First-generation epidermal growth factor receptor (EGFR) targeted kinase inhibitors (TKIs) are still used in selected non-small cell lung cancer (NSCLC) patients despite the resistance. Based on the correlation of programmed cell death receptor ligand 1 (PD-L1) and EGFR signaling pathway, whether continuous TKIs treatment will affect PD-L1 expression after disease progression remains unclear. To investigate the potential change of PD-L1 expression in TKI-resistant NSCLC after continuous TKIs treatment, we treated H1975 and HCC827 for more than one month and explored the possible effect on immune cells as well as underlying biological mechanisms. We found that continuous exposure to TKIs induced upregulation of PD-L1 in H1975 and HCC827. Moreover, PD-L1 upregulation significantly inhibited proliferation and slightly promoted apoptosis of T cells. We observed the activation of STAT3 and ERK1/2 along with the PD-L1 upregulation. With the pathway inhibitors, we found ERK1/2 pathway involved in inducing PD-L1 in resistant lung cancer. This study provides preclinical evidence that continuous TKIs treatment may induce PD-L1 expression in resistant NSCLC, resulting in the suppression of T cell function and immune escape. ERK1/2 pathway inhibitors, PD-L1/PD-1 inhibitors or combination strategies should be considered to reverse the resistance to TKIs in NSCLC patients. It is still urgent to optimize therapy strategies for patients with advanced disease. Currently, 83% of lung cancers are classified as non-small cell lung cancer (NSCLC), most of which are at an advanced stage when the first diagnosis is performed. Chemotherapy with or without radiation therapy used to be the standard resolution, but in recent decade targeted kinase inhibitors (TKIs) are proved to be superior, especially in the oncogene-driven tumors, such as epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK)5. EGFR, also named HER1/erbB1, is a critical member of the HER/erbB family of receptor tyrosine kinases (RTKs). About 85–90% mutations in the TK domain of EGFR are exon 19 deletions and exon 21 L858R mutations, resulting in constitutive phosphorylation of important tyrosine residues and activation of downstream signaling pathways (such as mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription(STAT))7. Tumors bearing these EGFR mutations are particular sensitive to EGFR TKIs compared to those with wild-type EGFR8. EGFR TKIs reversibly inhibit EGFR activity through competing with adenosine triphosphate (ATP) for binding to the receptor’s kinase pocket, thus blocking EGFR auto-phosphorylation. Unfortunately, widespread acquired resistance to TKIs always takes place within 6 to 12 months, which greatly restricts the long-term efficacy of these drugs. The most common mechanism of acquired resistance is a second EGFR mutation on threonine 790 in the ATP binding pocket, named T790M9. The T790M mutation increases the ATP affinity of the oncogenic L858R mutant and sterically interferences the binding of TKIs10.Lung cancer remains the leading health challenge to humanity worldwide, with the second highest incidence and the highest mortality in both males and females12. However, whether initial TKI therapy should be continued in resistant NSCLC has been debated. The IMPRESS trial indicated the continuation of Gefitinib failed to prolong progression-free survival in resistant NSCLC when combined with platinum-based doublet chemotherapy13. On the contrary, a retrospective study showed survival benefit from EGFR-TKIs beyond progressive disease compared to cytotoxic chemotherapy14. The ASPIRATION trial suggested Erlotinib was feasible for selected patients after progression15. As a result, Gefitinib and Erlotinib are still utilized in some TKI-resistant NSCLC in spite of possible limited benefit.Currently, new generation TKIs (such as Afatinib and Osimertinib), toxic therapy, immunotherapy or combination strategies are advocated to deal with this intricate situation16. The PD-1/PD-L1 pathway transfers inhibitory immune signals, which can limit tumor-infiltrating CD4+ and CD8+ T cells and contribute to immune evasion17. Accordingly, Gefitinib and Erlotinib may have a notable influence on the PD-L1 expression through changing the downstream signal pathways of EGFR, such as MAPK, PI3K, Janus kinase (JAK)/STAT). However, despite the initial inhibition of PD-L1 in EGFR-driven tumor, very limited information is known about the effect of continuous TKIs treatment on PD-L1 expression when NSCLC become resistant to TKIs. Based on STAT3 activation after continuous TKI treatment in our previous research18, we hypothesized that PD-L1 expression will increase in resistant NSCLC with the continuation of TKIs. To test the hypothesis, we treated H1975 and HCC827 for more than one month and tended to explore the possible effect on immune cells and the underlying biological mechanisms. Identifying the possible change of immune checkpoint will provide important information before clinical treatment strategies are made.As reported recently, the expression of programmed cell death receptor ligand 1 (PD-L1) could be induced by the oncogenic EGFR mutation and reduced apparently by EGFR TKIs in EGFR-driven tumorTo examine the influence of continuous exposure to TKIs on the PD-L1 expression in TKI-resistant NSCLC, H1975 cells which harbor the EGFR L858R/T790M double mutations were treated with Gefitinib or Erlotinib for 5 weeks. PD-L1 expression gradually upregulated with Gefitinib and Erlotinib treatment as showed by western blot Fig. . Though 19. To investigate the effect of acquired resistance on PD-L1 expression in NSCLC, HCC827 (EGFR Del 19), which is sensitive to TKIs therapy, was exposed to Gefitinib (2 μM) for more than 6 months. Finally, HCC827 became resistant to Gefitinib with increased IC50 values of 15.22 μM  were isolated from healthy donor and expanded 827 Fig. . When T 827 Fig. . Given t827 Fig. , the par20. To elucidate the mechanism involved in PD-L1 upregulation, we assessed the STAT3 signaling pathway. The level of total STAT3 and p-STAT3 (Tyr705) exhibited increase trend during the TKIs treatment  was another critical pathway for PD-L1 regulationime Fig. . Likewisime Fig. . The res22. Along with the development of new effective agents targeting specific resistance mechanisms, a correlation between immune suppression and TKIs resistance is gradually being understood, indicating that immunotherapy can be a promising way to deal with the situation. Here, our study showed that PD-L1 expression was upregulated by continuous TKI treatment in resistant NSCLC resulting in persistent immune suppression.In the past decade, EGFR-TKIs yield significant success in the treatment of lung cancer. Routine test for EGFR mutation status characterizes NSCLC patients into different subgroups, resulting in encouraging efficacy and survival benefit in selected ones. However, primary or secondary drug resistance usually inevitably emerge over time, limiting the duration of response25. However, Jiang, X.et al. demonstrated that resistance to selective BRAF inhibition increased PD-L1 expression in melanoma19, which means there is a dynamic change of PD-L1 expression during the TKIs treatment. In our study, enduring use of EGFR TKIs in NSCLC induced not only drug resistance but also the increase of PD-L1. Notably a new correlation was revealed between the resistance of EGFR TKIs and PD-L1 expression in the long-term treatment setting.An increasing body of evidence indicates that the activation of common oncogenic drivers in NSCLC  is correlated with the induction of PD-L1 and corresponding inhibitors will reduce PD-L1 expression in initial treatment28. So far, immune checkpoint inhibitor therapy has been considered a revolutionary treatment in lung cancer. After the success of CTLA-4 inhibitors in metastatic melanoma30, PD-L1 inhibitors show unprecedented responses in numerous clinical trials. PD-L1 expresses on a wide range of hematopoietic and non-hematopoietic cells including human cancer cells. High expression of PD-L1 