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Promoting the balanced development of regional basic education and finally realizing education equity is an important task for my country's current educational development. To this end, the balanced development of informatization and basic education has carried out experiments and practical explorations of informatization to promote the balanced development of regional basic education. Through synchronized interactive hybrid classrooms, the sharing of high-quality urban teachers to rural teaching sites has helped them to implement national regulations. Course. Using the SERVQUAL model for data analysis, synchronous interactive hybrid classrooms, as a new form of high-quality teacher sharing, are of great significance to promoting the balanced development of basic education in my country. It has attracted great attention from education researchers and is considered to be a solution area. Effective means for the problem of imbalanced sex education resources. Therefore, the VR teaching experience, the use of wearable sports equipment, the construction of a physical education network platform, and the four basic sports professional courses of physical education and university education interactive strategies are proposed, and the effects of the interactive strategies are analyzed. The experimental results show that the students' evaluation of the traditional basic physical education teaching mode is low, and the score is basically below 5 points. After the interaction between basic physical education and university education when applying the SERVQUAL model, the VR teaching experience strategy has a significant impact on basic physical education. VR teaching experience strategy basic physical education students achieve a score of 10, and their interest has increased by 34%. Sports performance under this interactive strategy increased by 51%. The strategy of interaction between basic physical education and university education has increased students' interest in basic physical education courses, improved students' performance in basic physical education courses, and promoted the development of physical education to a certain extent.

Background Tumor mutational burden (TMB) is a promising biomarker for stratifying patient subpopulation who would benefit from immune checkpoint blockade (ICB) therapies. Although great efforts have been made for standardizing TMB measurement, mutation calling and TMB quantification can be challenging in samples with low tumor content including liquid biopsies. The effect of varying tumor content on TMB estimation by different assay methods has never been systematically investigated. Method We established a series of reference standard DNA samples derived from 11 pairs of tumor–normal matched human cell lines across different cancer types. Each tumor cell line was mixed with its matched normal at 0% (control), 1%, 2%, 5%, and 10% mass‐to‐mass ratio to mimic the clinical samples with low tumor content. TMB of these reference standards was evaluated by both ∼1000× whole‐exome sequencing (wesTMB) and targeted panel sequencing (psTMB) at four different vendors. Both regression and classification analyses of TMB were performed for theoretical investigation and clinical practice purposes. Results Linear regression model was established that demonstrated in silico psTMB determined by regions of interest (ROI) as a great representative of wesTMB based on TCGA dataset. It was also true in our reference standard samples as the predicted psTMB interval based on the observed wesTMB captured the intended 90% of the in silico psTMB values. Although ∼1000× deep WES was applied, reference standard samples with less than 5% of tumor proportions are below the assay limit of detection (LoD) of wesTMB quantification. However, predicted wesTMB based on observed psTMB accurately classify (>0.97 AUC) for TMB high and low patient stratification even in samples with 2% of tumor content, which is more clinically relevant, as TMB determination should be a qualitative assay for TMB high and low patient classification. One targeted panel sequencing vendor using an optimized blood psTMB pipeline can further classify TMB status accurately (>0.82 AUC) in samples with only 1% of tumor content. Conclusions We developed a linear model to establish the quantitative correlation between wesTMB and psTMB. A set of DNA reference standards was produced in aid to standardize TMB measurements in samples with low tumor content across different targeted sequencing panels. This study is a significant contribution aiming to harmonize TMB estimation and extend its future application in clinical samples with low tumor content including liquid biopsy., Established 11 sets of reference standard samples with variable tumor proportions for evaluating TMB estimation. In silico and experimentally verified the linear regression model between the TMB analyzed by deep whole‐exome sequencing and targeted panel sequencing in reference standard samples. Targeted panel sequencing method might outperform WES for TMB estimation and categorization in samples with low tumor proportion.

