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Abstract Background The impact of prior SARS-CoV-2 infection on postoperative recovery of patients who underwent liver resection for hepatocellular carcinoma (HCC) remains uncertain given the lack of sufficient evidence. Aim To investigate the impact of prior SARS-CoV-2 infection on postoperative recovery of patients who underwent liver resection for hepatocellular carcinoma (HCC). Methods Patients who were pathologically diagnosed with HCC and underwent elective partial hepatectomy in Guangdong Provincial People’s Hospital between January 2022 and April 2023 were enrolled in this retrospective cohort study. The patients were divided into two groups based on their history of SARS-CoV-2 infection. Rehabilitation parameters, including postoperative liver function, incidence of complications, and hospitalization expenses, were compared between the two groups. Propensity score matching (PSM) was performed to reduce confounding bias. Results We included 172 patients (58 with and 114 without prior SARS-CoV-2 infection) who underwent liver resection for HCC. No significant differences in the rehabilitation parameters were observed between the two groups. After PSM, 58 patients were selected from each group to form the new comparative groups. Similar results were obtained within the population after PSM. Conclusion Prior SARS-CoV-2 infection does not appear to affect postoperative rehabilitation, including liver function, postoperative complications, or hospitalization expenses among patients with HCC after elective partial hepatectomy.

Abstract Background Immune checkpoint inhibitors (ICIs) have become the recommended first-line treatment for advanced non-small cell lung cancer (NSCLC) without driver gene mutations. However, data on the failure patterns of first-line ICIs monotherapy is limited, and the optimal strategy for subsequent treatment remains controversial. Methods Advanced NSCLC patients receiving first-line ICIs monotherapy at Guangdong Lung Cancer Institute between December 2017 and October 2021 were identified. The progressive sites were recorded to analyze failure patterns. Post-progression survival (PPS) was compared between different treatment regimens. Results A total of 121 patients receiving first-line ICIs monotherapy were identified, with a median progression-free survival of 8.6 months. Sixty-five patients had available imaging at diagnosis as well as progressive disease, with 56.9% showing oligoprogression. For those with progression in existing lesions, the most common sites were the liver (77.8%) and lung parenchyma (62.5%), while progression with new lesions frequently occurred in the liver (32.0%). Fifty patients with recorded subsequent treatment were included in the analysis of subsequent treatment patterns. Patients treated with anti-angiogenesis therapy could get better PPS (HR: 0.275, P = 0.013). Isolated oligoprogression occurred most often in the lung parenchyma and intracranial lesions. More than half of these patients continued immunotherapy after local treatment, with a 2.5-year PPS rate of 51.4%. Conclusion The liver was the most common site of progression on first-line ICIs monotherapy. Anti-angiogenesis-based therapy might be an optimal regimen at the time of progression. Patients with isolated oligoprogressive could still benefit from immunotherapy after local treatment.

Introduction: Transformation to SCLC is a resistance mechanism to tyrosine kinase inhibitor in EGFR-mutated lung adenocarcinoma (LUAD). Nevertheless, the clinical and molecular features of SCLC transformation in LUAD with leptomeningeal metastases (LM) are scarce. Methods: We retrospectively collected 237 patients with NSCLC who underwent lumbar puncture owing to suggestion of LM. All SCLC transformation in cerebrospinal fluid (CSF) was confirmed by two experienced pathologists using cytologic evaluation. CSF circulating tumor DNA (ctDNA) was tested by next-generation sequencing. Results: Tumor cells in CSF samples were found in 111 patients (111 of 237, 46.8%), and eight cases (eight of 111, 7.2%) were identified as having SCLC cells in CSF. Seven patients carried the EGFR mutation, including four patients with EGFR exon 19 deletion and three patients with EGFR exon 21 L858R mutation. Another patient harbored ERBB2 insertion. Seven of these patients were resistant to targeted therapy. CSF ctDNA analysis reported that TP53 and RB1 mutations were common. The median time from the diagnosis of advanced NSCLC to SCLC transformation found in CSF was 9.7 months (95% confidence interval [CI]: 4.0–17.5 mo). The median overall survival since the initial diagnosis of metastatic NSCLC was 15.3 months (95% CI: 1.2–29.4 mo). The median overall survival after SCLC transformation detected in CSF was 5.0 months (95% CI: 4.0–5.9 mo). Conclusions: SCLC transformation may be revealed in CSF by both cytologic evaluation and ctDNA, not just in tissue that underwent rebiopsy. SCLC transformation of CSF is informative for resistance mechanism in patients with LUAD with LM on tyrosine kinase inhibitor progression, which was associated with poor survival.

Abstract Background Tertiary lymphoid structures (TLSs) affect the prognosis and efficacy of immunotherapy in patients with non‐small cell lung cancer (NSCLC), but the underlying mechanisms are not well understood. Methods TLSs were identified and categorized online from the Cancer Digital Slide Archive (CDSA). Overall survival (OS) and disease‐free survival (DFS) were analyzed. GSE111414 and GSE136961 datasets were downloaded from the GEO database. GSVA, GO and KEGG were used to explore the signaling pathways. Immune cell infiltration was analyzed by xCell, ssGSEA and MCP‐counter. The analysis of WGCNA, Lasso and multivariate cox regression were conducted to develop a gene risk score model based on the SU2C‐MARK cohort. Results TLS‐positive was a protective factor for OS according to multivariate cox regression analysis (p = 0.029). Both the TLS‐positive and TLS‐mature groups exhibited genes enrichment in immune activation pathways. The TLS‐mature group showed more activated dendritic cell infiltration than the TLS‐immature group. We screened TLS‐related genes using WGCNA. Lasso and multivariate cox regression analysis were used to construct a five‐genes (RGS8, RUF4, HLA‐DQB2, THEMIS, and TRBV12‐5) risk score model, the progression free survival (PFS) and OS of patients in the low‐risk group were markedly superior to those in the high‐risk group (p < 0.0001; p = 0.0015, respectively). Calibration and ROC curves indicated that the combined model with gene risk score and clinical features could predict the PFS of patients who have received immunotherapy more accurately than a single clinical factor. Conclusions Our data suggested a pivotal role of TLSs formation in survival outcome and immunotherapy response of NSCLC patients. Tumors with mature TLS formation showed more activated immune microenvironment. In addition, the model constructed by TLS‐related genes could predict the response to immunotherapy and is meaningful for clinical decision‐making.