is now regarded as a poor prognostic factor, contributing to immune escape and disease progression31. We found that PD-L1 upregulation mainly inhibited proliferation rather than promoted apoptosis of T cell, which would greatly restrain the antitumor immunity. The result suggested upregulation of PD-L1 is able to induce the immune suppress, indicating optional immune targeted therapy.Early studies reported that PD-L1 on tumor cells was sufficient for tumor immune evasion through directly inhibiting CD8+ T cell responses. Therapeutically blocking the PD-1 pathway leaded to immune reinvigoration and tumor regression33. Our findings indicated that the increased phosphorylation of ERK1/2 pathway was engaged in inducing PD-L1 in resistant lung cancer. ERK1/2 pathway participates in cell proliferation, differentiation, and development and is critical for tumor survival. Meanwhile, previous study showed that activation of ERK1/2 signaling implicated in the resistance to EGFR kinase inhibitors34, suggesting an overlap in signaling mechanisms between TKI resistance and PD-L1 modulation. Moreover, clinical trials combining BRAF and MEK inhibitors with immune checkpoint blockades have been shown manageable safety profile and enhanced antitumor activity in melanoma36. As patients benefit temporarily from all the TKIs because of the acquired resistance, therapeutic synergy between EGFR-TKIs and PD-L1/PD-1 blockades or targeting downstream ERK1/2 signaling may prolong responses to these agents and delay the salvage treatment38.A number of preclinical studies have explored the possible mechanisms of PD-L1 modulation. The reported molecular mechanisms include MAPK pathway, PI3K-mTOR-S6 pathway, PI3K/PKB pathway, and JAK/STAT pathway, most of which have been extensively investigated in melanoma39. It was reported previously that STAT3 could bind to the PD-L1 promoter and modulated PD-L1 expression at the transcriptional level, and its inhibitors could decrease PD-L1 level in both parental and resistant melanoma19. Interestingly, the STAT3 inhibitor had no suppression effect on PD-L1 expression according to our study, conversely, strongly induced it, while ERK1/2 inhibitor suppressed STAT3 signaling at the same time but decreased PD-L1 expression successfully. Given that STAT3 siRNAs decreased PD-L1 expression by only 10–32% in two KRAS-mutant lung adenocarcinoma cell lines23, it is possible that STAT3 only partially involved. Considering the complexity of transcription and signaling networks, further investigation is needed to confirm the function of STAT3 in the regulation of PD-L1 in resistant lung cancer.STAT3 belongs to the STAT protein family, which involves various biological processesIn conclusion, continuous TKIs treatment upregulates PD-L1 expression in resistant NSCLC, contributing to the suppression of T cell function and immune escape. ERK1/2 pathway plays a vital role in the regulation of PD-L1. Thus, reassessment of genetic alterations and signaling pathways after clinical progression is necessary for subsequent therapy. Based on our preclinical study, ERK pathway inhibitors, PD-L1/PD-1 inhibitors or combination strategies should be considered to overcome the TKI resistance and improve outcomes in NSCLC patients.2 at 37 °C. Gefitinib was bought from AstraZeneca ; Erlotinib was kindly provided by Roche Pharmaceuticals , while inhibitors including S3I-201 and FR180204 were bought from Selleck Chemicals. These drugs were dissolved in dimethyl sulfoxide (DMSO) and diluted in culture medium before use. Besides, primary rabbit antibodies against PD-L1 (CST#13684), ERK1/2 (CST#4695) and p-ERK1/2 (CST#4376) were from Cell Signaling Technology; rabbit antibodies to STAT3 (ab68153), p-STAT3 (Tyr705) (ab76315) were from Abcam while mouse antibody to β-actin was from Santa Cruz biotechnology.Human NSCLC cell lines including H1975 (EGFR L858R/T790M) and HCC827 (EGFR Del 19) were obtained from State Key Laboratory of Biotherapy, Sichuan University, and maintained with RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin  under a moist atmosphere of 5% CO5 cells/ml) continuously to 5 μM Gefitinib or 3 μM Erlotinib for 5 weeks. Total proteins were collected every week. 5 weeks later, the expression of PD-L1 was tested by flow cytometry while the total RNA was extracted as well. Meanwhile, HCC827 was constantly incubated (1 × 105 cells/ml) with 2 μM Gefitinib for 6 months.H1975 was exposed  containing protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride. And the protein concentration was detected by bicinchoninic acid (BCA) protein assay. After cell lysates were separated in the SDS-PAGE Gels, they were transferred to the polyvinylidene difluoride (PVDF) membrane , which was then blocked with 5% non-fat milk. After the membrane was cut into small pieces, they were incubated in different primary antibodies at a 1:1000 dilution overnight at 4 °C. Then the membranes reacted with the anti-rabbit or mouse IgG conjugated with horseradish peroxidase (HRP) . Finally, visible bands were obtained through the enhanced chemiluminescence (ECL) detection.TM RT reagent Kit . Quantitative real-time PCR was conducted with Bio-rad CFX manager and SsoAdvanced SYBR Green Supermix  as the detection reagent. All samples were analyzed in triplicate for each primer set. The primer sequences of PCR were as follows: hPD-L1, sense 5-GGAATTGTCTCAGAATGGTC-3 and antisense 5-GTAGTTGCTTCTAGGAAGGAG-3; hGAPDH, sense 5-GAAGGTGAAGGTCGGAGT-3 and antisense 5-GAAGATGGTGATGGGATTTC-3. RT-PCR amplification was performed 40 cycles with DNA denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 20 s. Target gene expression was quantified with the relative quantification method.Total RNA was extracted and purified using RNA simple Total RNA Kit  according to the manufacturer’s instructions. The RNA samples were reverse-transcribed into cDNA with the PrimeScriptCells were collected and dissociated as single cells, then washed with 0.1% bovine serum albumin in PBS (PBSA) and stained with APC-conjugated anti-human CD274 antibody  or isotype-matched control antibody . After 30 minutes of incubation, cells were washed and resuspended in PBS, then performed on a BD Biosciences FACSCalibur cytometer. Data were analyzed using FlowJo software program.7 beads/mL) (bead-to-cell ratio 1:1). H1975, which were already treated with Gefitinib or Erlotinib for 5 weeks, and Gefitinib-resistant HCC827 cells were seeded in 24-well plates (5 × 104 cells/well) with parental cells as control and cultured in plain medium (with serum) overnight. Then isolated T cells were added to each well at a ratio of 2:1 (T cells: tumor cells) and co-cultured for 24 hours. Parental tumor cells were set as control.Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy volunteer donors by Ficoll Paque density centrifugation. T cells were selected by magnet  and stimulated with recombinant human IL-2  and Dynabeads Human T-activator CD3/CD28 beads  for 30 minutes on ice. Then washed T cells were counterstained with FITC Annexin V and PI for 15 min at room temperature (25 °C) according to the manufacturer’s instructions of FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). Finally the FITC Annexin V and PI signal of T cells were analyzed by flow cytometry.Expanded T cells were labeled with carboxyfluorescein diacetate succinimidyl ester  prior to co-culture with tumor cells (described above). After 48 hours, suspended cells were harvested and counterstained with anti-human CD3-APC antibody. The CFSE signal of T cells was analyzed by flow cytometry.3 cells per well. Cells were allowed to grow for 24 hours and then treated with Gefitinib at various concentrations , in six replicates. After 72 hours of treatment, 20 μL MTT -2,5 diphenyltetrazolium bromide) (Sigma-Aldrich) (5 mg/mL) was added to each well, incubated at 37 °C for 4 hours and terminated with 150 μL of dimethyl sulfoxide (Sigma-Aldrich). Cell viability was finally determined with an ELISA reader (Bio-Rad) at 570 nm. The 50% inhibitory concentration (IC50) values were calculated using a proper analysis (nonlinear regression-curve fit).HCC827 were plated in 96-well at a concentration of 2 × 10t test for two groups and one-way ANOVA Tukey test followed by Dunnett’s multiple comparison test for multiple groups were performed using GraphPad Prism 7. All data were summarized and presented as means ± SD. Statistical significance was defined as: “ns”p > 0.05, “*”p < 0.05, “**”p < 0.01, “***”p < 0.001 and “****”p < 0.0001.Experiments were all repeated in triplicate. For significance analysis of PD-L1 expression , western blot analysis and proliferation and apoptosis of T cells, All experiments involving human tissue samples were performed in accordance with relevant guidelines and regulations approved by medical ethics committee of Sichuan University. Informed consent was obtained from all healthy donors.supplementary figure S1–6


