefovir for patients with virus breakthrough or resumption of LAM for patients with virus relapse and none of them developed HBV reactivation. In contrast, of the nine patients who did not receive antiviral modification, six developed HBV reactivation and two died. In conclusion, this study demonstrated that virus breakthrough and relapse were the critical causative factors of HBV reactivation in patients receiving chemotherapy and prophylactic LAM. An optimized antiviral modification strategy could effectively prevent HBV reactivation in patients with virus breakthrough or relapse.

Beclin 1 activation enhances chemosensitivity and predicts a favorable outcome for primary duodenal adenocarcinoma.

We and others had proven that hypoxia-induced autophagy was essential to regulate cancer cell destiny under anticancer therapeutic stress. Here, we addressed the clinicopathologic effect of HIF-1α and autophagic Beclin 1 in primary duodenal adenocarcinoma (PDA). HIF-1α and Beclin 1 expression level were semi-quantitatively evaluated using tissue microarrays and immunohistochemistry (IHC) staining in 141 PDA patients. Among these patients, 77 acted as training set to select HIF-1α and Beclin 1 IHC cutoff score for patient outcome, and 64 cases were used as testing set to evaluate their prognostic effect. We found that Beclin 1 was cytoplasmic overexpressed, defined by training set fixed cutoff point, in 49.6 % PDA tissue, compared to 46.8 % patients had HIF-1α high expression. In testing set, Beclin 1 overexpression predicted a superior 5-year overall survival (OS) in both univariate (P = 0.010) and multivariate (P = 0.017) analyses. However, we did not detect any correlation between HIF-1α level and patient prognosis (P = 0.989). Significantly, among Beclin 1 overexpressed patients, radical surgery plus adjuvant chemotherapy had a 23.1-month OS improvement than given radical surgery alone (59.2 vs 36.1 months; P = 0.01). For Beclin 1 lowly expressed patients, radical surgery plus adjuvant chemotherapy and given radical surgery alone had the similar OS (P = 0.283). Contrary to previous studies, we failed to detect any correlation between Beclin 1 and HIF-1α levels in PDA (correlation coefficient 0.217, P = 0.099). In conclusions, our results confirmed that Beclin 1 was a favorable prognostic biomarker for PDA, and might be used to identify particular patients for more selective therapy.

TREM2 Inhibits Tau Hyperphosphorylation and Neuronal Apoptosis via the PI3K/Akt/GSK-3ß Signaling Pathway In vivo and In vitro.

Triggering receptor expressed on myeloid cells-2 (TREM2), a cell surface receptor mainly expressed on microglia, has been shown to play a critical role in Alzheimer's disease (AD) pathogenesis and progression. Our recent results showed that overexpression of TREM2 inhibited inflammatory response in APP/PS1 mice and BV2 cells. Several studies indicated that TREM2 ameliorated tau hyperphosphorylation might be ascribed to the inhibition of neuroinflammation. However, the precise signaling pathways underlying the effect of TREM2 on tau pathology and neuronal apoptosis have not been fully elucidated. In the present study, upregulation of TREM2 significantly inhibited tau hyperphosphorylation at Ser199, Ser396, and Thr205, respectively, as well as prevented neuronal loss and apoptosis. We also found that upregulation of TREM2 alleviated behavioral deficits and improved the spatial cognitive ability of APP/PS1 mice. Further study revealed that TREM2 could activate phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, resulting in an inhibitory effect on glycogen synthase kinase-3ß (GSK-3ß), which is a major kinase responsible for tau hyperphosphorylation in AD. In line with in vivo findings, TREM2-overexpressing BV2 microglia following ß-amyloid (Aß) stimulation led to a significant increase in the phosphorylation of PI3K, Akt, and GSK-3ß, accompanied by a decrease in tau hyperphosphorylation and apoptosis in co-cultured SH-SY5Y cells. Furthermore, LY294002, a specific PI3K inhibitor, was observed to abolish the beneficial effects of TREM2 on tau hyperphosphorylation, neuronal apoptosis, and spatial cognitive impairments in vivo and in vitro. Thus, our findings indicated that TREM2 inhibits tau hyperphosphorylation and neuronal apoptosis, at least in part, by the activation of the PI3K/Akt/GSK-3ß signaling pathway. Taken together, the above results allow us to better understand how TREM2 protects against tau pathology and suggest that upregulation of TREM2 may provide new ideas and therapeutic targets for AD.

Enhancement of CAR-T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins.

BACKGROUND: Existing treatments for cholangiocarcinoma have poor efficacy. However, chimeric antigen receptor-T (CAR-T) cells are emerging as a potential therapeutic strategy. Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR-T cell infiltration and function. This study aimed to improve the function of CAR-T cells through knock down immune checkpoints and immunosuppressive molecular receptors. METHODS: We evaluated the expression of epidermal growth factor receptor (EGFR) and B7 homolog 3 protein (B7H3) antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry. Subsequently, we engineered CAR-T cells targeting EGFR and B7H3 antigens. We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR-T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR-T cells for antitumor activity both in vitro, using tumor cell lines and cholangiocarcinoma organoid models, and in vivo, using humanized mouse models. RESULTS: We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues. EGFR-CAR-T and B7H3-CAR-T cells demonstrated specific anti-tumor activity. We found an abundance of programmed cell death protein 1 (PD-1), T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and T cell immunoglobulin and ITIM domain (Tigit) on infiltrated CD8+ T cells in the cholangiocarcinoma microenvironment. We then decreased the expression of these 3 proteins on the surface of CAR-T cells, named PTG-scFV-CAR-T cells. Furthermore, we knocked-down the expression of transforming growth factor beta receptor (TGFßR), interleukin-10 receptor (IL-10R), and interleukin-6 receptor (IL-6R) of PTG-scFV-CAR-T cells. Those cells, named PTG-T16R-scFV-CAR-T cells, potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoid model. Finally, the PTG-T16R-scFv-CAR-T cells showed greater inhibitory effect on tumor growth in vivo, and were superior in prolonging the survival of mice. CONCLUSIONS: Our results revealed that PTG-T16R-scFV-CAR-T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long-term efficacy both in vitro and in vivo. This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.