Abstract Background The effects of gut microbiota and metabolites on the responses to immune checkpoint inhibitors (ICIs) in advanced epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) have been studied. However, their effects on EGFR-mutated (EGFR +) NSCLC remain unknown. Methods We prospectively recorded the clinicopathological characteristics of patients with advanced EGFR + NSCLC and assessed potential associations between the use of antibiotics or probiotics and immunotherapy efficacy. Fecal samples were collected at baseline, early on-treatment, response and progression status and were subjected to metagenomic next-generation sequencing and ultra-high-performance liquid chromatography-mass spectrometry analyses to assess the effects of gut microbiota and metabolites on immunotherapy efficacy. Results The clinical data of 74 advanced EGFR + NSCLC patients were complete and 18 patients’ fecal samples were dynamically collected. Patients that used antibiotics had shorter progression-free survival (PFS) (mPFS, 4.8 vs. 6.7 months; P = 0.037); probiotics had no impact on PFS. Two dynamic types of gut microbiota during immunotherapy were identified: one type showed the lowest relative abundance at the response time point, whereas the other type showed the highest abundance at the response time point. Metabolomics revealed significant differences in metabolites distribution between responders and non-responders. Deoxycholic acid, glycerol, and quinolinic acid were enriched in responders, whereas L-citrulline was enriched in non-responders. There was a significant correlation between gut microbiota and metabolites. Conclusions The use of antibiotics weakens immunotherapy efficacy in patients with advanced EGFR + NSCLC. The distribution characteristics and dynamic changes of gut microbiota and metabolites may indicate the efficacy of immunotherapy in advanced EGFR + NSCLC.

ABSTRACTMetabolism reprogramming within the tumor microenvironment (TME) can have a profound impact on immune cells. Identifying the association between metabolic phenotypes and immune cells in lung adenocarcinoma (LUAD) may reveal mechanisms of resistance to immune checkpoint inhibitors (ICIs). Metabolic phenotypes were classified by expression of metabolic genes. Somatic mutations and transcriptomic features were compared across the different metabolic phenotypes. The metabolic phenotype of LUAD is predominantly determined by reductase-oxidative activity and is divided into two categories: redoxhigh LUAD and redoxlow LUAD. Genetically, redoxhigh LUAD is mainly driven by mutations in KEAP1, STK11, NRF2, or SMARCA4. These mutations are more prevalent in redoxhigh LUAD (72.5%) compared to redoxlow LUAD (17.4%), whereas EGFR mutations are more common in redoxlow LUAD (19.0% vs. 0.7%). Single-cell RNA profiling of pre-treatment and post-treatment samples from patients receiving neoadjuvant chemoimmunotherapy revealed that tissue-resident memory CD8+ T cells are responders to ICIs. However, these cells are significantly reduced in redoxhigh LUAD. The redoxhigh phenotype is primarily attributed to tumor cells and is positively associated with mTORC1 signaling. LUAD with the redoxhigh phenotype demonstrates a lower response rate (39.1% vs. 70.8%, p = 0.001), shorter progression-free survival (3.3 vs. 14.6 months, p = 0.004), and overall survival (12.1 vs. 31.2 months, p = 0.022) when treated with ICIs. The redoxhigh phenotype in LUAD is predominantly driven by mutations in KEAP1, STK11, NRF2, and SMARCA4. This phenotype diminishes the number of tissue-resident memory CD8+ T cells and attenuates the efficacy of ICIs.

Summary: Leptomeningeal metastases (LMs) remain a devastating complication of non-small cell lung cancer (NSCLC), particularly following osimertinib resistance. We conducted single-cell RNA sequencing on cerebrospinal fluid (CSF) from EGFR-mutant NSCLC with central nervous system metastases. We found that macrophages of LMs displayed functional and phenotypic heterogeneity and enhanced immunosuppressive properties. A population of lipid-associated macrophages, namely RNASE1_M, were linked to osimertinib resistance and LM development, which was regulated by Midkine (MDK) from malignant epithelial cells. MDK exhibited significant elevation in both CSF and plasma among patients with LMs, with higher MDK levels correlating to poorer outcomes in an independent cohort. Moreover, MDK could promote macrophage M2 polarization with lipid metabolism and phagocytic function. Furthermore, malignant epithelial cells in CSF, particularly after resistance to osimertinib, potentially achieved immune evasion through CD47-SIRPA interactions with RNASE1_M. In conclusion, we revealed a specific subtype of macrophages linked to osimertinib resistance and LM development, providing a potential target to overcome LMs.