The primary outcome was 3-year all-cause mortality and the secondary outcome was 3-year major adverse cardiovascular events (MACEs). Low L1 SMI was present in 141 (29.7%) of 475 patients. The incidence of all-cause mortality  and MACEs  was significantly higher in patients with low L1 SMI than in those with high L1 SMI. In multivariate analysis, low L1 SMI was an independent predictor of higher risk of all-cause mortality (hazard ratio (HR): 4.07; 95% confidence interval (CI): 1.95–8.45; p  < 0.001) and MACEs . These findings remained consistent after propensity score-matched analysis with 91 patient pairs (C-statistic = 0.848). CT-diagnosed low skeletal muscle mass is a powerful predictor of adverse outcomes in patients with CAD undergoing PCI.The impact of sarcopenia on atherosclerotic cardiovascular disease remains unclear. We aimed to investigate the prognostic impact of sarcopenia on coronary artery disease (CAD). A total of 475 patients with CAD who underwent successful percutaneous coronary intervention (PCI) and computed tomography (CT) were enrolled. The cross-sectional area of skeletal muscle at the first lumbar (L1) vertebral level was measured, and sex-specific cut-off values of L1 skeletal muscle index (L1 SMI; male <31.00 cm Sarcopenia is characterized by age-related generalized loss of skeletal muscle mass (SMM) and muscle strength, and frequently accompanies metabolic, physical, and functional disabilities ,2,3,4. APrevious studies have reported sarcopenia as a potential predictor of the prevalence and prognosis of atherosclerotic cardiovascular disease (ASCVD) ,15,16,17This study investigated the long-term prognostic impact of CT-diagnosed low SMM in patients with CAD who underwent successful percutaneous coronary intervention (PCI). We identified the sex-specific cut-off values of SMM at the first lumbar (L1) vertebral level to predict all-cause mortality and validated its prognostic significance.The PCI registry of Korea University Guro Hospital  is a single-center, prospective, all-comer cohort that consecutively enrolls patients undergoing PCI for CAD. The study population was enrolled from the KUGH PCI registry between January 2004 and April 2014, and was analyzed retrospectively. The study protocol complied with the Declaration of Helsinki and was approved by the Ethics Committee and Institutional Review Board at KUGH (2018GR0352). From the KUGH PCI registry, 788 patients who underwent PCI for CAD with stent implantation and performed CT scans within 180 days of PCI were selected. The following were excluded sequentially: (1) Patients without successful PCI; (2) patients who did not undergo CT within 30 days of PCI; and (3) CT exams without cross-sectional images at the L1 level. Finally, a total of 475 patients were included in the study .All participants in the present study underwent successful PCI. PCI was considered successful if the final angiography showed residual stenosis of <30% with achievement of Thrombolysis in Myocardial Infarction grade 3 flow. The morphology of the coronary artery lesion type was determined according to the American College of Cardiology and American Heart Association classification . MultiveAt initial admission for PCI, demographic features and underlying cardiovascular risk factors were recorded through detailed patient interviews, and baseline laboratory results including lipid profiles were obtained. The Canadian Study of Health and Aging (CSHA) Clinical Frailty Scale (CFS)  was usedAfter discharge, a regular outpatient clinic visit was scheduled at the end of the first month and every 3 to 6 months thereafter. If a patient did not show up for a scheduled visit, a telephone interview was conducted as an alternative approach for assessment of adverse outcomes. The follow-up duration was commenced with the date of successful PCI.The primary outcome was 3-year all-cause mortality, and the secondary outcome was 3-year major adverse cardiovascular events (MACEs), a composite of all-cause mortality, non-fatal myocardial infarction (MI), and repeat revascularization. Mortality was classified as cardiovascular or non-cardiovascular. MI was defined as a significant elevation of cardiac biomarkers accompanied by concomitant symptoms or electrocardiographic findings indicative of ischemia . Repeat CT measurement of the skeletal muscle area (SMA) of a specific body part accurately reflects the muscle mass of that region , and the2) of skeletal muscles on CT images were measured manually by utilizing the freehand module of the PACS workstation  by two independent observers (one radiologist and one cardiologist). Both observers were blinded to the clinical information during the measurement period. Repeat measurements were performed at least 4 weeks after the initial measurement to assess intra- and inter-observer variability with 50 randomly selected CT images. The cross-sectional areas were normalized for height (cm2/m2) and presented as the L1 skeletal muscle index (L1 SMI). Due to the absence of established reference values for defining sarcopenia with the L1 SMI in an Asian population, sex-specific cut-off point analysis was performed with the L1 SMI to determine the best reference value for predicting the primary outcome episodes. Based on the determined reference values, patients were divided into two groups by the presence of low SMM. Transverse CT images obtained at the level of the L1 transverse process were assessed in each patient. The CT Hounsfield unit ranges of −29 to 150 and −190 to –30 were considered to represent skeletal muscle and adipose tissue, respectively. The total SMA at the L1 level was composed of the following four components: Paraspinal muscle, extracostal abdominal wall muscle , intercostal muscle (including transversus abdominis muscle), and psoas muscle and diaphragm. Cross-sectional areas  curve. The cumulative incidence rate in each group was analyzed using the Kaplan–Meier method with the log-rank test. Multiple stepwise Cox-proportional hazards regression analysis was conducted to test the prognostic impact of low L1 SMI and frailty, and four models were established using subsequent adjustment of confounding factors. To adjust for additional confounding factors, propensity score-matched analysis was performed using a logistic regression model. The following factors, which showed significant differences at baseline or had proper relevance, were included in the model: Age, sex, body mass index (BMI), presentation as MI, hypertension, diabetes, dyslipidemia, previous malignancy, clopidogrel and statins at discharge, multivessel disease, creatinine clearance <60 mL/min, LVEF <50%, total cholesterol, number of treated lesions and treated vessels, number of implanted stents, average stent diameter, total stent length, and implantation of second generation drug-eluting stents. The logistic model estimating the propensity score revealed a good predictive value (C-statistic = 0.848). Intra- and inter-observer variability was quantified using the intra-class correlation coefficient (ICC) and visualized with Bland–Altman plots. Significant differences were defined at a p-value of <0.05, and all analyses were two-tailed. