TLR3 activation enhances abscopal effect of radiotherapy in HCC by promoting tumor ferroptosis.

Radiotherapy (RT) has been reported to induce abscopal effect in advanced hepatocellular carcinoma (HCC), but such phenomenon was only observed in sporadic cases. Here, we demonstrated that subcutaneous administration of Toll-like receptor 3 (TLR3) agonist poly(I:C) could strengthen the abscopal effect during RT through activating tumor cell ferroptosis signals in bilateral HCC subcutaneous tumor mouse models, which could be significantly abolished by TLR3 knock-out or ferroptosis inhibitor ferrostatin-1. Moreover, poly(I:C) could promote the presentation of tumor neoantigens by dendritic cells to enhance the recruitment of activated CD8+ T cells into distant tumor tissues for inducing tumor cell ferroptosis during RT treatment. Finally, the safety and feasibility of combining poly(I:C) with RT for treating advanced HCC patients were further verified in a prospective clinical trial. Thus, enhancing TLR3 signaling activation during RT could provide a novel strategy for strengthening abscopal effect to improve the clinical benefits of advanced HCC patients.

Personalized neoantigen vaccine enhances the therapeutic efficacy of bevacizumab and anti-PD-1 antibody in advanced non-small cell lung cancer.

Clinically, a considerable number of non-small cell lung cancer (NSCLC) patients are unable to receive or resist chemotherapy, and the efficacy of non-chemotherapy treatment strategies based on anti-angiogenic agents combined with immune checkpoint blockade is still unsatisfactory. Neoantigen vaccine, based on personalized tumor DNA mutations, could elicit tumor specific T cell infiltration into the tumor site, exerting potent anti-tumor efficacy. Here, we evaluated the feasibility and safety of a new antitumor strategy by adding neoantigen vaccine to the regimen of bevacizumab and anti-PD-1 antibody. Firstly, 7 novel immunogenic neoantigen peptides were identified and developed for neoantigen vaccine (LLCvac), which can elicit strong antitumor immune response in vivo. Then, in orthotopic lung cancer model, LLCvac further combining with bevacizumab and anti-PD-1 antibody exerted a stronger antitumor effect, exhibiting significant decrease of tumor volume without obvious toxicity. Furthermore, tumor immune microenvironment assessment also showed that the proportion of neoantigen-specific T cells in blood could be induced dramatically by the combined therapy. And a large amount of neoantigen-specific Ki67-positive CD8+ T cells were found in tumor tissues, which infiltrated tumor tissues effectively to kill tumor cells expressing identified neoantigens. Overall, these results suggested that this combined therapy could safely induce robust antitumor efficacy, serving as an effective chemotherapy-free strategy for NSCLC treatment.

Genome-Wide Association Analysis of Protein-Coding Variants in IgA Nephropathy.