Abstract Objective NF1 is a tumor suppressor gene that encodes the neurofibromin protein and negatively regulates Ras signaling. This study was aimed to investigate the molecular, clinical characteristics, and prognostic features of NF1 gene in EGFR mutant lung cancer patients. Method The next‐generation sequencing (NGS) was used to analyze the data from lung cancer patients in the Guangdong Lung Cancer Institute (GLCI) from June 2016 to December 2020. Results Somatic NF1 mutations were present in 4.2% (135/3220) of Chinese lung cancer patients. NF1 mutations where clearly enriched in older (p

Background The liver is a frequent site of metastases and liver metastases (LM) correlate with diminished immunotherapy efficacy in non-small cell lung cancer (NSCLC). This study aimed to analyze whether tumor response to immunotherapy differs between pulmonary lesions (PL) and LM in NSCLC and to explore potential mechanisms through multiomics analysis.Methods This observational longitudinal clinical cohort study included patients with NSCLC with LM receiving immunotherapy was conducted to evaluate organ-specific tumor response of PL and LM. We collected paired PL and LM tumor samples to analyze the organ-specific difference using whole-exome sequencing, RNA sequencing, and multiplex immunohistochemistry.Results A total of 52 patients with NSCLC with LM were enrolled to evaluate the organ-specific response of immunotherapy. The objective response rate (21.1% vs 32.7%) and disease control rate of LM were lower than that of PL (67.3% vs 86.5%). One-third of patients showed mixed response, among whom 88.2% (15/17) presented with LM increasing, but PL decreasing, while the others had the opposite pattern (p=0.002). In another independent cohort, 27 pairs of matched PL and LM tumor samples from the same individuals, including six simultaneously collected pairs, were included in the translational part. Genomic landscapes profiling revealed similar somatic mutations, tumor mutational burden, and neoantigen number between PL and LM. Bulk-RNA sequencing showed immune activation-related genes including CD8A, LCK, and ICOS were downregulated in LM. The antigen processing and presentation, natural killer (NK) cell-mediated cytotoxicity and T-cell receptor signaling pathway were enriched in PL compared with LM. Multiplex immunohistochemistry detected significantly lower fractions of CD8+ cells (p=0.036) and CD56dim+ cells (p=0.016) in LM compared with PL. Single-cell RNA sequencing also characterized lower effector CD8+ T cells activation and NK cells cytotoxicity in LM.Conclusions Compared with PL, LM presents an inferior organ-specific tumor response to immunotherapy. PL and LM showed limited heterogeneity in the genomic landscape, while the LM tumor microenvironment displayed lower levels of immune activation and infiltration than PL, which might contribute to developing precise immunotherapy strategies for patients with NSCLC with LM.

Introduction: BRAF variants were reported resistant mechanisms to EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant NSCLC. Nevertheless, characteristics and subsequent treatment strategies of such patients remain unclear. Methods: From October 2016 to May 2020, patients with advanced NSCLC for whom next-generation sequencing detected mutations of both BRAF and EGFR were retrospectively included. From June 2020 to January 2021, patients with EGFR-mutant NSCLC who acquired the BRAF V600E mutation after progression on osimertinib were prospectively enrolled to explore the efficacy and safety of EGFR plus BARF co-inhibition. Results: A total of 58 patients were retrospectively identified and five prospectively included. BRAF variants were acquired after a median time of 22.7 months from initial diagnosis. The frequency of variations in TP53, PIK3CA, RB1, MET, LRP1B, APC, CDKN2A, MYC, ERBB2, and SMAD4 was all more than 10%; these mutations affected the cell cycle or p53 pathway and the EGFR downstream and bypass pathways. The median progression-free survival was 5.0 months for patients on chemotherapy and 2.1 months for those on TKIs not targeting both of EGFR and BRAF (p = 0.019). The median PFS was 7.8 months in five patients who received EGFR plus BRAF co-inhibitory drugs. RAS signaling was activated on disease progression. Conclusions: Variations in the EGFR downstream and bypass pathways were frequent in patients with dual mutations of EGFR and BRAF. The efficacies of TKIs not targeting both EGFR and BRAF were inferior to chemotherapy. EGFR plus BRAF co-inhibition improved efficacy. Such treatment strategies should be further explored.

Abstract. Background. Mounting evidence, consistent with our previous study, showed that γ-aminobutyric acid type A receptor (GABAAR) played an indispensable role in airway inflammation and mucus hypersecretion in asthma. Monocyte chemotactic protein-inducing protein 1 (MCPIP1) was a key negative regulator of inflammation. Recent studies showed that inflammation was largely suppressed by enhanced MCPIP1 expression in many inflammatory diseases. However, the role and potential mechanism of MCPIP1 in airway inflammation and mucus hypersecretion in asthma were still not well studied. This study was to explore the role of MCPIP1 in asthmatic airway inflammation and mucus hypersecretion in both mice and BEAS-2B cells, and its potential mechanism. Methods. In vivo, mice were sensitized and challenged by ovalbumin (OVA) to induce asthma. Airway inflammation and mucus secretion were analyzed. In vitro, BEAS-2B cells were chosen. Interleukin (IL)-13 was used to stimulate inflammation and mucus hypersecretion in cells. MCPIP1 Lentiviral vector (LA-MCPIP1) and plasmid-MCPIP1 were used to up-regulate MCPIP1 in lung and cells, respectively. MCP-1, thymic stromal lymphopoietin (TSLP), mucin 5AC (MUC5AC), MCPIP1, and GABAARβ2 expressions were measured in both lung and BEAS-2B cells. Immunofluorescence staining was performed to observe the expression of GABAARβ2 in cells. Results. MCPIP1 was up-regulated by LA-MCPIP1 (P

Immune checkpoint inhibitors (ICIs), especially anti-programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) antibodies, have made dramatic progress in the treatment of lung cancer, especially for patients with cancers not driven by oncogenes. However, responses are limited to a subset of patients, and which subset of patients will optimally benefit from ICI remains unknown. With the advantage of being minimally invasive and dynamic, noninvasive biomarkers are promising candidates to predict response, monitor resistance, and track the evolution of lung cancer during ICI treatment. In this review, we focus on the application of circulating tumor DNA (ctDNA) in plasma in immunotherapy. We examine the potential of pre- and on-treatment features of ctDNA as biomarkers, and following multiparameter analysis, we determine the potential clinical value of integrating predictive liquid biomarkers of ICIs to optimize patient management. We further discuss the role of ctDNA in monitoring treatment resistance, as well as challenges in clinical translation.