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software, version 20.0 .Data are expressed as mean ± standard deviation (SD) or median with interquartile range (IQR) for continuous variables, and frequency (percent) for categorical variables. Differences between groups were analyzed using Student’s p  < 0.001; p = 0.045; 2/m2  in males and 25.00 cm2/m2  in females.The average duration from the date of PCI to CT scan was −3.3 ± 11.7 days (median −2.0 days). 2/m2 in males, respectively, and 21.77 ± 2.64 and 31.36 ± 4.02 cm2/m2 in females, respectively. The L1 SMI showed a weak correlation with BMI  of the 475 study patients. The measured L1 SMI values of the low L1 SMI and high L1 SMI groups were 26.52 ± 3.74 and 38.36 ± 5.24 cmwith BMI . Both inwith BMI . DetailsMean follow-up duration was 4.11 ± 3.02 years (median 4.04 years), and 82.3% of the study participants completed a 3-year follow-up. Baseline clinical characteristics, procedural characteristics, and treatment strategies are displayed in p < 0.001) and MACEs . Other patient-oriented outcome measures such as MI and repeat revascularization also showed significantly higher incidence in the low L1 SMI group than in the high L1 SMI group. Detailed information about the cause of non-cardiovascular death is provided in A comparison of 3-year clinical outcomes between the groups is displayed in p  < 0.001 and HR: 3.76; 95% CI: 2.27 to 6.23; p  < 0.001, respectively). The complete dataset for univariate analysis and stepwise multivariate analysis of the study population is displayed in p = 0.009; and 46.1% vs. 18.1%, p  < 0.001, respectively)   . Low L1 ctively) .p  < 0.001) and MACEs  than non-frail patients. In fully adjusted multivariate analysis, the presence of frailty was an independent predictor of 3-year all-cause mortality . However, the impact of frailty on 3-year MACEs was markedly attenuated .Detailed study results based on the presence of frailty are provided in n = 313) of patients were excluded from the initial enrollment, mostly due to the absence of CT scans within 30 days of PCI and unavailability of SMA data at the L1 level. Details of study participants and those excluded at initial enrollment are displayed in A modest proportion  of patients were excluded from the initial enrollment, mostly due to the absence of CT scans within 30 days of PCI and unavailability of SMA data at the L1 level. For balanced interpretation of the study results, detailed features of the excluded study population were provided and compared to those of the enrolled study participants in the Online First, the CT measurement of L1 SMI was not a primary goal. However, the image quality was optimal in most cases and L1 SMI measurement was highly reproducible. Under the widespread use of coronary CT in CAD, CT images to assess SMM at the L1 level could be easily acquired without additional radiation burden or extra payment in this population. Second, because of the intrinsic limitation of non-randomized registry data, the baseline differences between groups could have affected the outcome. To minimize such biases, multiple stepwise Cox regression and propensity score-matched analyses were performed. However, remaining confounders could have influenced the analyses. Third, a modest proportion n = 3; 39.7% oLow SMM is a powerful predictor of adverse clinical outcomes in patients with CAD who undergo successful PCI. CT-diagnosed low SMM may further aid in risk stratification and decision-making in patients with established ASCVD.


The data presented in this article are related to “Odd-even phonon transport effects in strained carbon atomic chains bridging graphene nanoribbon electrodes” . To provide the information that can be utilized in reproducing our results, we provide the fully optimized graphene nanoribbon (GNR)–carbon chain (CC)–GNR junction atomic configurations at different strain values, the computational setting for quantum phonon transport calculations, and several computational data that were not included in the main manuscript. Data on graphene–CC–graphene junctions are additionally presented. Specifications tableValue of the data•The data can be references to the atomistic modeling of carbon atomic chains bridging graphene and graphene nanoribbon electrodes with strain effects.•The data can be references to the quantum phonon transport calculations of carbon atomic chains, graphene nanoribbons, and their heterojunctions.•The data may be generally relevant for the researchers interested in the quantum transport properties of all-carbon devices.1displacement}.xyz”. The simulation cell information are provided in the comment lines within the xyz files. In In this data article, we present the data on the atomic configurations and corresponding lattice thermal conductances of carbon atomic chains (CCs) bridging graphene nanoribbon (GNR) and graphene (GRP) electrodes 22.1−6 eV, and atomic relaxations were performed until the Hellmann–Feynman forces on each atom fall below 10−3 eV/Å.For the junction models described in For the computation of ballistic phonon transport properties, we used an in-house code that implements the atomistic matrix Green’s function (MGF) formalism 2.2The geometries and quantum phonon transport properties of the GNR–CC–GNR and GRP–CC–GRP junction models were obtained following the procedure established earlier for the study of quantum electron transport properties in molecular ΔL = 0.4 Å and 0.8 Å and repeated energy minimization procedures.Expecting the CC parts will adopt similar configurations within the zGNR and aGNR electrodes, junction models based on 7aGNR electrodes were initially prepared by extracting the inner CC parts  in the 4zGNR–CC–4zGNR junction models and replacing the 4zGNRs by 7aGNRs. The scattering-region atoms were energy minimized once again. For the aGRP cases, we repeated a similar procedure to expedite model preparations. After obtaining the optimal geometries of the GNR–CC–GNR and GRP–CC–GRP junction models, we stretched junction model by moving GNR or GRP electrodes outward by


Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using “PD-L1” as a search term for 01/01/2015 to 31/08/2018, with limitations “English” and “human”. 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose. Clinical trials have shown that it is possible to successfully restore host immunity against various malignant neoplasms even in advanced stage disease by deploying drugs that target the PD-1/PD-L1 axis . In mostSeveral such fit-for-purpose immunohistochemistry kits are commercially available, but in clinical practice, and especially in publicly funded health care, it is challenging to make all such testing available to patients –9. BecauThe goal of this study is to assess the performance of PD-L1 immunohistochemistry assays based on their diagnostic accuracy at specific cut-points, as defined for specific immunotherapies according to the clinical efficacy demonstrated in their respective pivotal clinical trials.In other words, given an Food add Drug Administration-cleared assay, which other assays can be considered substantially equivalent for that specific purpose? Although comparison of immunohistochemistry assays for their analytical similarities is warranted and useful for clinical immunohistochemistry laboratories, it is an insufficient foundation on which to make an informed decision whether an Food and Drug Administration-approved companion diagnostic with a specific clinical purpose can be replaced by another assay, whether the substitute assay is an Food and Drug Administration-approved companion diagnostic for a different purpose or a laboratory developed test. The more appropriate approach for these qualitative assays would be comparing the results of the candidate assay for its diagnostic accuracy against a comparative method/assay or designated reference standard . We repoMethodology including data sources, study selection, data abstraction, and grading evidence, are detailed in Supplementary Files A systematic review of literature was conducted as a part of a national