SIGNIFICANCE STATEMENT: Genome-wide association studies have identified nearly 20 IgA nephropathy susceptibility loci. However, most nonsynonymous coding variants, particularly ones that occur rarely or at a low frequency, have not been well investigated. The authors performed a chip-based association study of IgA nephropathy in 8529 patients with the disorder and 23,224 controls. They identified a rare variant in the gene encoding vascular endothelial growth factor A (VEGFA) that was significantly associated with a two-fold increased risk of IgA nephropathy, which was further confirmed by sequencing analysis. They also identified a novel common variant in PKD1L3 that was significantly associated with lower haptoglobin protein levels. This study, which was well-powered to detect low-frequency variants with moderate to large effect sizes, helps expand our understanding of the genetic basis of IgA nephropathy susceptibility. BACKGROUND: Genome-wide association studies have identified nearly 20 susceptibility loci for IgA nephropathy. However, most nonsynonymous coding variants, particularly those occurring rarely or at a low frequency, have not been well investigated. METHODS: We performed a three-stage exome chip-based association study of coding variants in 8529 patients with IgA nephropathy and 23,224 controls, all of Han Chinese ancestry. Sequencing analysis was conducted to investigate rare coding variants that were not covered by the exome chip. We used molecular dynamic simulation to characterize the effects of mutations of VEGFA on the protein's structure and function. We also explored the relationship between the identified variants and the risk of disease progression. RESULTS: We discovered a novel rare nonsynonymous risk variant in VEGFA (odds ratio, 1.97; 95% confidence interval [95% CI], 1.61 to 2.41; P = 3.61×10 -11 ). Further sequencing of VEGFA revealed twice as many carriers of other rare variants in 2148 cases compared with 2732 controls. We also identified a common nonsynonymous risk variant in PKD1L3 (odds ratio, 1.16; 95% CI, 1.11 to 1.21; P = 1.43×10 -11 ), which was associated with lower haptoglobin protein levels. The rare VEGFA mutation could cause a conformational change and increase the binding affinity of VEGFA to its receptors. Furthermore, this variant was associated with the increased risk of kidney disease progression in IgA nephropathy (hazard ratio, 2.99; 95% CI, 1.09 to 8.21; P = 0.03). CONCLUSIONS: Our study identified two novel risk variants for IgA nephropathy in VEGFA and PKD1L3 and helps expand our understanding of the genetic basis of IgA nephropathy susceptibility.

Identification of tumor mutations in plasma based on mutation variant frequency change (MVFC).

To overcome the dependency of strategies utilizing cell-free DNA (cfDNA) on tissue sampling, the emergence of sequencing panels for non-invasive mutation screening was promoted. However, cfDNA sequencing with panels still suffers from either inaccuracy or omission, and novel approaches for accurately screening tumor mutations solely based on plasma without gene panel restriction are urgently needed. We performed unique molecular identifier (UMI) target sequencing on plasma samples and peripheral blood mononuclear cells (PBMCs) from 85 hepatocellular carcinoma (HCC) patients receiving surgical resection, which were divided into an exploration dataset (20 patients) or an evaluation dataset (65 patients). Plasma mutations were identified in pre-operative plasma, and the mutation variant frequency change (MVFC) between post- and pre-operative plasma was then calculated. In the exploration dataset, we observed that plasma mutations with MVFC < 0.2 were enriched for tumor mutations identified in tumor tissues and had frequency changes that correlated with tumor burden; these plasma mutations were therefore defined as MVFC-identified tumor mutations. The presence of MVFC-identified tumor mutations after surgery was related to shorter relapse-free survival (RFS) in both datasets and thus indicated minimum residual disease (MRD). The combination of MVFC-identified tumor mutations and Alpha Fetoprotein (AFP) could further improve MRD detection (P < 0.0001). Identification of tumor mutations based on MVFC was also confirmed to be applicable with a different gene panel. Overall, we proposed a novel strategy for non-invasive tumor mutation screening using solely plasma that could be utilized in HCC tumor-burden monitoring and MRD detection.

Dysregulation of global circular RNA abundance regulated by spliceosomes predicts prognosis in hepatocellular carcinoma.

CircRNAs have been reported to play crucial roles in tumor progression and recurrence, showing potential as biomarkers in cancer. However, the global abundance of circRNA and their involvement in hepatocellular carcinoma (HCC) development have not been fully explored. Whole transcriptome sequencing was performed on tumor and peritumor from 60 patients with HCC to quantify the expression of circRNAs, and the global circRNA abundance was calculated by circRNA index (CRI). Gene-set enrichment analysis and weighted gene co-expression network analysis were used to reveal the biological signaling pathways associated with the global circRNA abundance. The correlation between the global circRNA abundance and the infiltration level of CD8+ T cells was explored by immunohistochemical assays. Small interfering RNA was used to knock down the pre-messenger RNA spliceosome in HCC cell lines to verify the regulation of spliceosome in global circRNA abundance. We found that dysregulation of global circRNA abundance in both tumor and peritumor could lead to worse prognosis. The immunohistochemical assay further revealed that the dysregulation of global circRNA abundance in both tumor and peritumor would obstruct the CD8+ T cells from invading into the tumor, which might explain its correlation with HCC prognosis. We also demonstrated that the spliceosome genes were the main factors to regulate the global circRNA abundance in HCC, and these results were also confirmed by knockdown experiments. Conclusion: This study revealed the association between the global circRNA abundance and patients' prognosis and its underlying mechanism.