Osteosarcoma is a primary malignant bone tumor that has a high potential to metastasize to lungs. Little is known about the mechanisms underlying the dissemination of OS cancer cells to lungs. We performed whole exome sequencing of 13 OS primary tumors, with matched lung metastases and normal tissues. Phylogenetic analyses revealed that lung metastatic tumors often harbor clones that are nonexistent or rare in the matched primary OS tumors. Spatially and temporally separated lung metastases were from parallel seeding events with a polyphyletic pattern. Loss of TP53 or RB1 is among the early events during OS tumorigenesis, while loss of PTEN is involved at the later stages associated with lung metastases. Finally, KEAP1 was identified as a novel biomarker for increased metastatic risk. Patients whose primary tumors harbored KEAP1 amplification have significantly poorer lung-metastasis free survival. This finding was validated in two independent datasets. Further, in vitro experiments exhibited that KEAP1 depletion suppressed the invasion of OS cells. Our findings uncover the patterns of clonal evolution during OS progression and highlight KEAP1 as a novel candidate associated with the risk of lung metastasis in OS patients.

// Juan Du 1, * , Xue Wu 2, * , Xiaoling Tong 2 , Xiaonan Wang 3 , Jia Wei 1 , Yang Yang 1 , Zhili Chang 3 , Yu Mao 3 , Yang W Shao 2 , Baorui Liu 1 1 The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, Jiangsu, 210008, China 2 Geneseeq Technology Inc., Toronto, Ontario, M5G1L7, Canada 3 Nanjing Geneseeq Technology Inc., Sino-Danish Life Science Park, Nanjing, Jiangsu, 210032, China * These authors contributed equally to this work Correspondence to: Baorui Liu, email: baoruiliu@nju.edu.cn Yang W Shao, email: yang.shao@geneseeq.com Keywords: circulating tumor DNA, next generation sequencing, MET, crizotinib, drug resistance Received: April 19, 2016 Accepted: February 04, 2017 Published: February 17, 2017 ABSTRACT Crizotinib has been used to counter MET gene amplification in a number of different human malignancies. Transient response to crizotinib in MET -amplified gastric cancer has been reported, but the mechanisms of resistance are not well studied. Here, we reported a stage IV gastric cancer patient with high levels of MET amplification. The implementation of crizotinib treatment led to significant symptomatic improvement in the first 2 months, but was followed by rapid disease progression. Periodic mutation profiling of patient’s circulating tumor DNA (ctDNA) by next generation sequencing (NGS) revealed a number of genetic alterations including re-occurrence of MET amplification, multiple secondary MET mutations, a dramatic increase of FGFR2 gene relative copy number as well as mutations in other downstream and bypassing elements, which may collectively related to the patient’s cancer progression. Our results illustrate the complex and heterogeneous molecular mechanisms for crizotinib resistance in this patient, and demonstrate the great potential of ctDNA profiling for treatment decision-making and prognosis in clinical practice.

Background Microsatellite instability (MSI) is a molecular phenotype characterized by the lengthening or shortening of tandem DNA repeats within the human genome. MSI phenomenon has been reported in various solid tumors as the consequence of DNA mismatch repair deficiency. It is generally associated with good prognosis and effective immunotherapy treatment. The current MSI tests are based on various methodologies, including immunohistochemistry, polymerase chain reactions, or next-generation sequencing (NGS). Each testing methodology has its own technical advantages, but they either fail at liquid biopsy specimen or implement complicated and expensive experimental interventions. Methods A designated analysis pipeline was developed individually for tissue biopsy and liquid biopsy to evaluate the stability of MSI sites within a NGS panel testing targeting over 400 genes at a common depth of coverage ( Results For tissue biopsy specimen validation, the assay system displayed 0.97 area-under-the-curve (AUC) in receiver operative characteristics (ROC) curve analysis against the comparator assay, out-performing publicly available MSI analysis software. Unfortunately, the tissue MSI analysis pipeline and publicly available software fails with less than 10% sensitivity and 0.55 AUC when applied to liquid biopsy specimen with the patient-matched tissue MSI status used as the gold standard. In contrast, the dedicated cfDNA analysis pipeline significantly boosted the assay performance to 0.73 without additional experimental interventions, such as wild-type allele depletion or molecular barcode. Conclusions MSI testing in liquid biopsy specimen remains challenging due to the low fraction of tumor originated cfDNA and consequently weak signal intensity. The implementation of the signal enhancing MSI analysis algorithm demonstrated high proficiency at improving the signal-to-noise ratio. This bioinformatics approach offered a cost-effective and non-invasive alternative for MSI testing without significantly modifying the NGS library preparation protocols. Citation Format: Xiangyuan Ma, Hua Bao, Xiaoling Tong, Zhili Chang, Xunbiao Liu, Xiuyan Zhang, Ryan S. Robetyore, Xue Wu, Yang W. Shao. Validation of a NGS-based MSI testing workflow in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 730.