project for developing Canadian guidelines for PD-L1 testing. The Canadian Association of Pathologists – Association canadienne des pathologistes (CAP-ACP) National Standards Committee for High Complexity Testing initiated development of CAP-ACP Guidelines for PD-L1 testing to facilitate introduction of PD-L1 testing for various purposes to Canadian clinical immunohistochemistry laboratories. This review was also used to guide the selection of publications to be used in this meta-analysis.The purposes identified in the systematic review of published literature were based on either the clinical purpose that was specifically identified in the published study or the intended purpose for which the included specific companion diagnostic assay was clinically validated. Although a large number of potential purposes were identified, only few could be included in this meta-analysis. The selection was based on the type of data available, including which immunohistochemistry protocols and which readout was performed by the authors. The greatest limitation in the accrual of data from these published studies was based on the selection of the readout employed to assess the results; in most studies the readout was limited to tumor proportion score with 1% and 50% cut-offs, which is essentially based on the clinically meaningful cut-offs for pembrolizumab and nivolumab therapy. Hence, these two readouts were selected for our analysis and form the basis for outlining different purposes that are derived from the combination of the readouts and immunohistochemistry kits/protocols that use these readouts and are approved by regulatory agencies  for different clinical uses.Most published studies on PD-L1 test comparison did not include 2 × 2 tables that would allow calculations of either diagnostic sensitivity and specificity or positive percent agreement and negative percent agreement. The CAP-ACP National Standards Committee for High Complexity Testing requested this information from the authors of studies where it was evident that the authors generated such results, but did not include them in their published manuscript. Most studies required generation of multiple 2 × 2 tables, as each one was designed for a specific purpose and set of candidate’ and ‘comparator’ assays. For primary studies that provided sufficient detail, information on study setting, comparative method/reference standard and 2 x 2 tables for different tumor proportion score cut-offs were extracted, from which accuracy results were reported. Studies of PD-L1 immunohistochemistry assay comparisons that did not compare the performance of the assay to any designated or potential reference standard  –22 were Most studies evaluated PD-L1 immunohistochemistry in non-small cell lung cancer, resulting in 337 test comparisons. Comparisons of test performance in other tumors were much less common. These included analysis of urothelial carcinoma (20 test comparisons), mesothelioma (9 test comparisons) and thymic carcinoma (9 test comparisons).2 were used to examine heterogeneity among studies. Funnel plots and Egger’s test were applied to detect possible publication bias  from the individual studies were summarized. Random-effects models were fitted , 45. ForFor the purpose of this study, the immunohistochemistry candidate assays were considered to be acceptable for clinical applications if both sensitivity and specificity for the stated clinical purpose/application were ≥90% .Assuming that laboratories follow the instructions for use provided with Food and Drug Administration-approved or European CE-marked immunohistochemistry kits, the overall results of this meta-analysis could be considered highly representative and generalizable of Food and Drug Administration-approved assay performance and the diagnostic accuracy of that assay against a designated reference standard for the stated specific purpose in any laboratory. However, this assumption cannot be applied to the results of laboratory developed tests, because laboratory developed test immunohistochemistry protocol conditions were often different in different laboratories even when the same primary antibody was used, and the protocol was performed on the same automated instrument with the same detection system . When the results of the meta-analysis for laboratory developed tests were suboptimal, but one or more laboratories achieved ≥90% sensitivity and specificity; we cannot exclude the possibility that with appropriate immunohistochemistry protocol modification and assay validation, other laboratories could also achieve optimal results. This contrasts with the use of Food and Drug Administration-approved assays, where no protocol modifications are allowed. Therefore, where the results of laboratory developed tests are excellent, they are representative of what could be achieved by laboratory developed tests rather than that they are generalizable and that they will automatically be achieved in all laboratories.The number of studies comparing different assays in this meta-analysis was larger than the number of published manuscripts, due to the frequent inclusion of multiple test comparisons in single publication as well as use of different cut-off points for “positive” vs. “negative” test result. Table 2 for sensitivity and specificity across all studies are shown in Supplementary Files Table Cochran’s heterogeneity statistic Q and IWhere the number of studies was less than four or when the data were sparse due to the presence of a zero result in contingency tables , the models did not converge and did not allow for meta-analysis calculations. As summarized in Tables The highest diagnostic accuracy was shown for well-designed 22C3 laboratory developed tests compared to PD-L1 IHC pharmDx 22C3. The sensitivity and specificity were both 100% in 8/9 assays for the 50% tumor proportion score cut-off point Table , Table 2No other candidate assays reached 90% sensitivity and specificity in the meta-analysis for either the 50% or the 1% tumor proportion score cut-off for PD-L1 IHC 22C3 pharmDx Table  and 1B , 27.The highest results were achieved by the Ventana PD-L1 (SP263) assay; it had acceptable accuracy in the meta-analysis compared to PD-L1 IHC pharm Dx 28-8 at the 1% cut-off  Fig. , Table 1No candidate assays achieved required diagnostic accuracy for either the 1% or the 50% cut-off. Most candidate assays achieved acceptable specificity, but the sensitivity was too low for both cut-off points Tables .The most dominant result of this meta-analysis is that properly designed laboratory developed tests that are performed in an individual immunohistochemistry laboratory  and are developed for the same purpose as the relevant comparative reference method standard may perform essentially equally to the original Food and Drug Administration-approved assay, but also generally better than the Food and Drug Administration-approved companion diagnostics that were originally developed for different purposes. For example, to identify patients with non-small cell lung cancer for second line therapy with pembrolizumab where PD-L1 IHC pharmDx 22C3 is not available, the results of our study indicate that it is more likely that 22C3 or E1L3N well-developed, fit-for-purpose laboratory developed tests would identify the same patients as positive and/or negative as PD-L1 IHC pharmDx 22C3, rather than Ventana PD-L1 (SP263), Ventana PD-L1 (SP142), or PD-L1 IHC pharmDx 28-8, which were developed for different purposes –53.The accuracy of laboratory developed tests varied in our meta-analysis. 