This study aimed to investigate the effects of gender pairings on collaborative problem-solving performance, processes, and attitudes in a social learning context. Three types of pairings (i.e., male-male, female-female, and mixed pairings) were considered in an empirical study with 222 tenth-grade students. The selection of three different schools facilitated discussions regarding which schools were more divergent and competitive in a social learning context. The students were asked to solve computer science problems on a social media platform. The results revealed that (1) the single-gender groups had more focused discussions than the mixed-gender groups. Specifically, the male-male groups tended to develop and test their solutions directly without spending significant time on problem identification. Consequently, the single-gender groups exhibited superior performance compared to the mixed-gender groups in terms of applying their knowledge to problem solving. In terms of attitudes toward social learning, the female-female groups were more attentive to the benefits of social learning than the male-male groups. (2) The mixed gender groups had more diverse and divergent discussions compared to the single-gender groups. The educational implications of these findings are also discussed in this paper.

Background Tyrosine kinase domain (TKD) mutation and particularly exon 20 insertion mutations of ERBB2 have been extensively reported in non‐small cell lung cancer (NSCLC). Due to the increased accessibility of next‐generation sequencing, more ERBB2 mutations within the non‐TKD can be detected in clinical practice. Nevertheless, the clinical significance of non‐TKD mutations remains unknown. Hence, this study was designed to comprehensively outline the landscape and characteristics of ERBB2 mutations in NSCLC. Methods A total of 1934 patients with NSCLC from cBioPortal were included in the study. An ERBB2 mutation cohort was identified, while subsequent analyses revealed clinical and genomic characteristics. Results The frequency of ERBB2 mutation was 4.5%, and it was determined to be more likely to occur in never‐smokers. ERBB2 mutations occurring in the non‐TKD accounted for 57.5% of ERBB2 mutations. In the non‐TKD, missense mutation was the most recurrent mutation type, and S310F was the most recurrent mutation variant. ERBB2 mutations within non‐TKD also had a strong oncogenic ability where up to 37.5% of ERBB2 oncogenic mutations were within non‐TKD. The co‐mutation of EGFR or KRAS was higher in the non‐TKD mutation compared to the TKD mutation. Shorter overall survival was observed in ERBB2‐mutant patients compared with ERBB2 wild‐type patients. There was no significant difference in overall survival between patients with non‐TKD mutations and TKD mutations. Conclusions The present study showed that a considerable portion of non‐TKD mutations were oncogenic. ERBB2 mutation was a poor prognostic factor. The non‐TKD mutation might also be used as a therapeutic target in ERBB2‐directed target therapy. Key points • Significant findings of the study ERBB2 mutations were more abundant within a nontyrosine domain than those within the tyrosine domain. Up to 37.5% of ERBB2 oncogenic mutations were within the nontyrosine domain. ERBB2 mutation was a poor prognostic factor. • What this study adds The frequency of EGFR or KRAS co‐mutations were significantly higher in ERBB2 mutations within the nontyrosine kinase domain compared to ERBB2 mutations within the tyrosine kinase domain. Nontyrosine domain mutations confer equal overall survival to tyrosine domain mutations.

Abstract Background In previous studies, the predictive role of BIM deletion polymorphism with respect to responses to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) has been controversial. The potential reasons for these inconsistent findings were unknown. Methods Data from CTONG0901 clinical trial and medical records of Guangdong Lung Cancer Institute (GLCI) were retrospectively pooled. A total of 194 and 141 EGFR‐mutant non‐small cell lung cancer (NSCLC) patients treated with first‐ and second‐generation EGFR‐TKIs were examined in the CTONG0901 and GLCI cohorts, respectively. Sixty‐eight patients were treated with third‐generation EGFR‐TKIs in the GLCI cohort. The BIM gene status was examined by next‐generation sequencing. Results The frequency of BIM deletion polymorphism was 11.3% and 17.0% in CTONG0901 and GLCI cohorts, respectively. For first‐ and second‐generation EGFR‐TKIs in CTONG0901 cohort, objective response (ORR) was 54.5% in BIM deletion group versus 56.4% in wild‐type BIM group (P = .87); disease control rate (DCR) was 90.9% versus 88.4% (P = 1.00); progression‐free survival (PFS) was 10.5 versus 11.2 months (P = .59); and overall survival (OS) was 20.5 versus 20.5 months (P = .73). In GLCI cohort, ORR was 54.2% versus 60.7% (P = .55); DCR was 91.7% versus 96.6% (P = .27); PFS was 10.1 versus 11.6 months (P = .63); and OS was 58.5 versus 45.0 months (P = .93). For third‐generation EGFR‐TKIs, ORR was 18.2% versus 63.2% (P = .02); DCR was 81.8% versus 96.5%, (P = .12); PFS was 5.8 versus 9.0 months (P = .13); and OS was 30.0 versus 24.8 months (P = .85). Cox regression analysis showed that concomitant genetic alterations could adversely affect the response to EGFR‐TKIs, but not BIM deletion. Conclusions The presence of BIM deletion showed no relation to an impaired response to first‐, second‐, and third‐generation EGFR‐TKIs in NSCLC patients. The factors influencing the response of EGFR‐TKIs were concomitant genetic alterations, but not BIM deletion.