Personalized neoantigen vaccine combined with PD-1 blockade increases CD8+ tissue-resident memory T-cell infiltration in preclinical hepatocellular carcinoma models.

BACKGROUND: Personalized neoantigen vaccine could induce a robust antitumor immune response in multiple cancers, whose efficacy could be further enhanced by combining with programmed cell death 1 blockade (α-PD-1). However, the corresponding immune response and synergistic mechanisms remain largely unclear. Here, we aimed to develop clinically available combinational therapeutic strategy and further explore its potential antitumor mechanisms in hepatocellular carcinoma (HCC). METHODS: Neoantigen peptide vaccine (NeoVAC) for murine HCC cell line Hepa1-6 was developed and optimized by neoantigen screening and adjuvant optimization. Then the synergistic efficacy and related molecular mechanisms of NeoVAC combined with α-PD-1 in HCC were evaluated by orthotopic HCC mouse model, single-cell RNA sequencing, tetramer flow cytometry, immunofluorescence, etc. The tumor-killing capacity of CD8+ tissue-resident memory T cells (CD8+ TRMs) was assessed by orthotopic HCC mouse model, and autologous patient-derived cells. RESULTS: NeoVAC, which consisted of seven high immunogenic neoantigen peptides and clinical-grade Poly(I:C), could generate a strong antitumor immune response in HCC mouse models. Significantly, its efficacy could be further improved by combining with α-PD-1, with 80% of durable tumor regression and long-term immune memory in orthotopic HCC models. Moreover, in-depth analysis of the tumor immune microenvironment showed that the percentage of CD8+ TRMs was remarkedly increased in NeoVAC plus α-PD-1 treatment group, and positively associated with the antitumor efficacy. In vitro and in vivo T-cell cytotoxicity assay further confirmed the strong tumor-killing capacity of CD8+ TRMs sorting from orthotopic mouse HCC or patient's HCC tissue. CONCLUSIONS: This study showed that NeoVAC plus α-PD-1 could induce a strong antitumor response and long-term tumor-specific immune memory in HCC by increasing CD8+ TRMs infiltration, which might serve as a potential immune-therapeutic target for HCC.

Evaluation of an Artificial Intelligence System for the Detection of Diabetic Retinopathy in Chinese Community Healthcare Centers.

Objective: To evaluate the sensitivity and specificity of a Comprehensive Artificial Intelligence Retinal Expert (CARE) system for detecting diabetic retinopathy (DR) in a Chinese community population. Methods: This was a cross-sectional, diagnostic study. Participants with a previous diagnosis of diabetes from three Chinese community healthcare centers were enrolled in the study. Single-field color fundus photography was obtained and analyzed by the AI system and two ophthalmologists. Primary outcome measures included the sensitivity, specificity, positive predictive value, and negative predictive value with their 95% confidence intervals (CIs) of the AI system in detecting DR and diabetic macular edema (DME). Results: In this study, 443 subjects (848 eyes) were enrolled, and 283 (63.88%) were men. The mean age was 52.09 (11.51) years (range 18-82 years); 266 eyes were diagnosed with any DR, 233 with more-than-mild diabetic retinopathy (mtmDR), 112 with vision-threatening diabetic retinopathy (vtDR), and 57 with DME. The image ability of the AI system was as high as 99.06%, whereas its sensitivity and specificity varied significantly in detecting DR with different severities. The sensitivity/specificity to detect any DR was 75.19% (95%CI 69.47-80.17)/93.99% (95%CI 91.65-95.71), mtmDR 78.97% (95%CI 73.06-83.90)/92.52% (95%CI 90.07-94.41), vtDR 33.93% (95%CI 25.41-43.56)/97.69% (95%CI 96.25-98.61), and DME 47.37% (95%CI 34.18-60.91)/93.99% (95%CI 91.65-95.71). Conclusions: This multicenter cross-sectional diagnostic study noted the safety and reliability of the CARE system for DR (especially mtmDR) detection in Chinese community healthcare centers. The system may effectively solve the dilemma faced by Chinese community healthcare centers: due to the lack of ophthalmic expertise of primary physicians, DR diagnosis and referral are not timely.

