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Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 μg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.

Citrus exhibits unique nutritional values. Most citrus cultivars are derived from mutations. However, the effect of these mutations on fruit quality is unclear. We have previously found a yellowish bud mutant in the citrus cultivar ‘Aiyuan 38’. Therefore, this study aimed to determine the effect of the mutation on fruit quality. ‘Aiyuan 38’ (WT) and a bud mutant variant (MT) were used to analyze variations in fruit color variation and flavor substances using colorimetric instruments, high-performance liquid chromatography (HPLC), headspace solid-phase microextraction-gas chromatography–mass spectrometry (HS-SPME-GC–MS), and odor activity values (OAVs). The mutation in MT conferred yellowish characteristics to its peel. Although the differences in total sugar and acid content of the pulp were not statistically significant between WT and MT, the MT glucose content was significantly lower and the malic acid level was significantly higher. HS-SPME-GC–MS analysis revealed that the MT pulp released more types and contents of volatile organic compounds (VOCs) than the WT, whereas the opposite trend was observed for the peel. Analysis of the OAV revealed that the MT pulp contains 6 unique VOCs, whereas the peel contains only 1. This study provides a useful reference for the study of flavor substances associated with citrus bud mutations.

Protein complexes mediated by various post-translational modifications (PTMs) play important roles in almost every aspect of biological processes. PTM-mediated protein complexes often have weak and transient binding properties, which limit their unbiased profiling especially in complex biological samples. Here, we developed a plug-and-play chemical proteomic approach for high-throughput analyis of PTM-mediated protein complexes. Taking advantage of the glutathione-S-transferase (GST) tag, which is the gold standard for protein purification and has wide access to a variety of proteins of interest (POIs), a glutathione (GSH) group- and photo-cross-linking group-containing trifunctional chemical probe was developed to tag POIs and assembled onto a streptavidin-coated 96-well plate for affinity purification, photo-cross-linking, and proteomics sample preparation in a fully integrated manner. Compared with the previously developed photo-pTyr-scaffold strategy, by assembling the tyrosine phosphorylation (pTyr) binding domain through covalent NHS chemistry, the new plug-and-play strategy using a noncovalent GST-GSH interaction has comparable enrichment efficiency for EGF stimulation-dependent pTyr protein complexes. To further prove its feasibility, we additionally assembled four pTyr-binding domains in the 96-well plate and selectively identified their pTyr-dependent interacting proteins. Importantly, we systematically optimized and applied the plug-and-play approach for exploring protein methylation-mediated protein complexes, which are difficult to be characterized due to their weak binding affinity and the lack of efficient enrichment strategies. We explored a comprehensive protein methylation-mediated interaction network assembled by five protein methylation binding domains including the chromo domain of MPP8, tandem tudor domain of KDM4A, full-length CBX1, PHD domain of RAG2, and tandem tudor domain of TP53BP1 and validated the chromo domain- and tudor domain-mediated interaction with histone H3. Collectively, this plug-and-play approach provides a convenient and generic strategy for exploring PTM-dependent protein complexes for any POIs with the GST tag.

Background The main goals of treatment for advanced breast cancer were to prolong survival and improve quality of life. Paclitaxel is a commonly used chemotherapy drug for advanced breast cancer. The occurrence and development of tumors largely depend on the functional state of the immune system. NK cells and T cells are the two main cell subsets who play a key role in tumor immune surveillance and anti-tumor. Their functions mainly depend on the expression of receptors on their surface. This study mainly investigated the correlation between the expression levels of NK cell activating receptors and CD28 and PD-1 molecules on T cells in peripheral blood with prognosis in patients with advanced breast cancer undergoing paclitaxel chemotherapy. Methods Peripheral blood was detected by flow cytometry for immunophenotype and PCR technology for detection of NCR transcripts from 53 patients with advanced breast cancer. To compare the differences in immunophenotype and NCR3 transcripts among breast cancer patients with different molecular types. The correlation between immunophenotype and NCR3 splice isomer expression with tumor load, chemotherapy sensitivity to paclitaxel and survival were further analyzed. Results The ratio of CD28+/CD28− T cells and NCR3 transcript expression in peripheral blood of triple-negative breast cancer patients was higher than that of patients in other groups. Elevation of NCR3 transcripts mainly inhibits NKp30c. NKp30 on NK cells, NCR3 transcripts, the proportion of CD4+CD28− T cells and PD-1 on T cells were positively correlated with tumor burden in patients. The proportion of CD4+CD28+ T cells was negatively correlated with the tumor burden in patients. After paclitaxel chemotherapy, patients with high expression of CD28 and low expression of PD-1 on T cells before paclitaxel chemotherapy were more sensitive to chemotherapy; patients with low expression level of NKp30 inhibitory splice isomer in NK cells and high ratio of CD28+/PD-1+ T cells before paclitaxel chemotherapy had better PFS. Conclusion The expression of NCR3 in NK cells and the expression of CD28 and PD-1 molecules on T cells can be used as potential biomarkers for evaluating tumor progression, chemotherapy effect and prognosis in patients with advanced breast cancer.