Abstract Numerous studies have shown that learning is enhanced when students show interest in the subject matter. However, educators continue to grapple with the challenges, or simply do not recognize their potential roles, in the development of students’ academic interest and interest in learning in general. This conceptual paper is written under the auspices of the interest-driven creator (IDC) initiative, a theoretical synthesis effort carried out by a group of educational researchers in Asia. The intention is to co-construct a holistic developmental/design framework to guide the students in fostering their learning interests, capabilities in creation, and learning habits—the three anchored concepts of IDC theory. This paper focuses on delineating a three-component “interest loop” to guide the design of a coherent learning process that encompasses a series of learning tasks. The three components are triggering interest, immersing interest, and extending interest. Underpinned by the rich literature on interest development, we will propose suitable design strategies for each of the three components, namely, curiosity, flow, and meaningfulness, respectively. We will then explicate their respective design considerations/principles to maximize the intended effects.

The Qinghai–Tibet Plateau Area (QTPA) has a complex natural ecosystem, causing a greatly increased risk of spreading various tick-borne diseases including rickettsial infections, which are regarded as one of the oldest known vector-borne zoonoses. However, the information of one of its pathogen, spotted fever group Rickettsia (SFG Rickettsia), is limited in tick vectors and animals in this area. Therefore, this study focused on the investigation of SFG Rickettsia in tick vectors, yaks (Bos grunniens), and Tibetan sheep (Ovis aries) in the QTPA. A total of 1,000 samples were collected from nine sampling sites, including 425 of yaks, 309 of Tibetan sheep, 266 of ticks. By morphological examination, PCR, and sequencing, we confirmed the species of all collected ticks. All tick samples, all yak and Tibetan sheep blood samples were detected based on SFG Rickettsia ompA and sca4 gene. The results showed that all tick samples were identified to be Haemaphysalis qinghaiensis, and the positive rates of SFG Rickettsia were 5.9% (25/425), 0.3% (1/309), and 54.1% (144/266) in yaks, Tibetan sheep, and ticks, respectively. All positive samples were sequenced, and BLASTn analysis of the ompA gene sequences of SFG Rickettsia showed that all positive samples from animals and ticks had 99.04–100% identity with yak and horse isolates from Qinghai Province, China. BLASTn analysis of the sca4 gene sequences of SFG Rickettsia showed that all positive samples had 97.60–98.72% identity with tick isolates from Ukraine. In addition, the phylogenetic analysis showed that all the SFG Rickettsia ompA and sca4 sequences obtained from this study belong to the same clade as Rickettsia raoultii isolated from livestock and ticks from China and other countries. Molecularly, this study detected and characterized SFG Rickettsia both in the tick vectors and animals, suggesting that the relationship between SFG Rickettsia, tick species and animal hosts should be explored to understand their interrelationships, which provide a theoretical basis for preventing control of this pathogen.

Backgrounds Immunotherapy is less effective in patients with epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer (NSCLC). Lower programmed cell death-ligand 1 (PD-L1) expression and tumor mutation burden (TMB) are reported to be the underlying mechanism. Being another important factor to affect the efficacy of immunotherapy, tumor microenvironment (TME) characteristics of this subgroup of NSCLC are not comprehensively understood up to date. Hence, we initiated this study to describe the specific TME of EGFR-mutant lung adenocarcinoma (LUAD) from cellular compositional and functional perspectives to better understand the immune landscape of this most common subtype of NSCLC.Methods We used single-cell transcriptome sequencing and multiplex immunohistochemistry to investigate the immune microenvironment of EGFR-mutant and EGFR wild-type LUADs and determined the efficacy of immunotherapy. We analyzed single cells from nine treatment-naïve samples and compared them to three post-immunotherapy samples previously reported from single cell perspective using bioinformatics methods.Results We found that EGFR-mutant malignant epithelial cells had similar characteristics to the epithelial cells in non-responders. EGFR-mutant LUAD lacked CD8+ tissue-resident memory (TRM) cells, which could promote tertiary lymphoid structure generation by secreting CXCL13. In addition, other cell types, including tumor-associated macrophages and cancer-associated fibroblasts, which are capable of recruiting, retaining, and expanding CD8+ TRM cells in the TME, were also deficient in EGFR-mutant LUAD. Furthermore, EGFR-mutant LUAD had significantly less crosstalk between T cells and other cell types via programmed cell death-1 (PD-1) and PD-L1 or other immune checkpoints compared with EGFR wild-type LUAD.Conclusions Our findings provide a comprehensive understanding of the immune landscape of EGFR-mutant LUAD at the single-cell level. Based on the results, many cellular components might have negative impact on the specific TME of EGFR-mutant LUAD through influencing CD8+ TRM. Lack of CD8+ TRM might be a key factor responsible for the suppressive TME of EGFR-mutant LUAD.

Background: Treatment by immune checkpoint blockade (ICB) provides a remarkable survival benefit for multiple cancer types. However, disease aggravation occurs in a proportion of patients after the first couple of treatment cycles. Methods: RNA sequencing data was retrospectively collected. 6 tumour-immune related features were extracted and combined to build a lung cancer-specific predictive model to distinguish responses as progression disease (PD) or non-PD. This model was trained by 3 public pan-cancer datasets and a lung cancer cohort from our institute, and generated a lung cancer-specific integrated gene expression score, which we call LITES. It was finally tested in another lung cancer dataset. Results: LITES is a promising predictor for checkpoint blockade (area under the curve [AUC]=0.86), superior to traditional biomarkers. It is independent of PD-L1 expression and tumour mutation burden. The sensitivity and specificity of LITES was 85.7% and 70.6%, respectively. Progression free survival (PFS) was longer in high-score group than in low-score group (median PFS: 6.0 vs. 2.4 months, hazard ratio=0.45, P=0.01). The mean AUC of 6 features was 0.70 (range=0.61-0.75), lower than in LITES, indicating that the combination of features had synergistic effects. Among the genes identified in the features, patients with high expression of NRAS and PDPK1 tended to have a PD response (P=0.001 and 0.01, respectively). Our model also functioned well for patients with advanced melanoma and was specific for ICB therapy. Conclusions: LITES is a promising biomarker for predicting an impaired response in lung cancer patients and for clarifying the biological mechanism underlying ICB therapy.