Copy number profiling of circulating free DNA predicts transarterial chemoembolization response in advanced hepatocellular carcinoma.

Transarterial chemoembolization (TACE) is the most commonly used treatment for advanced hepatocellular carcinoma (HCC), but still lacks accurate real-time biomarkers for monitoring its therapeutic efficacy. Here, we explored whether copy number profiling of circulating free DNA (cfDNA) could be utilized to predict responses and prognosis in HCC patients with TACE treatment. In total, 266 plasma cfDNA samples were collected from 64 HCC patients, 57 liver cirrhosis (LC) patients and 32 healthy volunteers. We performed low-depth whole-genome sequencing (LD-WGS) on cfDNA samples to conduct copy number variant (CNV) analysis and tumour fraction (TFx) quantification. Then, the correlation between TFx/CNVs and therapeutic efficacy, treatment outcomes and lipiodol deposition were explored. The change in TFx during TACE treatment was associated with patients' tumour burden, and could accurately and earlier predict treatment response and prognosis, providing an alternative strategy other than mRECIST. Meanwhile, the chromosomal 16q/NQO1 amplification indicated worse therapeutic response; in patients who underwent multiple TACE sessions, TFx change during their first TACE treatment reflected the long-term survival; additionally, the copy number amplification of chromosome 1q, 3p, 6p, 8q, 10p, 12q, 18p or 18q affected lipiodol deposition. Overall, we have provided a new liquid biopsy approach for future TACE management of HCC patients.

Personalized neoantigen vaccine prevents postoperative recurrence in hepatocellular carcinoma patients with vascular invasion.

BACKGROUND: Clinically, prophylactic anti-recurrence treatments for hepatocellular carcinoma (HCC) patients after radical surgery are extremely limited. Neoantigen based vaccine can generate robust anti-tumor immune response in several solid tumors but whether it could induce anti-tumor immune response in HCC and serve as a safe and effective prophylactic strategy for preventing postoperative HCC recurrence still remain largely unclear. METHODS: Personalized neoantigen vaccine was designed and immunized for 10 HCC patients with high risk of postoperative recurrence in a prime-boost schedule. The safety and immune response were assessed through adverse events, tissue sequencing, ELISpot, TCR sequencing. The clinical response was evaluated by recurrence-free survival (RFS) and personalized circulating tumor DNA (ctDNA) sequencing. RESULTS: In the 10 enrolled patients, no obvious adverse events were observed during neoantigen vaccinations. Until the deadline of clinical trial, 8 of 10 patients were confirmed with clinical relapse by imaging, the other 2 patients remained relapse-free. From receiving first neoantigen vaccination, the median RFS of 10 patients were 7.4 months. Among 7 patients received all planned neoantigen vaccinations, 5 of them demonstrated neoantigen-induced T cell responses and have significantly longer RFS after radical surgery than other 5 patients without responsive neoantigens or only with prime vaccination and propensity scores matching control patients (p = 0.035). Moreover, tracking personalized neoantigen mutations in ctDNA could provide real-time evaluation of clinical response in HCC patients during neoantigen vaccination and follow up. CONCLUSION: Personalized neoantigen vaccine is proved as a safe, feasible and effective strategy for HCC anti-recurrence, and its progression could be sensitively monitored by corresponding neoantigen mutations in ctDNA, and thus provided solid information for individualized medicine in HCC. TRIAL REGISTRATION: This study was registered at Chinese Clinical Trial Registry; Registration number: ChiCTR1900020990 .