22C3 laboratory developed tests achieved the best results, with both sensitivity and specificity of 100% in 8/9 studies. E1L3N also showed excellent results, but in only 3/12 comparisons. Its success in 3 separate comparisons illustrates that it is possible to develop an acceptable laboratory developed test with this clone and that this antibody can be optimized for clinical applications for which the PD-L1 IHC 22C3 pharmDx was developed. The successful applications of some of the laboratory developed tests reinforce the importance of considering the original purpose of the immunohistochemistry assay, a point emphasized in the ISIMM and IQN Path series of papers entitled “Evolution of Quality Assurance for Immunohistochemistry in the Era of Personalized Medicine” –57. It sAs so far there are no tools to measure analytical sensitivity and specificity of immunohistochemistry assays; this presents a significant problem in assay development, methodology transfer, and daily monitoring of assay performance, as well as direct comparison of assay calibration. The lack of tools that could assess analytical sensitivity and specificity also hinders attempts of immunohistochemistry protocol standardization/harmonization for the PD-L1 assays; without such tools it is not possible to determine the desirable range of analytical sensitivity and specificity of relevance for diagnostic accuracy for any of the PD-L1 assays. This is one cause that we can identify as a potential source of the discrepancy between previously published works that suggested analytical interchangeability of the several Food and Drug Administration-approved PD-L1 assays, but did not necessarily lead to interchangeability based on calculated diagnostic accuracy as shown in our study.The Ventana PD-L1 (SP263) assay had very high diagnostic sensitivity against all other Food and Drug Administration-approved PD-L1 assays, but its diagnostic specificity was consequently lower. Although several of the studies included in this meta-analysis demonstrated substantial analytical similarity between PD-L1 IHC 22C3 pharmDx, PD-L1 IHC 28-8 pharmDx, and Ventana PD-L1 (SP263), our cumulative results suggest that the diagnostic sensitivity of these various assays  is ordered as follows: PD-L1 IHC 22C3 pharmDx < PD-L1 IHC 28-8 pharmDx < Ventana PD-L1 (SP263).The results of this meta-analysis confirm previous observations that the Ventana PD-L1 (SP142) assay’s analytical sensitivity is significantly lower than that of the three other Food and Drug Administration-approved PD-L1 assays and that the diagnostic sensitivity of Ventana PD-L1 (SP142) against PD-L1 IHC 22C3 pharmDx, PD-L1 IHC 28-8 pharmDx, and Ventana PD-L1 (SP263) assays is prohibitively low for both the 1% and the 50% tumor proportion score in non-small cell lung cancer and other tumor models.Several investigators have evaluated the so-called “interchangeability” of PD-L1 immunohistochemistry assays. The term “interchangeability” has also been used widely by the pharmacological industry to designate drugs that have demonstrated the following characteristics: same amount of the same active ingredients, comparable pharmacokinetics, same clinically significant formulation characteristics, and to be administered in the same way as the drug prescribed . BasicalAlthough they cannot be designated as “interchangeable”, the diagnostic accuracy of assays for a specific clinical purpose may be compared. In this manner, the comparison indirectly generates results that can be used to justify clinical usage of assays other than those included in the clinical trials. We employed ≥90% diagnostic sensitivity and ≥90% diagnostic specificity because these values are often used in other settings, including performance of immunohistochemistry assays –68. WhilThe strengths of this meta-analysis are the focus on diagnostic accuracy, fit-for-purpose approach, and the access to previously unpublished data from a large number of studies, which all resulted in pooled PD-L1 assay comparison in a way that has not been done before.The most significant limitation is that this is a meta-analysis of test comparisons where designated reference standards are other tests rather than clinical outcomes. However, to complete a meta-analysis with clinical outcomes may not be possible for many years, if ever. Other limitations of this meta-analysis are that only two cut-off points were assessed (1% and 50%), no assessment for readout that includes inflammatory cells was included, the impact of pathologists’ readout as potential source of variation between the studies was not assessed, and it is somewhat uncertain how the results apply to tumors other than non-small cell lung cancer due to the smaller number of such studies.The complexity of the PD-L1 immunohistochemistry testing cannot be safely simplified without consideration of the original test purpose. Determination of the diagnostic accuracy and indirect clinical validation of a candidate assay can be achieved by comparing the results of that assay to a previously designated reference standard assay, when direct access to clinical trial data or clinical outcomes is not possible.1) Well-designed, fit-for-purpose PD-L1 laboratory developed test candidate assays may achieve higher accuracy than PD-L1 Food and Drug Administration-approved kits that were designed and approved for a different purpose, when both are compared to an appropriate designated reference standard;2) More candidate assays achieved ≥ 90% sensitivity and specificity for 50% tumor proportion score cut-off than for 1% tumor proportion score cut-off;3) The overall diagnostic sensitivity and specificity analyses indicates that the relative analytical sensitivities of the Food and Drug Administration-approved kits for tumor cell scoring, most specifically in non-small cell lung cancer, are as follows: Ventana PD-L1 (SP142) << PD-L1 IHC 22C3 pharmDx < PD-L1 IHC 28-8 pharmDx < Ventana PD-L1 (SP263).Conflict of Interest (Appendix A)Meta-Analysis MethodologySupplementary Files Table 1Supplemetary Figures 2 - 13Funnel Plots


The final products as filler into carboxylated acrylonitrile‒butadiene rubber (XNBR) improve the dynamic mechanical properties. The improvement in the dynamic mechanical properties of the nanocomposite mainly depends on high interfacial interaction and graphene’s performance characteristics, as well as a good dispersion between functionalized graphene and the XNBR matrix.Graphene has been considered an ideal nanoscale reinforced phase for preparing high-performance composites, but the poor compatibility and weak interfacial interaction with the matrix have limited its application. Here a highly effective and environmentally friendly method for the functionalization of graphene is proposed through an interaction between as-exfoliated graphene and (3-aminopropyl) triethoxysilane (KH550), in which 1-butylsulfonate-3-methylimidazolium bisulfate (BSO Nowadays, damping materials have been widely applied in the mechanical vibration field. Among the numerous damping materials, polymer composites have been extensively researched for their excellent physical and chemical properties ,2. HowevIonic liquids (ILs) have attracted extensive attention due to their unique properties like low toxicity, nonflammability, and recyclability . Liu et 3HMIm)(HSO4) ILs-modified multilayers graphene (MLG) is an efficient platform for further grafting with KH550 to improve compatibility and interfacial interactions in the XNBR matrix. (3-aminopropyl) triethoxysilane (KH550) has been used to increase compatibility in polymer matrixes [3HMIm)(HSO4)/KH550-functionalized graphene was dispersed into a rubber matrix to estimate the dynamic mechanical properties.In this work, a new method for the preparation of functionalized multilayer graphene was proposed. (HSO4) ILs and high-purity graphite rods were obtained from Lanzhou Institute of Chemical Physics . Direct-current power supply was commercially obtained from HYELEC Co., Ltd. . XNBR (1027) with an acrylonitrile mass fraction of 27% was obtained from NANCAR Co., Ltd. . 