Abstract The interest-driven creator (IDC) theory is being developed as a group endeavor by Asian researchers to articulate a holistic learning design theory for future education in Asia. The theory hypothesizes that students, driven by interest, can be engaged in the creation of knowledge (generating ideas and artifacts). By repeating this creation process in their daily learning routines, they will excel in learning performance, develop twenty-first-century competencies, and form creation habits. We hope that with such practices in education, our future generations will ultimately become lifelong interest-driven creators. In IDC Theory, there are three anchored concepts, namely, interest, creation, and habit. Each anchored concept comprises three component concepts which form a concept loop. For example, the creation loop consists of three component concepts—imitating, combining, and staging. Imitating is concerned with taking in (or inputting) an abundant amount of existing knowledge from the outside world to form one’s background knowledge. Combining refers to delivering (or outputting) new ideas or artifacts prolifically by synthesizing existing information encountered in the world and thoughts arising from the students’ background knowledge. Staging relates to frequently demonstrating the generated ideas or artifacts to the relevant communities and receiving feedback from these communities to improve the novelty and value of the demonstrated outcomes while gaining social recognition and nurturing positive social emotions. This paper focuses on describing the three components of the creation loop. We provide three case studies to illustrate the creation loop at work, as well as how it intertwines with both the interest and habit loops in supporting students to develop their creation capabilities. In presenting this iteration of the creation concept, an anchored concept in IDC theory, we acknowledge the roles played of imitation, combination, and staging in different learning and education contexts—indeed, there are multiple theories that inform and intersect with it.

Background: Resistance to third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) presents a major clinical challenge in advanced non-small cell lung cancer (NSCLC). Here, we report resistance mechanisms to abivertinib, a novel third-generation EGFR TKI, from a phase I dose-escalation/expansion study (NCT02330367). Methods: Patients with EGFR T790M-positive advanced NSCLC and progression on prior EGFR TKIs received abivertinib in dose escalation (50–350 mg twice daily [BID]) or expansion (300 mg BID) cohorts. Patients enrolled at Guangdong Lung Cancer Institute who underwent next-generation sequencing (NGS)-based genomic profiling upon abivertinib progression (prior to October 30, 2018) were enrolled in this exploratory analysis. Findings: Thirty of 73 patients enrolled were eligible for resistance analysis. Upon abivertinib progression, 27 patients provided plasma samples (six patients also provided paired samples from the progression sites) and three patients only provided tissue samples from the progression sites for NGS. A heterogeneous landscape of resistance to abivertinib was observed: 15% (4/27) experienced EGFR T790M loss and 13% (4/30) developing EGFR tertiary mutations including C797S. EGFR amplification was observed in 11 patients (37%), and considered a putative resistance mechanism in seven (23%) patients. Other EGFR-independent resistance mechanisms involved CDKN2A, MET, PIK3CA, HER2, TP53, Rb1 and small-cell lung cancer transformation. Interpretation: Our findings reveal a heterogenous pattern of resistance mechanisms to abivertinib which is distinct from that previously reported with osimertinib. EGFR amplification was the most common resistance mechanism in this cohort. Fund: The National Key R&D Program of China (Grant No. 2016YFC1303800), Key Lab System Project of Guangdong Science and Technology Department – Guangdong Provincial Key Lab of Translational Medicine in Lung Cancer (Grant No. 2012A061400006/2017B030314120). Keywords: Abivertinib, Resistance mechanisms, EGFR T790M mutation, NSCLC, EGFR amplification

Abstract Conventional teacher-led instruction remains dominant in most elementary mathematics classrooms in Taiwan. Under such instruction, the teacher can rarely take care of all students. Many students may then continue to fall behind the standard of mathematics achievement and lose their interest in mathematics; they eventually give up on learning mathematics. In fact, students in Taiwan generally have lower interest in learning mathematics compared to many other regions/countries. Thus, how to enhance students’ mathematics achievement and interest are two major problems, especially for those low-achieving students. This paper describes how we designed a game-based learning environment, called Math-Island, by incorporating the mechanisms of a construction management game into the knowledge map of the elementary mathematics curriculum. We also report an experiment conducted with 215 elementary students for 2 years, from grade 2 to grade 3. In this experiment, in addition to teacher-led instruction in the classroom, students were directed to learn with Math-Island by using their own tablets at school and at home. As a result of this experiment, we found that there is an increase in students’ mathematics achievement, especially in the calculation and word problems. Moreover, the achievements of low-achieving students in the experimental school outperformed the low-achieving students in the control school (a control group in another school) in word problems. Moreover, both the low-achieving students and the high-achieving students in the experimental school maintained a rather high level of interest in mathematics and in the system.