Background Individual carrying germline mutations within BRCA1 or BRCA2 gene confers well-established increased risk of developing breast and ovarian cancers in the life-time than the general population. Recent advancement in clinical oncology and pharmacology discovered that poly ADP ribose polymerase (PARP) inhibitors are particularly effective in treating cancer that is homologous recombination deficient (HRD) through synthetic lethality, irrespective of the primary tumor site or the type of the BRCA1/2 inactivation variant. Methods A dedicated next-generation sequencing (NGS) panel was designed to interrogate the mutational status of BRCA1/2 and 18 other HRD-associated genes. Using a cohort of 522 clinical samples and artificially engineered cell line samples, we evaluated the mutation detection proficiency for single nucleotide variant (SNV) and small insertion or deletion (Indel) variants. BRCA1/2 inactivation is often the consequence of large genomic rearrangement (LGR) within the gene and phenotypically shown as exon-level copy number variant (CNV). Given the technical challenge of accurate exon-level CNV detection within a small panel, we developed a noise-reduction model based on a pool of normal (PON) samples for CNV result analysis. We further designed droplet digital polymerase chain reaction (ddPCR) for each exon of BRCA1/2 to verify the exonic CNV findings. Results Using a comparator assay as the gold standard, the BRCA1/2 panel achieved an accuracy of 96.7%, a precision of 98.9%, and a lower limit of detection of 3% variant allelic frequency (VAF) for SNV and Indel. The CNV pipeline achieved 97.8% sensitivity and 99.7% specificity for exonic CNV events that were supported by at least one piece of secondary evidence, out-performing publicly available software. The ddPCR verification further enhances assay accuracy and provides a significantly improved statistically confidence for the final CNV calls unparalleled by any other experimental methodologies. Conclusions Using a large cohort of clinical samples, the authors demonstrated the proficiency of a targeted NGS approach for variant and LGR detection of BRCA1/2 and the other genes of the DNA damage repair pathway. An effective method was developed to suppress the noise within the NGS-derived CNV profile, which is exaggerated by the small panel size and short targeted regions. The authors would also like to call the researcher's attention to a critical caveat within the CNV testing results generated from NGS-based methods. Citation Format: Xiangyuan Ma, Hua Bao, Zhili Chang, Yong Wu, Changwan Du, Rong Gao, Yu Song, Ryan S. Robetyore, Xue Wu, Yang W. Shao. Technical validation of a NGS-based analysis pipeline for BRCA1/2 variants and large genomic rearrangements detection [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4268.

The global epidemic of drug-resistant tuberculosis is a catastrophic example of how antimicrobial resistance is undermining the public health gains made possible by combination drug therapy. Recent evidence points to unappreciated bacterial factors that accelerate the emergence of drug resistance. In a genome-wide association study of Mycobacterium tuberculosis isolates from China, we find mutations in the gene encoding the transcription factor prpR enriched in drug-resistant strains. prpR mutations confer conditional drug tolerance to three of the most effective classes of antibiotics by altering propionyl-CoA metabolism. prpR-mediated drug tolerance is carbon-source dependent, and while readily detectable during infection of human macrophages, is not captured by standard susceptibility testing. These data define a previously unrecognized and clinically prevalent class of M. tuberculosis variants that undermine antibiotic efficacy and drive drug resistance.

Osteosarcoma is a primary malignant bone tumor that has a high potential to metastasize to lungs. Little is known about the mechanisms underlying the dissemination of OS cancer cells to lungs. We performed whole exome sequencing of 13 OS primary tumors, with matched lung metastases and normal tissues. Phylogenetic analyses revealed that lung metastatic tumors often harbor clones that are nonexistent or rare in the matched primary OS tumors. Spatially and temporally separated lung metastases were from parallel seeding events with a polyphyletic pattern. Loss of TP53 or RB1 is among the early events during OS tumorigenesis, while loss of PTEN is involved at the later stages associated with lung metastases. Finally, KEAP1 was identified as a novel biomarker for increased metastatic risk. Patients whose primary tumors harbored KEAP1 amplification have significantly poorer lung-metastasis free survival. This finding was validated in two independent datasets. Further, in vitro experiments exhibited that KEAP1 depletion suppressed the invasion of OS cells. Our findings uncover the patterns of clonal evolution during OS progression and highlight KEAP1 as a novel candidate associated with the risk of lung metastasis in OS patients.