Profiling of hepatocellular carcinoma neoantigens reveals immune microenvironment and clonal evolution related patterns.

OBJECTIVE: Neoantigens derived from tumor-specific genomic alterations have demonstrated great potential for immunotherapeutic interventions in cancers. However, the comprehensive profile of hepatocellular carcinoma (HCC) neoantigens and their complex interplay with immune microenvironment and tumor evolution have not been fully addressed. METHODS: Here we integrated whole exome sequencing data, transcriptome sequencing data and clinical information of 72 primary HCC patients to characterize the HCC neoantigen profile, and systematically explored its interactions with tumor clonal evolution, driver mutations and immune microenvironments. RESULTS: We observed that higher somatic mutation/neoantigen load was associated with better clinical outcomes and HCC patients could be further divided into two subgroups with distinct prognosis based on their neoantigen expression patterns. HCC subgroup with neoantigen expression probability high (NEP-H) showed more aggressive pathologic features including increased incidence of tumor thrombus (P=0.038), higher recurrence rate (P=0.029), more inclined to lack tumor capsule (P=0.026) and with more microsatellite instability sites (P=0.006). In addition, NEP-H subgroup was also characterized by higher chance to be involved in tumor clonal evolution [odds ratio (OR)=46.7, P<0.001]. Gene set enrichment analysis revealed that upregulation of MYC and its targets could suppress immune responses, leading to elevated neoantigen expression proportion in tumor cells. Furthermore, we discovered an immune escape mechanism that tumors could become more inconspicuous by evolving subclones with less immunogenicity. We observed that smaller clonal mutation clusters with higher immunogenicity in tumor were more likely to involve in clonal evolution. Based on identified neoantigen profiles, we also discovered series of neoantigenic hotspot genes, which could serve as potential actionable targets in future. CONCLUSIONS: Our results revealed the landscape of HCC neoantigens and discovered two clinically relevant subgroups with distinct neoantigen expression patterns, suggesting the neoantigen expression should be fully considered in future immunotherapeutic interventions.

Accurate transcriptome assembly by Nanopore RNA sequencing reveals novel functional transcripts in hepatocellular carcinoma.

The long reads of Nanopore sequencing permit accurate transcript assembly and ease in discovering novel transcripts with potentially important functions in cancers. The wide adoption of Nanopore sequencing for transcript quantification, however, is largely limited by high costs. To address this issue, we developed a bioinformatics software, NovelQuant, that can specifically quantify long-read-assembled novel transcripts with short-read sequencing data. Nanopore Direct RNA Sequencing was carried out on three hepatocellular carcinoma (HCC) patients' tumor, matched portal vein tumor thrombus, and peritumor to reconstruct the HCC transcriptome. Then, based on the reconstructed transcriptome, NovelQuant was applied on Illumina RNA sequencing data of 59 HCC patients' tumor and paired peritumor to quantify novel transcripts. Our further analysis revealed 361 novel transcripts dysregulated in HCC and that 101 of them were significantly associated with prognosis. There were 19 novel prognostic transcripts predicted to be long noncoding RNAs (lncRNAs), and some of them had regulatory targets that were reported to be associated with HCC. Additionally, 42 novel prognostic transcripts were predicted to be protein-coding mRNAs, and many of them could be involved in xenobiotic metabolism. Moreover, the tumor-suppressive roles of two representative novel prognostic transcripts, CDO1-novel (lncRNA) and CYP2A6-novel (protein-coding mRNA), were further functionally validated during HCC progression. Overall, the current study shows a possibility of combining long- and short-read sequencing to explore functionally important novel transcripts in HCC with accuracy and cost-efficiency, which expands the pool of molecular biomarkers that could enhance our understanding of the molecular mechanisms of HCC.