BackgroundEchinoderm microtubule-associated protein-like 4 (EML4) is the canonical anaplastic lymphoma kinase (ALK) fusion partner in non-small cell lung cancer (NSCLC), and ALK-positive patients showed promising responses to ALK tyrosine kinase inhibitors (TKIs). However, studies that comprehensively investigate ALK TKI treatment in patients with different ALK fusion patterns are still lacking.MethodsNinety-eight ALK-positive patients with advanced NSCLC were retrospectively studied for their response to crizotinib and subsequent treatments. Comprehensive genomic profiling (CGP) was conducted to divide patients into different groups based on their ALK fusion patterns. Non-canonical ALK fusions were validated using RNA-sequencing.Results54.1% of patients had pure canonical EML4-ALK fusions, 19.4% carried only non-canonical ALK fusions, and 26.5% harbored complex ALK fusions with coexisting canonical and non-canonical ALK fusions. The objective response rate and median progression-free survival to crizotinib treatment tended to be better in the complex ALK fusion group. Notably, patients with complex ALK fusions had significantly improved overall survival after crizotinib treatment (p = 0.012), especially when compared with the pure canonical EML4-ALK fusion group (p = 0.010). The complex ALK fusion group also tended to respond better to next-generation ALK TKIs, which were used as later-line therapies. Most identified non-canonical ALK fusions were likely to be expressed in tumors, and some of them formed canonical EML4-ALK transcripts during mRNA maturation.ConclusionOur results suggest NSCLC patients with complex ALK fusions could potentially have better treatment outcomes to ALK TKIs therapy. Also, diagnosis using CGP is of great value to identify novel ALK fusions and predict prognosis.

Abstract Purpose The aim of this was to discover disease‐causing gene mutations linked to genetic epilepsy with febrile seizures plus (GEFS+) in a family in the Southern Chinese Han population. Of a three‐generation pedigree of 18 members in this family, 4 were affected with GEFS+. Method Blood samples of 7 family members—3 affected and 4 unaffected individuals—were collected. Whole‐exome sequencing was performed to assess for genetic mutations in two of the affected individuals and two of the unaffected individuals. Results Fourteen potentially consequential mutations were found in the two affected individuals and were validated with the Sanger sequencing method. Blood DNA tested in polymerase chain reaction with KCNAB3 primers revealed that one novel missense mutation, c.773A>G (p.H258R) in the KCNAB3 gene, which encoded the potassium voltage‐gated channel subfamily A regulatory β subunit 3 (KCNAB3), was shared by all three affected and one unaffected family member. However, this mutation did not appear in 300 unrelated control subjects. According to the bioinformatics tools SIFT and PROVEAN, p.H258R was thought to affect protein function. Functional verification showed that the KCNAB3 mutation could accelerate the inactivation of potassium channels, thus inhibiting potassium current, increasing neuronal excitability, and promoting epileptic convulsion. Conclusions These results reveal that mutations in the KCNAB3 gene may be associated with GEFS+.

Background Immunotherapy has become an important treatment option for patients with advanced non-small cell lung cancer (NSCLC). At present, none of these existing biomarkers can effectively stratify true responders and there is an urgent need for identifying novel biomarkers. Exosomes derived from the serum of patients with cancer have been proven to be reliable markers for cancer diagnosis. Here, we explored the possibility of using plasma-derived exosomal microRNAs as potential biomarkers for optimal selection of patients with advanced EGFR/ALK negative NSCLC to immunotherapy.Methods From June 2017 to February 2019, 30 patients with advanced EGFR/ALK wild-type (WT) NSCLC who received PD-1/PD-L1 inhibitors were enrolled. The efficacy evaluation was conducted after every three cycles of treatment according to RECIST 1.1. Plasma samples of these patients were collected before the administration of PD-1/PD-L1 inhibitors as baseline, and after every three cycles if the patients achieved partial response (PR) or complete response. Plasma from seven healthy individuals was also collected as normal control. Exosomes were prepared by ultracentrifugation followed by total RNA extraction, and exosome-derived miRNAs were profiled using small RNA next-generation sequencing followed by differential expression analysis.Results In order to identify biomarker for better response, all five patients who achieved PR and four patients with progressive disease (PD) at efficacy evaluation were included for differential expression analysis. Based on unsupervised hierarchical clustering, exosomal miRNA expression profile was significantly altered in patients with NSCLC compared with normal controls with a total of 155 differentially expressed exosomal miRNAs. Interestingly, hsa-miR-320d, hsa-miR-320c, and hsa-miR-320b were identified significantly upregulated in the PD groups compared with the PR group at baseline before the treatment. In addition, we identified that hsa-miR-125b-5p, a T-cell suppressor, showed a trend of increased expression in the PD group at baseline and was significantly downregulated in the post-treatment plasma exosomes compared with pre-treatment samples of the PR patients.Conclusion Patients with NSCLC represent unique plasma exosomal miRNA profiles. Hsa-miR-320d, hsa-miR-320c, and hsa-miR-320b were identified as potential biomarkers for predicting the efficacy of immunotherapy in advanced NSCLCs. When T-cell suppressor hsa-miR-125b-5p was downregulated during the treatment, the patients may obtain increased T-cell function and respond well to immunotherapy.

Background: Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectrometry platform. Methods: A review of the literature revealed 60 mutation hotspots in seven target genes (EGFR, KRAS, PIK3CA, BRAF, ERBB2, NRAS, and BIM) that are closely related to EGFR TKI resistance to lung cancer. A total of 183 primers comprised 61 paired forward and reverse amplification primers, and 61 matched extension primers were designed using Assay Design Software. The detection method was established by analyzing nine cell lines, and by comparison with LungCarta™ kit in ten lung cancer specimens. EGFR, KRAS, and BIM genes in all cell lines and clinical samples were subjected to Sanger sequencing for confirming reproducibility. Results: Our data showed that designed panel was a high-throughput and robust tool, allowing genotyping for sixty hotspots in the same run. Moreover, it made efficient use of patient diagnostic samples for a more accurate EGFR TKIs resistance analysis. The proposed method could accurately detect mutations in lung cancer cell lines and clinical specimens, consistent with those obtained by the LungCarta™ kit and Sanger sequencing. We also established a method for detection of large-fragment deletions based on single-base extension technology of MassARRAY platform. Conclusions: We established an effective method for high-throughput detection of genetic mutations related to EGFR TKI resistance based on the MassARRAY platform, which could provide more accurate information for overcoming cancers with de novo or acquired resistance to EGFR-targeted therapies.