Background: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase related to the lipid kinases of the phosphoinositide 3-kinase (PI3K) family, which has been confirmed to be closely related to the development of a variety of human cancers. Onatasertib (ATG-008) is a 2nd generation mTOR inhibitor which inactivates both mTORC1 and mTORC2. Our previous clinical investigation (NCT01177397) has demonstrated preliminary evidence of antitumor activity of onatasertib across multiple solid and hematologic malignancies, with encouraging signals of activity in subjects with hepatitis B virus (HBV)+ unresectable, refractory HCC. Methods: Asian patients with advanced HBV+ HCC who had experienced progressive disease after at least 1 line of systemic therapy were enrolled in this study. Onatasertib was administered orally once a day (QD) at 3 dose levels (15 mg, 30 mg and 45 mg) or 20 mg twice daily (BID). The primary endpoints were pharmacokinetics (PK), safety and efficacy (NCT03591965). Results: As of July 11, 2022, 73 patients were enrolled and evaluated. The median age was 52.0 years and the median follow up duration was 26.5 months. The mean number of prior lines was 1.8. 63 patients (86.3%) had at least one Grade 3-4 treatment emergent adverse event (TEAE), and 25 (34.2%) had at least one serious adverse event (SAE). Among the 73 subjects, 3 achieved confirmed partial response (PR), all in the 45 mg QD cohort. 18 pts were enrolled in the 45 mg QD cohort, in which 11 (61.1%) pts had received at least 2 prior lines of systemic therapy and 15 (83.3%) pts had been exposed to an anti-PD-1/PD-L1 antibody. The overall response rate (ORR) in the 45 mg QD cohort was 16.7%. The median progression-free survival (mPFS) was 3.0 months (95% CI 1.9, 4.6) in the intention to treat (ITT) population and was 5.3 months (95% CI 1.9, 7.6) in the 45mg QD cohort. Median overall survival time (mOS) was not evaluable in the 45 mg QD cohort and the mOS was 13.4 months (95% CI 7.4, 19.8) in the ITT population. Onatasertib demonstrated linear pharmacokinetics between 15 mg QD and 45 mg QD in this Asian population and there was no significant exposure accumulation after multiple dosing, as previously seen in the US population. Conclusion: Onatasertib (ATG-008) showed encouraging single agent antitumor activity in patients with HBV+ advanced HCC after at least 1 prior systemic therapy, notably in the 45 mg QD dose level, in which most patients had been previously exposed to anti-PD-L1/PD-1 therapy. The results indicates that onatasertib has potential efficacy in HBV+ HCC patients who failed prior CPI treatment. Further study may be warranted, particularly in HBV+ HCC patients who have failed prior anti-VEGFR and anti-PDL1/PD-1 therapy. Citation Format: Shukui Qin, Xiaoyan Lin, Zhiqaing Meng, Zhenggang Ren, Yuxian Bai, Shanzhi Gu, Li Zheng, Qiu Li, Sun-Yong Oh, Yabing Guo, Yoong-Koo Kang, Wei-Yu Kao, Wei Li, Jung-Hwan Yoon, Helong Zhang, Pei-Jer Chen, Tsai-Sheng Yang, Jeong Heo, Zhendong Zheng, Hui Xie, Zhinuan Yu. Result of an open-label phase 2 trial of dual TORC1/TORC2 inhibitor onatasertib (ATG-008) in HBV+ advanced hepatocellular carcinoma (HCC) subjects who have received at least one prior line of systemic therapy (TORCH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT150.

By implementing dynamic circulating tumor DNA (ctDNA) analysis, we explored the impact of TP53 mutations on tumor evolution and resistance mechanisms to ensartinib in patients with ALK-positive NSCLC.In a multicenter phase 2 trial, patients with ALK-positive NSCLC who progressed on crizotinib were treated with ensartinib. Blood samples for ctDNA analysis were collected at baseline, cycle 3 day 1, and progression disease (PD) and analyzed with a 212-gene panel.A total of 440 samples were collected from 168 patients. Baseline TP53 mutations (20.2%) significantly correlated with inferior progression-free survival (4.2 mo versus 11.7 mo, p0.0001). Patients with TP53 mutations had higher mutation load than those without TP53 mutations at baseline (13.79 ± 3.72 versus 4.67 ± 0.39, p0.001). Although there was no significant difference in mutation load between these groups at cycle 3 day 1 (5.89 ± 2.25 versus 3.72 ± 0.62, p = 0.425), patients with mutated TP53 developed more mutations at PD (7.07 ± 1.25 versus 3.20 ± 0.33, p = 0.003). Frequency and abundance of secondary ALK mutations G1269A, G1202R, and E1210K increased markedly at PD than baseline. In patients without secondary ALK mutations, we identified ALK-independent resistance mechanisms including bypass signaling activation, downstream effector protein reactivation, epithelial-mesenchymal transformation, and epigenetic dysregulation.Our study highlighted the advantage of ctDNA analysis for monitoring tumor evolution. TP53 mutations promoted genetic evolution and accelerated occurrence of resistance. We also unveiled ALK-dependent resistance mechanisms, mainly by G1269A, G1202R, and E1210K mutations, and ALK-independent resistance mechanisms to ensartinib.