1-butylsulfonate-3-methylimidazolium bisulfate (BSO3HMIm)(HSO4)/KH550-functionalized graphene was prepared according to four steps. First, two high-purity graphite rods were inserted as electrodes into the solution of water and 1-butylsulfonate-3-methylimidazolium bisulfate with a volume ratio of 10:1. Two high-purity graphite rods were placed parallel to each other and 2 cm apart in a 50-mL beaker. Direct-current power supply was explored to provide a static potential of 5 V. After 4 h, the electrolyte became black due to the exfoliation from the anode graphite surface. Secondly, the black solution with exfoliated graphene was centrifuged at a speed of 4000 rpm. The black precipitate was washed with absolute ethanol and deionized water several times. The as-obtained ILs-modified multilayer graphene was denoted as MLG1. Thirdly, KH550 was used as a grafting agent at 60 °C for 5 h and the product was isolated by centrifugation at 4000 rpm. Finally, the functionalized multilayer graphene was thoroughly washed with deionized water to remove KH550 monomers and dried in vacuum drying oven, and the as-prepared sample was denoted as MLG1-KH550. In order to estimate the reaction efficiency between (BSO3HMIm)(HSO4) and KH550, (BSO3HMIm)(HSO4) with different concentrations was added to regulate its density on MLG’s surface. MLG2-KH550 and MLG3-KH550 were obtained by the same process with mixtures of water and (BSO3HMIm)(HSO4) at 10:2 and 10:3, respectively.. X-ray photoelectron spectroscopy  was employed to explore the chemical state of typical elements on the functionalized multilayer graphene. The morphology of MLG-KH550 and XNBR/MLG-KH550 composites were determined by transmission electron microscopy . The degree of defects in carbon materials was evaluated by Raman microscopy (Horiba LabRam HR800). The thermogravimetric curves of samples were investigated with a Thermal Analyzer  at a heating rate of 20 °C/min in flowing N2. Dynamic thermomechanical analysis was explored by DMA . A twin-roller laboratory mill was employed to mix the rubber and filler. First, the MLG1-KH550 with additional content of 1.0 wt % was added to XNBR and they were mixed at room temperature until the filler had uniform distribution in the matrix. Secondly, the rubber mixture was placed in a metal mold for vulcanization. The vulcanization temperature was set at 145 °C for 10 min and the pressure dependence was under 10 MPa. Finally, the composites with different addition content  were prepared and to explore the performance characteristics. The composites with different content were denoted as XNBR/MLG1-KH550-0.2wt %, XNBR/MLG1-KH550-0.5wt %, XNBR/MLG1-KH550-1.0wt %, and XNBR/MLG1-KH550-2.0wt %, respectively.3H-functionalized ILs can react with amines to stabilize amino silane (KH550) in A possible reaction pathway has been proposed ,33. As s– anions were effectively intercalated into the interlayer spacing of graphite [3HMIm)+ cations in ILs play an important role in the formation of free radicals, like 1-octyl-3-methylimidazolium free radicals that can connect on graphene via physicochemical interaction [The preparation procedure of functionalized-MLG-enhanced XNBR nanocomposites is shown in graphite . (BSO3HMeraction  and coul3HMIm)(HSO4), the surface morphology of graphene can be definitively identified by transmission electron microscopy (TEM). 3HMIm)(HSO4) layer. The diffraction pattern of single-layer graphene shows a typical six-fold symmetry. Observing the TEM images of exfoliated functionalized graphene (as shown in 3HMIm)(HSO4) on the graphene surface (in 3HMIm)(HSO4), the amorphous structure are definitively identified and the families of spots disappear (HSO4) ILs, new absorption peaks at 1230 and 1566 are assigned to O–SO2–O stretch vibration and H–C=C–H scissoring vibration, respectively [−1 are assigned to imidazolium framework vibration, which belong to (BSO3HMIm)(HSO4) ILs. Compared with the spectra of MLG1 and MLG1-KH550, the peak at 2875 cm−1 is assigned to methylene stretch vibration [−1 due to Si–O–C and Si–O–Si bonds [−1 is assigned to the NH3+ stretching mode, indicating that ammonium groups can react with (BSO3HMIm)(HSO4) ILs [FTIR spectra were employed to explore the chemical structure of functionalized MLG. ectively ,35. The ibration ; the strSi bonds , suggestSO4) ILs .3HMIm)(HSO4) were successfully introduced on the interlayer/basal plane of multilayer graphene [3HMIm)(HSO4) and KH550. XPS was used to further analyze the chemical state of typical elements for functionalized graphene. The full-range XPS spectrum of original graphite displays only C 1s peak in graphene ,38,39. C3HMIm)(HSO4), indicating that the KH550 layer is partly covered on the graphene surface. From MLG1-KH550 to MLG3-KH550, the N/O ratio increases from 0.267 to 0.373, close to the theoretical value of 0.33 for KH550. Meanwhile, the Si/C ratio increases from 0.065 to 0.106, close to the theoretical value of 0.111 for KH550. Therefore, it can be assumed that a high concentration of ILs can effectively increase the thickness of the KH550 grafted layer.The elemental ratio of MLG1-KH550, MLG2-KH550, and MLG3-KH550 was explored by XPS spectra (as shown in 2 hybridized carbon [−1 further verifies that (BSO3HMIm)(HSO4) ILs are successful adhered to graphene via physicochemical interaction. Moreover, the 2D band can be used to confirm the layers of graphene, which is sensitive to graphene thickness due to the dispersion of π electrons [Raman spectroscopy plays an important role in the characterization of the features of spd carbon . Raman sd carbon . In Figulectrons . As the lectrons ,43. In Flectrons . It is wlectrons . 3HMIm)(HSO4) are shown in 3HMIm)(HSO4), while pure (BSO3HMIm)(HSO4) shows a serious decomposition (weight loss of approximately 98 wt %), suggesting that the higher the density of (BSO3HMIm)(HSO4) there is on graphene, the more weight loss occurs. After grafting with KH550, the weight loss of MLG1-KH550 is 19 wt %, indicating higher thermal stability of MLG1-KH550 under the same calcination procedure.Thermogravimetric (TG) curves of graphite, MLG1, MLG1-KH550, and (BSO3HMIm)(HSO4) ILs and further grafting with KH550, which could offer excellent dispersion into the XNBR matrix and could enhance the interface interaction between graphene and the XNBR matrix, thereby improving the performance characteristics for practical applications.The combination of the above experimental results confirms that graphene has been functionalized by (BSOg). From g). As shown in DMA is a useful tool to evaluate the interfacial interaction of polymer composites . Thus weg of XNBR/MLG1 and XNBR/MLG1-KH550 are obviously shifted to a lower temperature from g of XNBR/MLG2-KH550 and XNBR/MLG3-KH550 are obviously higher than that of pure XNBR. In previous works [From us works ,48, simiThe dispersity of the functionalized graphene sheets in the XNBR matrix was characterized by TEM images. The darker lines are viewed as a layer structure of functionalized graphene sheets, while the gray regions are identified as the XNBR matrix. As shown in 3HMIm)(HSO4) via an electrochemical exfoliation of the graphite procedure and can be further grafted with KH550 via (BSO3HMIm)(HSO4) ILs as a high-performance platform for interactions. The efficient modification of graphene improves its compatibility in the XNBR matrix. The functionalized multilayer graphene greatly improves the dynamic mechanical properties of XNBR, which we can attribute to the enhancement in the interaction between functionalized multilayer graphene and the XNBR matrix.To allow for practical application of high-performance rubbers, a high-efficiency and environmentally friendly method has been proposed to prepare functionalized multilayer graphene as a filler. This work demonstrates that multilayer graphene can be modified by  Functionalized graphene prepared by electrochemical exfoliation of graphite in an ionic liquids solution and further grafting of KH550 is an environmentally friendly and high-efficiency method for the improvement and development of graphene-like materials;(b) KH550-grafted graphene with a higher density can effectively enhance the dynamic mechanical properties, mainly due to the beneficial interactions of functionalized graphene and the XNBR matrix;(c) KH550-grafted graphene as a nanoscale reinforcement can overcome stacking problems and enhance the dynamic mechanical properties. So, it is of great significance for preparing a high-performance nanocomposite for real-world applications.