Cancer is a disease of complex genetic alterations, and comprehensive genetic diagnosis is beneficial to match each patient to appropriate therapy. However, acquisition of representative tumor samples is invasive and sometimes impossible. Circulating tumor DNA (ctDNA) is a promising tool to use as a non-invasive biomarker for cancer mutation profiling. Here we implemented targeted next generation sequencing (NGS) with a customized gene panel of 382 cancer-relevant genes on 605 ctDNA samples in multiple cancer types. Overall, tumor-specific mutations were identified in 87% of ctDNA samples, with mutation spectra highly concordant with their matched tumor tissues. 71% of patients had at least one clinically-actionable mutation, 76% of which have suggested drugs approved or in clinical trials. In particular, our study reveals a unique mutation spectrum in Chinese lung cancer patients which could be used to guide treatment decisions and monitor drug-resistant mutations. Taken together, our study demonstrated the feasibility of clinically-useful targeted NGS-based ctDNA mutation profiling to guide treatment decisions in cancer.

As a useful heavy metal ion, chromium has seen its applications in various fields. While it is also a toxic contaminant in water and may cause serious threats to the environment and human health. To develop a novel material with good adsorption capacity and easy solid-liquid separation strategy was necessary and significant. In this paper, the β-cyclodextrin (β-CD) functionalized three-dimensional structured graphene foam (CDGF) was successfully synthesized with the facile and one-step hydrothermal method. The SEM, BET, XRD, FT-IR and XPS analysis were carried out and the results confirmed the successfully grafting of β-CD onto GF. The batch adsorption of Cr(VI) was also taken out and the CDGF possessed good selectivity compared with other metal ions at pH = 3. The adsorption capacity reduced gradually as the initial pH of the Cr(VI) solution grew higher, which was because the anionic species of Cr(VI) were partial to the positively charged surface of CDGF. The easy separation strategy of the CDGF was also demonstrated and the CDGF could be taken out easily with a tweezer after the adsorption of Cr(VI), which significantly simplified the separation procedure and reduced time. By comparing the FT-IR and XPD analysis results, the adsorption mechanism was explored and the hydroxyl groups on CDGF played the main role in the adsorption process. This work brings a novel material for the adsorption of Cr(VI) from water and provides an innovative direction for the easy and fast solid-liquid separation strategy in the adsorption and other application fields.

Cell-free DNA (cfDNA) is short, extracellular, fragmented double-stranded DNA found in plasma. Plasma of patients with solid tumor has been found to show significantly increased quantities of cfDNA. Although currently poorly understood, the mechanism of cfDNA generation is speculated to be a product of genomic DNA fragmentation during cellular apoptosis and necrosis. Sequencing of cfDNA with tumor origin has identified tumor biomarkers, elucidating molecular pathology and assisting in accurate diagnosis. In this study, we performed whole-genome sequencing ofcfDNA samples with matching tumor and whole blood samples from five patients diagnosed with stage IV gastric or lung cancer. We analyzed the coverage spectrum of the human genome in our cfDNA samples. cfDNA exhibited no large regions with significant under-coverage, although we observed unbalanced coverage depth in cfDNA at transcription start sites and exon boundaries as a consequence of biased fragmentation due to ordered nucleosome positioning. We also analyzed the copy number variant status based on the whole-genome sequencing results and found high similarity between copy number profile constructed from tumor samples and cfDNA samples. Overall, we conclude that cfDNA comprises a good representation of the tumor genome in late stage gastric and lung cancer.