Circular RNA CircEPB41L2 Functions as Tumor Suppressor in Hepatocellular Carcinoma Through Sponging miR-590-5p.

BACKGROUND: Circular RNAs (circRNAs) could interact with miRNAs to regulate gene expression, participating in hepatocellular carcinoma (HCC) initiation and development. This work aimed to determine the potential function and molecular mechanism of circEPB41L2 (hsa_circ_0077837) during HCC progression. MATERIALS AND METHODS: The expression of circEPB41L2 in HCC tissues and HCC cell lines was quantified using real-time quantitative PCR (qRT-PCR). CCK-8 assays and colony formation assays were utilized to detect the proliferation of HCC cells. Wound healing assay and transwell assay were performed to determine the capability of migration and invasion for HCC cells. Western blot was conducted to determine gene expression on protein levels. The effect of circEPB41L2 on HCC in vivo was investigated via xenograft experiment. Interaction between circEPB41L2 and miR-590-5p was predicted through bioinformatics methods and confirmed via luciferase reporter assay. RESULTS: Extensive analysis of circRNA profiles in tumor and matched para-tumor tissues collected from 61 HCC patients identified that circEPB41L2 was significantly down-regulated in HCC, which was further confirmed in another HCC group by qRT-PCR analysis. The clinicopathological analysis revealed that down-regulation of circEPB41L2 was negatively associated with tumor size, vascular invasion and alpha-fetoprotein, while positively correlated with HCC prognosis. The biological function experiments showed that overexpression of circEPB41L2 could obviously inhibit the proliferation and metastasis of HCC cells in vitro, while knockdown of circEPB41L2 induced opposite results. Moreover, we also found that circEPB41L2 inhibited HCC migration and invasion though EMT signaling pathway. Similarly, overexpression of circEPB41L2 can also significantly inhibit the proliferation of HCC cells in vivo. Bioinformatic analysis and luciferase reporter assay revealed that circEPB41L2 interacts directly with miR-590-5p and the corresponding biological functions were also verified in miRNA rescue experiments. CONCLUSION: Our results suggest that circEPB41L2 might function as a tumor suppressor during HCC progression by sponging miR-590-5p.

An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage.

Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R2 = 0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

Genomic and Transcriptomic Landscape of Tumor Clonal Evolution in Cholangiocarcinoma.

Cholangiocarcinoma remained a severe threat to human health. Deciphering the genomic and/or transcriptomic profiles of tumor has been proved to be a promising strategy for exploring the mechanism of tumorigenesis and development, which could also provide valuable insights into Cholangiocarcinoma. However, little knowledge has been obtained regarding to how the alteration among different omics levels is connected. Here, using whole exome sequencing and transcriptome sequencing, we performed a thorough evaluation for the landscape of genome and transcriptome in cholangiocarcinoma and illustrate the alteration of tumor on different biological levels. Meanwhile, we also identified the clonal structure of each included tumor sample and discovered different clonal evolution patterns related to patients' survival. Furthermore, we extracted subnetworks that were greatly influenced by tumor clonal/subclonal mutations or transcriptome change. The topology relationship between genes affected by genomic/transcriptomic changes in biological interaction networks revealed that alteration of genome and transcriptome was highly correlated, and somatic mutations located on important genes might affect the expression of numerous genes in close range.

Dataset for quantitative phospho-proteomics analysis of a serial hepatoma cell lines with increasing invasion and metastasis potential.

Hepatoma is one of the most common malignant tumor, and most patients have very poor prognosis. Early prediction and intervention of the hepatoma recurrence/metastasis are the most effective way to improve the patients' clinical outcomes. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) based quantitative phospho-proteomics approach to identify biomarkers associated with hepatoma recurrence/metastasis in hepatoma cell lines with increasing metastasis ability. In total, 75 phosphorylated peptides corresponding to 60 phosphoproteins were significantly dysregulated. Bioinformatics analysis (GO, KEGG and IPA) allowed these data to be organized into distinct categories. These data represent the first in-depth proteomics analysis of a serial hepatoma cell lines with increasing invasion and metastasis potential. The data are related to (Xing et al., 2019).