Disulfiram (DSF) is an anti-alcoholism medication with superior antitumor activity and clinical safety; its antitumor mechanisms in gastric cancer (GC) have not been fully explored. In the present work, low nontoxic concentrations of copper (Cu) ions substantially enhanced DSF’s antitumor activity, inhibiting the proliferation and growth of GC cell lines. DSF/Cu elevated the generation of reactive oxygen species (ROS), and apoptosis was induced in an ROS-dependent manner. This process might involve primary inhibition GC by DSF/Cu through induction of apoptosis via the ROS/mitogen-activated protein kinase pathway. Disordering transportation of ubiquitinated protein may also fuel the process. In summary, we found that DSF exerts antitumor effects on GC. DSF/Cu should be considered as adjunctive therapy for GC.

Increasing evidence suggests that many adipokines are involved in cancer-related anorexia and cachexia syndrome (CACS), although the underlying mechanism remains to be clarify. Asprosin is a new peptide hormone mainly secreted by white adipose tissues that can increase appetite and body weight. In this cross-sectional study, we tested whether asprosin may intervene in the development of CACS. The fasting plasma asprosin levels were determined via enzyme-linked immune-sorbent assay. Anorexia was determined using the anorexia/cachexia subscale (A/CS) of the functional assessment of anorexia/cachexia therapy (FAACT) questionnaire. The body composition was assessed using bioelectrical impedance analysis. The association of plasma asprosin with anorexia, cachexia, and nutritional status was analyzed. One hundred twenty treatment-naive patients with pathological confirmed gastrointestinal or lung cancer and 14 mild gastritis patients were recruited. We found no significant difference in asprosin levels between subgroups of patients by age, sex, cancer types or stage. Correlation analysis suggested that asprosin levels were positively associated with body fat mass (r = 0.248, p = 0.043). No correlations were found between asprosin levels and hemoglobin, white blood cell count, blood platelet count, albumin, C-reactive protein, glucose, cholesterol, triglyceride, high density lipoprotein, low density lipoprotein, body mass index, body fat percentage, protein, skeletal muscle, muscle mass, lean body mass, and basal metabolic rate. Furthermore, asprosin levels were not significantly different between patients with or without cachexia. However, patients with anorexia had significantly lower asprosin levels compared with patients without anorexia. No significant difference in asprosin levels between gastritis and gastric cancer patients. Similarly, no significant change of asprosin levels occurred postoperatively in 10 gastric cancer patients. Patients with anorexia had significantly lower asprosin levels compared with patients without anorexia. We therefore speculated that asprosin might intervene in the development of cancer anorexia and serve as a potential therapeutic target.

Summary Background Ensartinib is a potent new-generation ALK inhibitor with high activity against a broad range of known crizotinib-resistant ALK mutations and CNS metastases. We aimed to assess the efficacy and safety of ensartinib in ALK-positive patients with non-small-cell lung cancer (NSCLC), in whom crizotinib therapy was unsuccessful. The associations between ensartinib efficacy and crizotinib-resistant mutations were also explored. Methods We did a single-arm, open-label, phase 2 study at 27 centres in China. Patients were aged 18 years or older, had stage IIIb or stage IV ALK-positive NSCLC that had progressed while they were on crizotinib therapy, an Eastern Cooperative Oncology Group performance status of 2 or less, had measurable disease, and had received fewer than three previous treatments. Patients with CNS metastases were included if these metastases were asymptomatic and did not require steroid therapy. All patients received 225 mg ensartinib orally once daily on a continuous dosing schedule. The primary outcome was the proportion of patients with an objective response according to the Response Evaluation Criteria in Solid Tumors (version 1.1), as assessed by an independent review committee in all patients who received at least one dose of ensartinib with no major violations of the inclusion criteria (ie, the full analysis set). Safety was assessed in all enrolled patients who received at least one dose of ensartinib. This trial was registered with ClinicalTrials.gov , NCT03215693 . Findings Between Sept 28, 2017, and April 11, 2018, 160 patients were enrolled and had at least one dose of ensartinib (safety analysis set). Four patients had inclusion violations and were excluded from the efficacy analysis, which thus included 156 patients (full analysis set). 97 (62%) patients in the full analysis set had brain metastases. 76 (52% [95% CI 43–60]) of 147 patients in the full analysis set, with responses that could be assessed by the independent review committee, had an objective response. 28 (70% [53–83]) of 40 patients with measurable brain metastases as assessed by the independent review committee had an intracranial objective response. 145 (91%) of 160 patients had at least one treatment-related adverse event, which were mostly grade 1 or 2. The most common treatment-related adverse events were rash (89 [56%]), increased alanine aminotransferase concentrations (74 [46%]), and increased aspartate aminotransferase concentrations (65 [41%]). Interpretation Ensartinib has activity and is well tolerated in patients with crizotinib-refractory, ALK-positive NSCLC, including those with brain metastases. The role of ensartinib in patients in whom other second-generation ALK inhibitors have been unsuccessful warrants further studies. Funding Betta Pharmaceuticals.