Aurora-A activation, correlated with hypoxia-inducible factor-1α, promotes radiochemoresistance and predicts poor outcome for nasopharyngeal carcinoma.

Previously, we and others showed that hypoxia-inducible factor-1α (HIF-1α) and transcriptionally upregulated Aurora-A were required for disease progression in several tumors. Here, we address the clinicopathologic value of Aurora-A and HIF-1α in locally advanced nasopharyngeal carcinoma (NPC). Aurora-A and HIF-1α expression was semiquantitatively evaluated by immunohistochemistry staining in 144 cases from a randomized controlled trial. Of these patients, 69 received neoadjuvant chemotherapy plus concurrent chemoradiotherapy, and acted as the training set, and 75 cases treated with neoadjuvant chemotherapy plus radiotherapy were used as the testing set to validate the prognostic effect of Aurora-A and HIF-1α. We found that Aurora-A and HIF-1α were highly expressed in NPC, but were deficient in normal adjacent epithelia. In the testing set, Aurora-A overexpression predicted a shortened 5-year overall survival (59.1% vs 82.5%, P = 0.024), progression-free survival (44.8% vs 79.8%, P = 0.004), and distant metastasis-free survival (43.0% vs 17.3%, P = 0.016). Multivariate regression analysis confirmed that Aurora-A was indeed an independent prognostic factor for death, recurrence, and distant metastasis both in the testing set and overall patients. Moreover, a positive correlation between Aurora-A and HIF-1α was detected (P = 0.037). Importantly, although HIF-1α did not show any prognostic effect for patient outcome, the subset with Aurora-A and HIF-1α co-overexpression had the poorest overall, progression-free, and distant metastasis-free survival (all P < 0.05). Our results confirmed that Aurora-A was an independent prognostic factor for NPC. Aurora-A combined with HIF-1α refined the risk definition of the patient subset, thus potentially directing locally advanced NPC patients for more selective therapy.

Physiological and omics analysis of maize inbred lines during late grain development.

BACKGROUND: There were significant differences in the change of moisture content and grain composition at the late stage of grain development among different maize varieties, but the regulation mechanism is not clear. OBJECTIVE: To explore the key genes causing the variation in physiological traits of two typical maize inbred lines in late grain development. METHODS: The grains at different development stages were selected as materials to determine the content of water, sucrose, starch and ABA. Transcriptomic and proteomic analysis of the materials were performed to screen relevant genes. RESULTS: The grain dehydration rate and the content of sucrose, starch and ABA were showed significant differences between two varieties in the late stage of grain development. The enrichment analysis of common differentially expressed genes (proteins) showed that most of the genes (proteins) were enriched in the extracellular region. The downregulated genes were mainly concentrated in carbohydrate metabolism and lipid metabolism, while the upregulated genes were mainly in response to stress. Furthermore, this study also identified many key candidate genes (dehydrin genes, pathogenesis-related genes, sucrose synthase and secondary metabolites related genes) related to late grain development of maize. CONCLUSIONS: The suggested genes related to late grain development of maize can be candidates for further functional study.

Downregulation of TREM2 expression exacerbates neuroinflammatory responses through TLR4-mediated MAPK signaling pathway in a transgenic mouse model of Alzheimer's disease.

Activation of glial cells and neuroinflammation play an important role in the onset and development of Alzheimer's disease (AD). Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor in the brain that is involved in regulating neuroinflammation. However, the precise effects of TREM2 on neuroinflammatory responses and its underlying molecular mechanisms in AD have not been studied in detail. Here, we employed a lentiviral-mediated strategy to downregulation of TREM2 expression on microglia in the brain of APPswe/PS1dE9 (APP/PS1) transgenic mice and BV2 cells. Our results showed that downregulation of TREM2 significantly aggravated AD-related neuropathology including Aß accumulation, peri-plaque microgliosis and astrocytosis, as well as neuronal and synapse-associated proteins loss, which was accompanied by a decline in cognitive ability. The further mechanistic study revealed that downregulation of TREM2 expression initiated neuroinflammatory responses through toll-like receptor 4 (TLR4)-mediated mitogen-activated protein kinase (MAPK) signaling pathway and subsequent stimulating the production of pro-inflammatory cytokines in vivo and in vitro. Moreover, blockade of p38, JNK, and ERK1/2 inhibited the release of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) induced by Aß1-42 in TREM2-knocked down BV2 cells. Taken together, these findings indicated that TREM2 might be a potential therapeutic target for AD and other neuroinflammation-related diseases.