Comprehensive Liquid Profiling of Circulating Tumor DNA and Protein Biomarkers in Long-Term Follow-Up Patients with Hepatocellular Carcinoma.

PURPOSE: Circulating tumor DNA (ctDNA) provides a novel approach for detecting tumor burden and predicting clinical outcomes of hepatocellular carcinoma (HCC). Here, we performed a thorough evaluation of HCC circulating genetic features and further fully integrated them to build a robust strategy for HCC monitoring and prognostic outcome assessment. EXPERIMENTAL DESIGN: We performed target sequencing and low-coverage whole-genome sequencing on plasma samples collected from 34 long-term follow-up patients with HCC to capture tumor somatic SNVs and CNVs, respectively. Clinical information was also obtained to evaluate the prognostic performance of ctDNA comparing with clinically applied protein biomarkers. RESULTS: All plasma samples before surgery showed somatic genetic variations resembling corresponding tumor tissues. During follow-up, SNVs and CNVs dynamically changed correlating to patients' tumor burden. We integrated the comprehensive ctDNA mutation profiles to provide a robust strategy to accurately assess patients' tumor burden with high consistence comparing with imaging results. This strategy could discover tumor occurrence in advance of imaging for an average of 4.6 months, and showed superior performance than serum biomarkers AFP, AFP-L3%, and Des-Gamma-Carboxy Prothrombin (DCP). Furthermore, our strategy could precisely detect minimal residual disease (MRD) in advance and predict patients' prognostic outcomes for both relapse-free survival (P = 0.001) and overall survival (P = 0.001); further combining ctDNA with DCP could increase the sensitivity for MRD detection. CONCLUSIONS: We demonstrated that plasma CNV and SNV levels dynamically correlated with patients' tumor burden in HCC. Our strategy of comprehensive mutation profile integration could accurately and better evaluate patients' prognostic risk and detect tumor occurrence in advance than traditional strategies.

ANXA2Tyr23 and FLNASer2152 phosphorylation associate with poor prognosis in hepatic carcinoma revealed by quantitative phosphoproteomics analysis.

Hepatoma is one of the most common malignant tumors, and most patients have very poor prognosis. Early prediction and intervention of the hepatoma recurrence/metastasis are the most effective way to improve the patients' clinical outcomes. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) based quantitative phospho-proteomics approach to identify biomarkers associated with hepatoma recurrence/metastasis in hepatoma cell lines with increasing metastasis ability. In total, 75 phosphorylated peptides corresponding to 60 phosphoproteins were significantly dysregulated and the participated biological processes of these phosphoproteins were tightly associated with tumor metastasis. Further signaling pathway analysis revealed that key signaling pathways which play crucial roles in cancer metastasis have been significantly over activated in the highly metastatic cells. Furthermore, the phosphorylation of FLNASer2152 and ANXA2Tyr23 were validated to be significantly up regulated in the high-metastatic cells comparing with the low-metastatic cells. By further investigation the clinical significance of the phosphorylation of FLNASer2152 and ANXA2Tyr23 in large-scale clinical samples, revealed that the over phosphorylation of FLNASer2152 and ANXA2Tyr23 were associated with poor prognosis and might be potential prognostic biomarkers for the primary hepatoma. When FLNASer2152 combined with ANXA2Tyr23, it had a better prognostic value for both OS and TTR.

Genomic and transcriptional Profiling of tumor infiltrated CD8+ T cells revealed functional heterogeneity of antitumor immunity in hepatocellular carcinoma.