Background: N -methyladenosine (m A) methylation is a common class of RNA modification. Similar to DNA methylation, m 6 A methylation regulates most mRNA expressions. At present, most research has found that m 6 A methylation is related to tumorigenesis and development; however, there are few studies about hepatocellular carcinoma (HCC). This study aimed to analyze the expression level of m 6 A methylation regulators and their correlation on the clinical features in HCC. Methods: A total of 13 m 6 A methylation regulators were evaluated. mRNA data and clinical information were obtained from the Cancer Genome Atlas (TCGA). The Wilcoxon test was utilized to analyze the differences between m 6 A RNA methylation regulators, and Pearson’s test was used to test the correlation between them. We constructed a tumor subgroup model based on the 13 molecules used for the analysis of the correlations with the clinical features. Two genes ( ZC3H13 and YTHDF2 ) screened by Cox and LASSO regression were used to construct a tumor risk model for analyzing the correlations with clinical features. Finally, we verified the expression of the two molecules in liver cancer and adjacent tissues by Western blot and real-time polymerase chain reaction (PCR) (n=6). P Results: Eleven of the 13 molecules were higher in the liver cancer tissues than the adjacent tissues (P vs . 77%) with significant differences (P vs . 31%, 5-year survival rate: 12% vs . 17%). The Western blot test showed that the expression of YTHDF2 in the liver cancer tissues was greater than that in the precancerous tissues (P ZC3H13 was not significant. Real-time PCR showed that the expression of YTHDF2 mRNA in liver cancer tissues were higher than that in adjacent tissues (7.64±0.44 vs . 4.99±0.61, P=0.006), while the expression of ZC3H13 mRNA had no statistical difference (5.56±0.18 vs . 5.42±0.33, P>0.05). The results of the in vitro experiment were consistent with bioinformatic analysis. Conclusions: The abnormal expression of m 6 A methylation regulators in the liver tissues suggest that m 6 A may play an important role in the development of HCC. Tumor models we constructed that could effectively predict the prognosis of patients, and the clinical correlation results were consistent with clinical practices. Our research is expected to provide a reference for the prognostic stratification and treatment strategy development of HCC.

MicroRNAs (miRNAs) are involved in tumorigenesis, progression, recurrence, and drug resistance of hepatocellular carcinoma (HCC). However, few miRNAs have been identified and entered clinical practice. Herein, we report that microRNA (miR)-552 is upregulated in HCC tissues and has an important function in liver tumor-initiating cells (T-ICs). Functional studies revealed that a forced expression of miR-552 promotes liver T-IC self-renewal and tumorigenesis. Conversely, miR-552 knockdown inhibits liver T-IC self-renewal and tumorigenesis. Mechanistically, miR-552 downregulates phosphatase and tensin homolog (PTEN) via its mRNA 3′ UTR and activates protein kinase B (AKT) phosphorylation. Our clinical investigations elucidated the prognostic value of miR-552 in HCC patients. Furthermore, miR-552 expression determines the responses of hepatoma cells to sorafenib treatment. The analysis of patient cohorts and patient-derived xenografts (PDXs) further demonstrated that miR-552 may predict sorafenib benefits in HCC patients. In conclusion, our findings revealed the crucial role of the miR-552 in liver T-IC expansion and sorafenib response, rendering miR-552 an optimal target for the prevention and intervention in HCC.