Little is known regarding the psychometric properties of computerized long-menu formats in comparison to classic formats. We compared single-best-answer (Type A) and long-menu formats using identical question stems during the computer-based, summative, intermediate clinical-clerkship exams for nine disciplines.In this randomised sequential trial, we assigned the examinees for every summative exam to either the Type A or long-menu format . The primary outcome was the power of discrimination. The study was carried out at the Faculty of Medicine, University of Geneva, Switzerland, and included all the students enrolled for the exams that were part of the study. Examinees were surveyed about the long-menu format at the end of the trial.p = 0.7948) after 22 exams including 88 experimental items. The long-menu format had a similar discriminatory power but was more difficult than the Type A format . Over half of the options (54.4%) chosen by the examinees in long-menu formats were not proposed as distractors in the Type A formats. Most examinees agreed that their reasoning strategy was different.The trial was stopped for futility (In a non-selected population of examinees taking summative exams, long-menu questions have the same discriminatory power as classic Type A questions, but they are slightly more difficult. They are perceived to be closer to real practice, which could have a positive educational impact. We would recommend their use in the final years of the curriculum, within realistic key-feature problems, to assess clinical reasoning and patient management skills. Computer-based assessment facilitates prompt and timely feedback to both students and teachers , it is aPsychometric properties do not differ significantly between the two formats , and morIn addition to the long-menu format, computer-based exams allow the use of other formats, such as key-feature problems where sequential questions need to be answered in a specific order. The sequence mimics real patient management more closely, moving from history-taking to diagnosis and then to treatment. These series of questions can be viewed as simulations  falling p = .005) and more discriminating . However, the retrospective observational design was a limitation to this study: since different questions were used in different formats, the contents and underlying learning objectives were likely to have had an impact on both difficulty and discrimination. Furthermore, all the items were related to a single medical speciality.In a retrospective study assessing the psychometric performance of 553 items used in 13 computer-based paediatrics exams , we founTo the best of our knowledge, little is known about the specific psychometric added value of a long-menu format, compared to a single best answer format, when a teacher chooses how the examinee should answer an exam question: The present study’s main objectives were to compare the level of difficulty and power of discrimination of long-menu questions against single-best-answer multiple choice questions in real-life conditions, i.e. summative undergraduate examinations, by applying both formats simultaneously to the same question stem. The experimental items would thus only differ in the answering modalities: long-menu questions on one hand, and single best answer (best choice), typically from a list of five alternatives , on the other.This prospective study was carried out at the Faculty of Medicine of the University of Geneva, Switzerland. Apart from the selection exam at the beginning of the curriculum, all the written exams for the medical students have been computer-based since 2011, first using desktop computers and then tablets. We included all the written exams taken during the first and second clinical years of the curriculum . In each exam, we identified four Type A questions which could be modified and transformed into long-menu questions (or long-menu questions that could be transformed into Type A questions for the paediatrics exams). For each exam, all enrolled examinees were eligible and randomly divided into two approximately equal sized groups with two different versions of the exam. One group had two questions in the Type A format and the other two questions in the long-menu format, and vice versa for the second group. The question stems for the four questions were kept rigorously identical. The other questions were identical in both versions of the exam, and all questions were included in the computation of the exam score.The administrative staff randomly assigned (computerized procedure) an examination room seat number to every enrolled examinees. Independently and in parallel, the IT staff randomly uploaded  one of the two versions of the exam to every desktop or tablet in the examination room.We used the Item Management System provided by UCAN  combined with CAMPUS (Desktop-based exams) or tEXAM (Tablet-based written exams), which are provided by the same consortium.The primary outcome was the question’s power of discrimination, expressed by the point biserial correlation: this evaluates the item’s ability to differentiate among students on the basis of how well they perform during the exam. It can be viewed as an estimator of the degree to which a single item measures the same underlying construct as all the other items in the exam.The secondary outcome was the difficulty of the question, defined as the average capacity of students to find the correct answer, i.e. for a single best answer the relative frequency of choice of the unique correct answer. By extension, it was for the long-menu format the relative frequency of choice of the correct answer in the long-menu list .For each long-menu question, we determined which distractors the examinees chose. In order to evaluate their perception of the long-menu format, all the examinees received a four-item, online, self-administered questionnaire at the end of the study. The items, measured on a four-point Likert scale  were: “The long-menu format is more difficult than the other formats”; “My reasoning is different whenever I have to answer a long-menu format”; “The situation I am put in when I have to answer a long-menu format is closer to real life than when I have to answer a classic MCQ item”; and “It is more difficult to answer a long-menu question than an open free-text item. We added the latter item although it is not directly related to the main objective of the study because the long-menu format is sometimes criticized as it does not allow, unlike the open-free text, to validate any chain of characters entered through the keyboard or keypad.”Study design followed a sequential procedure . Indeed,Repeated sequential paired Student’s t-test were performed using for every item the difference between the estimates  computed from the two versions of the exam Fig. . With a Unless specified, data were summarised as mean ± standard deviation (S.D.). The difficulty and the power of discrimination were provided either by the item management system, or by some in-house validated programs. No manual data handling or computation was required. Student’s t-tests were used to compare two groups of continuous variables. All analyses were run on TIBCO Spotfire S +® 8.1 for Windows , with the additional S + SeqTrial module.n = 305; 60% of women; mean age ± S.D.: 24 ± 2.4). The mean total number of items per exam was 58 ± 26, with a mean of 107 ± 42 candidates taking an exam. Most Type A questions  included five response options, 14 questions included six options and three included four options. The likelihood estimates had already hit the defined stopping boundaries by the second interim analysis, but the Rao–Blackwell adjusted estimate had not, so it was decided to continue the experiment. The study was stopped after the third interim analysis (88 items from 22 different exams), and we concluded that there was no significant difference regarding the discriminatory powers of the type A and long-menu formats , the long-menu versions were more difficult: − 6.34% .The powers of discrimination and difficulties of both formats are shown in Fig. p-value Student’s t-test = 0.1688 for the comparison with the long-menu format; = 0.3648 with the Type A format). The average percentage of correct answers was 78.1% ± 20.3%, similar to the Type A experimental items , but higher than the long-menu experimental items (p = 0.010).The mean discriminatory power of all the other exam items, i.e. all the items in every exam apart from the four experimental items included either as Type A or long-menu questions, was 0.182 ± 0.202 and showed no evidence of difference with the experimental items  were distractors listed in the Type A questions, and 736 (54.4%) were not proposed as distractors in the Type A questions.The results of the survey conducted at the end of the study  can be found in Table To the best of our knowledge, this study is the first randomised controlled trial in real-life conditions of summative assessment to compare long-menu and Type A formats using the same question items. The long-menu format used to record the examinees’ answers had an equivalent discriminatory power to the classic Type A format and it was more difficult. This higher level of difficulty could be explained, in part, by the straightforward mathematical construction of the long-menu format, which reduces the probability of choosing the right answer at random. This was also observed by Schuwirth  et al. iOur findings did not support the conclusions of our previous retrospective study that had shown a higher discriminatory power for long-menu questions than for other classic formats . The context of our retrospective study was clearly different: psychometric properties were computed from different groups of examinees, and more importantly, the question stems were different, meaning that not only the answer formats were compared but also the topics themselves and the underlying learning objectives. These are likely to be more important elements than the answer format itself , both inIt is interesting to note that more than three quarters of the responding examinees thought that the long-menu format was closer to real life. This assertion is supported by the physicians who often face a long list of alternative diagnoses in their daily practice, but it is also asserted by our results: more than half of the incorrect options chosen by students who had the long-menu format were not among the distractors listed in the type A format. This corroborates the finding of Huwendiek et al.  that thiA vast majority of responding students acknowledged that they had used a different reasoning approach when trying to answer long-menu questions. We may hypothesise that the long-menu format encourages retrieval practice. Therefore, the cognitive effort required by the long-menu format may support mid- and long-term learning and retention of knowledge. However, future research would be needed to address this issue. The students’ perceptions and their observed increased difficulty with long-menu questions also might bring some evidence of a positive educational effect .Almost two thirds of the responding examinees did not find the format more difficult than the free-text open question format, which not only requires a long process of manual marking but is also more liable to a subjective evaluation, and it has never been clearly proven superior to classic multiple choice  or long-Our study has limitations. It was carried out in a single faculty and the results might not be generalizable to other faculties. However, students’ results at the Swiss Federal Licensing Exam have been similar across all faculties in the past few years, showing that there were no significant differences in teaching at the various faculties of medicine and in the students’ performances. Moreover, we included all the students, hence avoiding the selection bias of studies conducted on volunteer students. The two formats were compared in many different medical specialities, decreasing the likelihood that the results were influenced by topics and content. Another limitation was the choice of exam items for our study. The items were not designed especially for the study: in each exam, with the exception of paediatrics, we identified Type A questions which could be modified into long-menu questions. This could have led to a selection bias based, for example, on our knowledge of existing long-menu lists that could be easily re-used and adapted, or on the fact that we could not make any significant changes to the existing question items. Finally, the short survey was conducted to measure the examinees’ general perception regarding some item formats: this does not constitute evidence about differences in psychometric properties or reasoning processes between the item formats, but may represent interesting topics for future studies.They is no evidence that the long-menu questions and Type A question differ regarding the discriminatory power, but the long-menu questions they are slightly more difficult. Long-menu questions are perceived to be closer to real practice, which could have a positive educational impact. Taking into account the increased time required to develop long-menu items when compared to Type A items, we would recommend their use in the final years of the curriculum, within more realistic key-feature problems, which focus on assessing clinical reasoning and patient management skills.