2572 Background: Third generation epithelial growth factor receptor ( EGFR) tyrosine kinase inhibitor (TKI) osimertinib (AZD9291) has proven effective in Non-small cell lung cancer (NSCLC) patients who have developed EGFR T790M-mediated resistance to other EGFR TKIs. Unfortunately, a majority of patients still undergo progressed disease after receiving osimertinib treatment. Acquired EGFR C797S mutation has been identified as one major mechanism; however, resistance mechanisms of remaining cases are still largely unknown. Methods: Using next generation sequencing (NGS) targeting 416 cancer-relevant genes, we analyzed the mutation profiles of 99 NSCLC patients that were clinically resistant to osimertinib. Results: In addition to the notable EGFR C797 variants (22%), L792 mutations were identified in 10% of patients, and a further 7% cases carry L718 mutations. Further analysis of 14 patients with paired pre-treatment samples confirmed that these EGFR mutations were acquired during treatment. Interestingly, all L792 mutations are in cis with T790M and in trans with C797 mutations (when present in the same patient). 2 out of 10 L792-positive patients and 6 out of 7 L718-positive patients did not have co-existing C797 mutations, suggesting C797-, L792- and L718-mutated cells may represent different resistant clones. In vitro experiments demonstrated that L792 and L718 mutants also increase osimertinib IC50, and therefore confer their resistance. Besides secondary EGFR mutations, alterations in other key genes such as MET, KRAS, ERBB2 and PIK3CA may also contribute to osimertinib resistance. Notably, MET and KRAS amplifications are present only in patients without above EGFR secondary mutations. Conclusions: In this study, we identified secondary mutations on C797, L792 or L718 residues of EGFR in 29% of osimertinib-resistant patients. Combined with in vitro study, our data strongly suggest that L792 and L718 mutations are likely to alternatively cause osimertinib resistance. Furthermore, MET and KRAS amplification may serve as bypass resistance mechanisms in patients who are EGFR C797-, L792- and L718-wild type.

1581Background: Cancer is a disease of genetic alterations. Comprehensive genetic diagnosis is needed to match each patient to appropriate cancer therapy. However, acquisition of representative tumor samples is invasive and often infeasible. As tumor-derived fragmented DNA, circulating tumor DNA (ctDNA) is becoming a promising non-invasive biomarker for cancer mutational profiling. However, most recent studies focused on utilizing ctDNA in monitoring tumor burden; and none of them have systematically evaluated its clinical application on mutational profiling to guide personalized treatments. Methods: We implemented targeted next generation sequencing (NGS) with a customized pan-cancer gene panel of 382 cancer-relevant genes and 16 genes frequently rearranged in solid tumors on 605 prospective clinical ctDNA samples of multiple cancer types. A two-tiered study was designed with a training cohort containing patient ctDNA samples and matched archived formalin-fixed paraffin-embedded (FFPE) or frozen tissue s...

BACKGROUND: Shigella, the causative agent of shigellosis, is a major global public health concern, particularly in developing countries with poor sanitation. A comprehensive and current understanding of the prevalent species and serotypes of shigellosis is essential for both disease prevention and vaccine development. However, no current data are available on the causative species/serotypes of shigellosis in mainland China during the past decade. METHODS AND FINDINGS: Relevant studies addressing the prevalent species of shigellosis in mainland China from January 2001 to December 2010 were identified from PubMed and the Chinese BioMedical Literature Database (in Chinese) until April 2012. A total of 131 eligible articles (136 studies) were included in this review. Meta-analyses showed that the prevalences of S. flexneri and S. sonnei were 76.2% (95% CI, 73.7%-78.5%) and 21.3% (95% CI, 19.0%-23.7%), respectively. Stratified analyses indicated a decrease in the prevalence of S. flexneri cases and an increase in the prevalence of S. sonnei cases concurrent with the rapid economic growth experienced by China in recent years. Moreover, significantly higher rates of S. sonnei were observed in the East, North and Northeast regions of China, as compared to the rest of the country. These phenomena imply the possible association between the prevalent species of Shigella and regional economic status; however, additional factors also exist and require further investigations. Moreover, the two major serotypes S. flexneri 2a and 4c accounted for 21.5% (95% CI, 16.7%-27.4%) and 12.9% (95% CI 9.8%-16.9%) of S. flexneri infections, respectively, in the past decade. However, these results were found to be frequently heterogeneous (p for Q tests