Background The epidemiological characteristics of combined small cell lung cancer (C-SCLC) are still unclear. Therefore, in this study, we aimed to investigate the prognostic factors and treatment modalities for C-SCLC. Methods The clinical data from patients with primary C-SCLC from 2004 to 2015 were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. Next, we applied the 8th edition of the TNM classification. A Cox regression model was used to identify the relevant variables and treatment modalities which affected the overall survival (OS) and cancer-specific survival (CSS). Results In total, 1,010 patients were included in the analysis. The median OS and median CSS were 9 months and 10 months, respectively. Individuals aged 65 and younger, female, and classified as being in an earlier stage were independent predictors of an improved OS and CSS. For patients with stage I, surgery alone significantly improved the OS (HR, 0.371; 95% CI, 0.180–0.769) and CSS (HR, 0.367; 95% CI, 0.162–0.892). For patients with stage II, surgery combined with radiotherapy significantly improved the OS (HR, 0.237; 95% CI, 0.063–0.890). For patients with stage III, radiotherapy alone was an independent predictor of OS (HR, 0.464; 95% CI, 0.316–0.618) and CSS (HR, 0.452; 95% CI, 0.305–0.670). For patients with stage IV, surgery combined with radiotherapy significantly improved OS (HR, 0.315; 95% CI, 0.181–0.547) and CSS (HR, 0.329; 95% CI, 0.189–0.573), and surgery alone also improved OS (HR, 0.257; 95% CI, 0.144–0.461) and CSS (HR, 0.280; 95% CI, 0.156–0.501). Undergoing chemotherapy was also an independent predictor of an improved OS (HR, 0.619; 95% CI, 0.419–0.915) in patients with stage III, and OS (HR, 0.283; 95% CI, 0.228–0.350) and CSS (HR, 0.289; 95% CI, 0.232–0.259) of patients with stage IV. Conclusions These findings may help to determine possible treatment choices and aid further research on this rare disease.

Disulfiram (DSF) is an approved drug for the treatment of alcohol dependence. Accumulating evidence indicates that DSF, alone or in combination with copper (Cu), possesses strong antitumor activity in various malignancies. This study investigated the effects of DSF on gastric cancer (GC) and the potential mechanisms involved.GC cell proliferation and apoptosis upon treatment with DSF with or without copper were analyzed using CCK-8 assay, colony formation assay, and flow cytometry. Glucose metabolism was investigated using glucose consumption and lactate production assays. The expression of caspase-3, Bcl-2, LC-3, P62, S6K1, c-Myc, GLUT1, PKM2, and LDHA was analyzed using western blot assay. In vivo nude mice studies were performed to verify the findings from in vitro analyses.Our study showed that DSF was highly toxic to GC cells in a Cu-dependent manner. Nontoxic concentrations of Cu enhanced the inhibitory effects of DSF on cell viability and colony formation. DSF also induced apoptotic and autophagic cell death in the presence of Cu. In addition, DSF/Cu inhibited glycolysis and xenograft growth of GC cells by suppressing the expression of S6K1, c-Myc, and their downstream molecules, including GLUT1, PKM2, and LDHA.Our study demonstrated that DSF/Cu exerted antitumor activity against GC cells both in vitro and in vivo. DSF/Cu may represent a promising therapeutic strategy for the treatment of GC.

BACKGROUND: Methylation is one of the common forms of RNA modification, which mainly include N6-methyladenosine (m6A), C5-methylcytidine (m5C), and N1-methyladenosine (m1A). Numerous studies have shown that RNA methylation is associated with tumor development. We aim to construct prognostic models of gastric cancer based on RNA methylation regulators. METHODS: The transcriptome and clinical data of gastric cancer and normal samples were obtained from the National Cancer Institute Genome Data Commons (NCI-GDC). Use Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression analysis to construct risk models for different types of RNA methylation. Receiver operating characteristic (ROC) curves were generated to evaluate the predictive efficiency of risk characteristics. Cluster heat maps are used to assess the correlation with clinical information. Univariate and multivariate Cox analyses were used to analyze prognostic effects of risk scores. Gene Set Enrichment Analysis (GSEA) analyzes the functional enrichment of RNA methylation genes. And make a separate analysis of the data of Asians. RESULTS: The expression of most of the 30 RNA methylation regulators were significantly different in cancer and paracancerous tissues (P