Abstract Background Detecting epidermal growth factor receptor (EGFR) activating mutations in plasma could guide EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment for advanced non-small cell lung cancer (NSCLC). However, dynamic quantitative changes of plasma EGFR mutations during the whole course of EGFR-TKI treatment and its correlation with clinical outcomes were not determined. The aim of this study was to measure changes of plasma EGFR L858R mutation during EGFR-TKI treatment and to determine its correlation with the response and resistance to EGFR-TKI. Methods This study was a pre-planned exploratory analysis of a randomized phase III trial conducted from 2009 to 2014 comparing erlotinib with gefitinib in advanced NSCLC harboring EGFR mutations in tumor (CTONG0901). Totally, 256 patients were enrolled in CTONG0901 and randomized to receive erlotinib or gefitinib. One hundred and eight patients harbored L858R mutation in their tumors and 80 patients provided serial blood samples as pre-planned scheduled. Serial plasma L858R was detected using quantitative polymerase chain reaction. Dynamic types of plasma L858R were analyzed using Ward’s hierarchical clustering method. Progression-free survival (PFS) and overall survival (OS) were compared between different types. Results As a whole, the quantity of L858R decreased and reached the lowest level at the time of best response to EGFR-TKI. After the analysis of Ward’s hierarchical clustering method, two dynamic types were found. In 61 patients, L858R increased to its highest level when disease progressed (ascend type), while in 19 patients, L858R maintained a stable level when disease progressed (stable type). Median PFS was 11.1 months (95 % CI, 6.6–15.6) and 7.5 months (95 % CI, 1.4–13.6) in patients with ascend and stable types, respectively (P = 0.023). Median OS was 19.7 months (95 % CI, 16.5–22.9) and 16.0 months (95 % CI, 13.4–18.5), respectively (P = 0.050). Conclusions This is the first report finding two different dynamic types of plasma L858R mutation during EGFR-TKI treatment based on a prospective randomized study. Different dynamic types were correlated with benefits from EGFR-TKI. The impact of plasma L858R levels at disease progression on subsequent treatment strategy needs further exploration. Trial registration ClinicalTrials.gov, NCT01024413

Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs.

AIMS: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells. METHODS: We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry. RESULTS: In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels. CONCLUSION: Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.

Bioluminescence reporter proteins have been widely used in the development of tools for monitoring biological events in living cells. Currently, some assays like flow cytometry analysis are available for studying DNA synthetic phase (S-phase) targeted anti-cancer drug activity in vitro; however, techniques for imaging of in vivo models remain limited. Cyclin A2 is known to promote S-phase entry in mammals. Its expression levels are low during G1-phase, but they increase at the onset of S-phase. Cyclin A2 is degraded during prometaphase by ubiquitin-dependent, proteasome-mediated proteolysis. In this study, we have developed a cyclin A2-luciferase (CYCA-Luc) fusion protein targeted for ubiquitin-proteasome dependent degradation, and have evaluated its utility in screening S-phase targeted anti-cancer drugs. Similar to endogenous cyclin A2, CYCA-Luc accumulates during S-phase and is degraded during G2/M-phase. Using Cdc20 siRNA we have demonstrated that Cdc20 can mediate CYCA-Luc degradation. Moreover, using noninvasive bioluminescent imaging, we demonstrated accumulation of CYCA-Luc in response to 10-hydroxycamptothecin (HCPT), an S-phase targeted anti-cancer drug, in human tumor cells in vivo and in vitro. Our results indicate that a CYCA-Luc fusion reporter system can be used to monitor S-phase of cell cycle, and evaluate pharmacological activity of anti-cancer drug HCPT in real time in vitro and in vivo, and is likely to provide an important tool for screening such drugs.

BACKGROUND: Appropriate patient selection is needed for targeted therapies that are efficacious only in patients with specific genetic alterations. We aimed to define subgroups of patients with candidate driver genes in patients with non-small cell lung cancer. METHODS: Patients with primary lung cancer who underwent clinical genetic tests at Guangdong General Hospital were enrolled. Driver genes were detected by sequencing, high-resolution melt analysis, qPCR, or multiple PCR and RACE methods. RESULTS: 524 patients were enrolled in this study, and the differences in driver gene alterations among subgroups were analyzed based on histology and smoking status. In a subgroup of non-smokers with adenocarcinoma, EGFR was the most frequently altered gene, with a mutation rate of 49.8%, followed by EML4-ALK (9.3%), PTEN (9.1%), PIK3CA (5.2%), c-Met (4.8%), KRAS (4.5%), STK11 (2.7%), and BRAF (1.9%). The three most frequently altered genes in a subgroup of smokers with adenocarcinoma were EGFR (22.0%), STK11 (19.0%), and KRAS (12.0%). We only found EGFR (8.0%), c-Met (2.8%), and PIK3CA (2.6%) alterations in the non-smoker with squamous cell carcinoma (SCC) subgroup. PTEN (16.1%), STK11 (8.3%), and PIK3CA (7.2%) were the three most frequently enriched genes in smokers with SCC. DDR2 and FGFR2 only presented in smokers with SCC (4.4% and 2.2%, respectively). Among these four subgroups, the differences in EGFR, KRAS, and PTEN mutations were statistically significant. CONCLUSION: The distinct features of driver gene alterations in different subgroups based on histology and smoking status were helpful in defining patients for future clinical trials that target these genes. This study also suggests that we may consider patients with infrequent alterations of driver genes as having rare or orphan diseases that should be managed with special molecularly targeted therapies.