As key players in HCC antitumor response, the functions of tumor infiltrated CD8+ T cells are significantly affected by surrounding microenvironment. A detailed profiling of their genomic and transcriptional changes could provide valuable insights for both future immunotherapy development and prognosis evaluation. We performed whole exome and transcriptome sequencing on tumor infiltrated CD8+ T cells and CD8+ T cells isolated from other tissue origins (peritumor tissues and corresponding PBMCs) in eight treatment-naive HCC patients. The results demonstrated that transcriptional changes, rather than genomic alterations were the main contributors to the functional alterations of CD8+ T cells in the process of tumor progression. The origins of CD8+ T cells defined their transcriptional landscape, while the tumor infiltrated CD8+ T cells shared more similarity with peritumor-derived CD8+ T cells compared with those CD8+ T cells in blood. In addition, tumor infiltrated CD8+ T cells also showed larger transcriptional heterogeneity among individuals, which was modulated by clinical features such as HBV levels, preoperative anti-viral treatment and the degree of T cell infiltration. We also identified multiple inter-connected pathways involved in the activation and exhaustion of tumor infiltrated CD8+ T cells, among which IL-12 mediated pathway could dynamically reflect the functional status of CD8+ TILs and activation of this pathway indicated a better prognosis. Our results presented an overview picture of CD8+ TILs' genomic and transcriptional landscape and features, as well as how the functional status of CD8+ TILs correlated with patients' clinical course.

Circular RNA profiling identifies circADAMTS13 as a miR-484 sponge which suppresses cell proliferation in hepatocellular carcinoma.

Circular RNA (circRNA) can participate in various biological processes, including tumorigenesis, through their microRNA response elements. Alterations in circRNA profiles during hepatocellular carcinoma (HCC) progression and their clinical significance remain unclear. Here, we present extensive analysis of circRNA profiles in tumor and matched peritumor tissues collected from 10 HCC patients, conducted to identify circRNA related to HCC progression. A total of 42 dysregulated circRNA (38 down-regulated and 4 up-regulated) were identified in HCC tumor tissues compared with matched peritumor tissues, revealing the heterogeneity of circRNA profiles in HCC. CircADAMTS13, derived from Exon 13-14 of the ADAMTS13 gene, was significantly downregulated in HCC tumor tissues. Furthermore, clinicopathological analysis revealed that up-regulation of circADAMTS13 was negatively associated with tumor size but positively associated with prognosis. In addition, overexpression of circADAMTS13 could markedly inhibit HCC cell proliferation in vitro. Bioinformatic analysis and luciferase reporter assays further revealed that circADAMTS13 directly interacts with microRNA (miR)-484. Rescue experiments showed that miR-484 mimics can reverse the tumor-suppressing roles of circADAMTS13 in HCC. Therefore, our results demonstrated that circADAMTS13 can serve as a tumor suppressor during HCC progression via the functional pathway of sponging miR-484.

Effects of the portage early education program on Chinese children with global developmental delay.

Children with global developmental delay (GDD) were trained with the Portage Guide to Early Education (PGEE) program.In the treatment group, the PGEE program was performed on children with GDD (45 cases) through a combination of family and hospital interventions, in a 1-to-1 ratio. The Gesell Infant Development Scale (GESELL) developmental quotient (DQ) and social adaptability were measured before and 6 months after PGEE implementation in the treatment group. These parameters were also evaluated in a control group (30 cases) during an initial visit and 6 months later.Before the PGEE intervention, no significant differences were observed between the general characteristics of children in the control and treatment groups. Six months after the PGEE intervention, the DQ values of the children with GDD in the treatment group (64.7 ±â€Š9.5) were significantly higher than those before treatment (54.6 ±â€Š9.3) and those of the control group (58.3 ±â€Š10.2) (P < .05). The PGEE intervention significantly increased the DQ values on 5 aspects, including gross motor, fine motor, adaptability, language, and personal social activity abilities, and the scores on the Infants-Junior Middle School Students' Social-Life Abilities Scales (SM scales), as compared with the control group (P < .05).The PGEE program improves the DQ, social adaptability, and prognosis of children with GDD.