PURPOSE Donafenib, a novel multikinase inhibitor and a deuterated sorafenib derivative, has shown efficacy in phase Ia and Ib hepatocellular carcinoma (HCC) studies. This study compared the efficacy and safety of donafenib versus sorafenib as first-line therapy for advanced HCC. PATIENTS AND METHODS This open-label, randomized, parallel-controlled, multicenter phase II-III trial enrolled patients with unresectable or metastatic HCC, a Child-Pugh score ≤ 7, and no prior systemic therapy from 37 sites across China. Patients were randomly assigned (1:1) to receive oral donafenib (0.2 g) or sorafenib (0.4 g) twice daily until intolerable toxicity or disease progression. The primary end point was overall survival (OS), tested for noninferiority and superiority. Efficacy was primarily assessed in the full analysis set (FAS), and safety was assessed in all treated patients. RESULTS Between March 21, 2016, and April 16, 2018, 668 patients (intention-to-treat) were randomly assigned to donafenib and sorafenib treatment arms; the FAS included 328 and 331 patients, respectively. Median OS was significantly longer with donafenib than sorafenib treatment (FAS; 12.1 v 10.3 months; hazard ratio, 0.831; 95% CI, 0.699 to 0.988; P = .0245); donafenib also exhibited superior OS outcomes versus sorafenib in the intention-to-treat population. The median progression-free survival was 3.7 v 3.6 months ( P = .0570). The objective response rate was 4.6% v 2.7% ( P = .2448), and the disease control rate was 30.8% v 28.7% (FAS; P = .5532). Drug-related grade ≥ 3 adverse events occurred in significantly fewer patients receiving donafenib than sorafenib (125 [38%] v 165 [50%]; P = .0018). CONCLUSION Donafenib showed superiority over sorafenib in improving OS and has favorable safety and tolerability in Chinese patients with advanced HCC, showing promise as a potential first-line monotherapy for these patients.

Low-abundance phosphotyrosine (pTyr)-mediated signaling protein complexes play critical roles in cancer signaling. The precise and comprehensive profiling of these pTyr-mediated protein complexes remains challenging because of their dynamic nature and weak binding affinity. Taking advantage of the SH2 domains modified with trifunctional chemical probes and genetic mutations (termed Photo-pTyr-scaffold), we developed a Photo-pTyr-scaffold-based forward-phase protein array that can be used to specifically capture complexes by developing an engineered SH2 domain, photoaffinity cross-linking, and antibody-based measuring weak pTyr-mediated protein complexes from complex biological samples in a 96-well microplate format. This platform demonstrated good precision for quantitation (R2 = 0.99) and high sensitivity by which only 5 μg of whole cell lysates is needed. We successfully applied the technology for profiling the dynamic EGF-stimulation-dependent EGFR signaling protein complexes across four different time courses (i.e., 0, 2, 5, 10, and 30 min) in a high-throughput manner. We further evaluated the modulation of EGFR-GRB2-SHC1 protein complexes by FDA-approved EGFR kinase inhibitor erlotinib, demonstrating the feasibility of this approach for high-throughput drug screening. The Photo-pTyr-scaffold-based forward-phase protein array could be generically applicable for exploring the dynamic pTyr signaling complexes in various biological systems and screening for related drugs in a high-throughput manner.

e16197 Background: Donafenib (Dona), a multikinase inhibitor recently marketed in China for advanced HCC with its superiority in overall survival (OS) over Sorafenib from a large head-to-head Phase 3 study. The clinical experience of triple therapy (TKIs + ICIs + TACE) in the treatment of patients (pts) with uHCC is limited. We assessed the safety and efficacy of triple therapy [Dona +anti-PD-1+ TACE] in uHCC in this Phase 1 study. Methods: The objective of this Phase 1, open-label, multicenter, dose-escalation and expansion study was to assess the MTD/recommended dose for Dona in combination of a fixed-dose anti-PD-1 plus TACE in uHCC. The primary endpoint was DLT occurring in the first treatment cycle. Secondary endpoints included the overall safety and tolerability, ORR and PFS by mRECIST. Qualified pts (age 18-75) were enrolled if uHCC without macrovascular invasion (VP3/4) or extrahepatic spread; measurable disease by mRECIST; ECOG 0 to 1; Child-Pugh A; systemic therapy naive. Results: At the cut-off date (Dec 31 2021), data of 25 pts who underwent triple therapy from 6 cancer centers in China were analyzed (Table). The median duration of follow-up was 105 days (1-358 days). No DLTs were observed [Dona 50 mg Bid (n=3); 150 mg Qd (n=3); 100 mg Bid (n=6)]. Additional 13 pts were treated with the recommended dose of Dona 100 mg Bid. Treatment-related adverse events (TRAE) occurred in 24 pts (Grade 3, 36%; no Grade 4). 16 pts had at least one response assessment, the ORR was 62.5%; in 12 pts with BLCL B, the ORR was 83.3%. mPFS was not reached. Conclusions: Combination of Dona, anti-PD-1 and TACE showed a high rate of tumor response and good safety profile in uHCC. Clinical trial information: NCT04605185. [Table: see text]

Phosphotyrosine (pTyr) signaling complexes are important resources of biomarkers and drug targets which often need to be profiled with enough throughput. Current profiling approaches are not feasible to meet this need due to either biased profiling by antibody-based detection or low throughput by traditional affinity purification-mass spectrometry approach (AP-MS), as exemplified by our previously developed photo-pTyr-scaffold approach. To address these limitations, we developed a 96-well microplate-based sample preparation and fast data independent proteomic analysis workflow. By assembling the photo-pTyr-scaffold probe into a 96-well microplate, we achieved steric hindrance-free photoaffinity capture of pTyr signaling complexes, selective enrichment under denaturing conditions, and efficient in-well digestion in a fully integrated manner. EGFR signaling complex proteins could be efficiently captured and identified by using 300 times less cell lysate and 100 times less photo-pTyr-scaffold probe as compared with our previous approach operated in an Eppendorf tube. Furthermore, the lifetime of the photo-pTyr-scaffold probe in a 96-well microplate was significantly extended from 1 week up to 1 month. More importantly, by combining with high-flow nano LC separation and data independent acquisition on the Q Exactive HF-X mass spectrometer, LC-MS time could be significantly reduced to only 35 min per sample without increasing sample loading amount and compromising identification and quantification performance. This new high-throughput proteomic approach allowed us to rapidly and reproducibly profile dynamic pTyr signaling complexes with EGF stimulation at five time points and EGFR inhibitor treatment at five different concentrations. We are therefore optimized for its generic application in biomarkers discovery and drug screening in a high-throughput fashion.

Since the first discovery of old yellow enzyme 1 (OYE1) from Saccharomyces pastorianus in 1932, biocatalytic asymmetric reduction of activated alkenes by OYEs has become a valuable reaction in organic synthesis. To access stereocomplementary C=C-bond bioreduction, the mining of novel OYEs and especially the protein engineering of existing OYEs have been performed, which successfully achieved the stereocomplementary reduction in several cases and further raise the potential of applications. In this review, we analyzed the structures, active sites, and substrate recognition of OYEs, which are the bases for their substrate specificity and stereospecificity. Sequence similarity network of OYEs superfamily was also constructed to investigate the scope of characterized OYEs. The structure-guided engineering to switch the stereoselectivity of OYEs and thus access stereocomplementary bioreduction over the last decade (2009-2020) was then reviewed and discussed, which might give new insights into the mining and engineering of related biocatalysts. KEY POINTS: • The sequence similarity network of OYEs superfamily was constructed and annotated. • The structures and active sites of OYEs from different classes were compared. • "Left/right" binding mode was used to explain the stereopreferences of OYEs. • Structure-guided engineering of OYEs to switch their stereoselectivity was reviewed.

Fruquintinib is an orally active kinase inhibitor that selectively targets the vascular endothelial growth factor (VEGF) receptor. A Phase II trial has demonstrated a significant benefit in progression-free survival (PFS) for fruquintinib-treated patients with locally advanced/metastatic nonsquamous non-small-cell lung cancer (NSCLC) who have progressed after second-line chemotherapy. This Phase III trial is a randomized, double-blind, multicenter trial to confirm fruquintinib's efficacy in the same patient population.From December 2015 to February 2018, 730 patients were screened, of whom 527 were enrolled into the study. Participants were randomized 2:1 to receive fruquintinib (n = 354) or placebo (n = 173) once daily for 3 weeks on-treatment, and 1 week off-treatment. Patients were stratified according to epidermal growth factor receptor mutation status and prior use of VEGF inhibitors. Primary endpoint was overall survival (OS).Median OS was 8.9 months for the fruquintinib group and 10.4 months for placebo group (hazard ratio [HR] 1.02; 95 % confidence interval [CI], 0.82-1.28; P = 0.841), with median PFS of 3.7 months and 1.0 months, respectively (HR 0.34; 95 % CI, 0.28-0.43; P 0.001). Objective response rate and disease control rate were 13.8 % and 66.7 % for fruquintinib, and 0.6 % and 24.9 % for placebo, respectively (P 0.001). Hypertension was the most frequent treatment-emergent adverse event (≥grade 3) observed in fruquintinib-treated patients (21.0 %). Post hoc analysis revealed that fruquintinib prolonged the median OS for patients who did not receive subsequent antitumor therapy: 7.0 months versus 5.1 months for placebo (HR 0.65; 95 % CI, 0.46-0.91; P = 0.012). Patients receiving fruquintinib also reported improvements in quality of life for most functional scales measured by EORTC QLQ-C30 and LC13 questionnaires.Although the study did not meet its primary endpoint, fruquintinib could be effective in combination with other agents for the treatment of patients with NSCLC who have failed second-line chemotherapy.

BACKGROUND Before tyrosine kinase inhibitor (TKI) therapy can be administered in patients with advanced non-small cell lung cancer (NSCLC), EGFR mutation testing is required. However, few studies have evaluated the extent of EGFR testing in real-world practice in China. METHODS A multicenter, observational study of EGFR testing in NSCLC patients in North China was conducted. Treatment-naive patients or those with postoperative recurrent stage IIIB/IV NSCLC were enrolled. The primary objective was EGFR testing rate. Secondary objectives included EGFR mutation status, EGFR testing methods and specimens, factors associated with EGFR testing, and overall survival with or without EGFR testing. RESULTS Overall, 2809 patients with stage IIIB/IV NSCLC were enrolled; 90.78% had adenocarcinoma. The EGFR screening rate was 42.54%. EGFR testing rates were higher in tumor samples obtained by lymph node puncture, and in patients with urban medical insurance, adenocarcinoma, non-smokers, or those located in developed cities (all P < 0.001). The EGFR mutation rate was 46.44%. The most commonly used specimens for EGFR testing were biopsy tumor samples (67.53%). PCR-based methods (72.05%), Sanger sequencing (5.36%), and Luminex liquid chip (5.10%) were the most frequently used testing platforms. Similar positive EGFR mutation rates were achieved with different platforms. TKI therapy was the first-line treatment administered to most EGFR-positive patients (56.22%), and chemotherapy in EGFR-negative patients (84.88%). Overall survival was higher in EGFR-tested than in untested patients (27.50 vs. 19.73 months; P = 0.007). CONCLUSION Real-world EGFR testing rates for NSCLC in North China were relatively low because of clinical and social factors, including medical insurance coverage.

Embryonal rhabdomyosarcoma (ERMS) has a low prevalence, poor prognosis, and limited treatment efficacy. We report a case of an 18-year-old male whose disease relapsed in the abdominal cavity after a testicular ERMS curative resection. The patient received eight sequential cycles of rescue therapy using cisplatin and isocyclophosphamide in combination with a vascular targeted drug, Endostar. The therapeutic effect of the combination regimen has been evaluated for complete response. This is the first case to report using Endostar and chemotherapy in relapsed ERMS, and the curative effect results in complete response. Endostar, a new vascular targeted drug, combined with chemotherapy may play a synergistic role and provide a reference for the treatment of ERMS.

With the continuous development of computer education, programming teaching as a core course of computer elementary science education is receiving more and more attention. Colleges and universities have started to combine the Online Judge System (OJ) to develop programming skills of students. OJ platforms have been domestically and internationally developed, and there is any corresponding problem solving reports on the Internet. However, these resources currently are not well organised and used. Therefore, it could be meaningful to cluster these dissociative problem solution reports. The OJ problem, short text classification is usually used to refer to the algorithm. In this paper, the clustering algorithm is utilized to transform the problem into a succinct text clustering problem. The research results can not only serve as an effective guide for OJ problem solving, but also provide a more scientific match for learning algorithms, and can provide a valuable reference for participants in various programming competitions.

Hepatocellular carcinoma (HCC) and primary pancreatic cancer are common malignant tumors of the digestive system. However, there are significant differences in treatment methods, medication types, and survival prognoses. For patients whose imaging findings suggest there to be a significant pancreatic mass and multiple masses in the liver, it can be easily misdiagnosed as a primary pancreatic cancer with liver metastasis in the clinic instead. Therefore, patients with a high likelihood of primary pancreatic cancer based on their clinical data, the pathological diagnosis should be confirmed through a needle biopsy as early as possible to avoid a misdiagnosis and possible mistreatment. In this study, our department admitted a patient with HCC that was characterized by a pancreatic occupation. The clinical data originally highly suggested primary pancreatic cancer with liver metastasis, but the final pathological puncture results had confirmed pancreatic metastasis of HCC. This case reminds us that a pathological biopsy should still be used as the final means of definite diagnosis and should be widely popularized for pancreatic nodules.

Transcatheter arterial chemoembolization (TACE) plays an important role in the treatment of unresectable liver cancer. We conducted this meta-analysis to compare the clinical safety and efficacy of conventional TACE (C-TACE) and drug-eluting beads (DEB)-TACE. A search for those procedures was performed using the PubMed, EMBASE, and Cochrane Library databases. A meta-analysis of patients who underwent C-TACE or DEB-TACE was conducted. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. Of 334 studies, 30 were analyzed. The complete response rate, disease control rate, objective response rate, 3-year survival rate, and non-response rate were significantly higher in patients who underwent DEB-TACE than those in patients who underwent C-TACE. The 1-year survival rate, 2-year survival rate, 30-day mortality rate, complete response rate, disease control rate, complete necrosis rate, non-response rate, objective response rate, progressive disease rate, and recurrence did not differ significantly between patients who underwent C-TACE and patients who underwent DEB-TACE. Patients who undergo DEB-TACE might have a higher complete response rate, disease control rate, and 3-year survival rate than patients who undergo C-TACE. Safety did not differ significantly between C-TACE and DEB-TACE.