The clinical application of immune checkpoint inhibitors (ICIs) has led to dramatic changes in the treatment strategy for patients with advanced non-small cell lung cancer (NSCLC). Despite the observation of improved overall survival in NSCLC patients treated with ICIs, their efficacy varies greatly among different immune and molecular profiles in tumors. Particularly, the clinical significance of ICIs for oncogene-driven NSCLC has been controversial. In this review, we provide recent clinical and preclinical data focused on the relationship between oncogenic drivers and immunological characteristics and discuss the future direction of immunotherapy in NSCLC patients harboring such genetic alterations EGFR activating mutations [BRAF V600E mutations [ALK [ROS1 [RET [Lung cancer is the leading cause of cancer-related death worldwide . Non-smautations ,4,5,6, Butations , and oncons [ALK ,11,12,13LK [ROS1  and RET OS1 [RET ,16. HoweThe recent development of immune-oncology has drastically changed NSCLC treatment. The receptors of anti-program death 1 (PD-1) and anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) are expressed on activated T cells that normally downregulate excessive immune responses, while tumor cells and tumor-infiltrating immune cells express programmed death-ligand 1 (PD-L1) to evade antitumor immunity through the interaction with these molecules . Immune Several phase III trials have demonstrated that ICIs significantly prolong the OS of NSCLC patients compared with chemotherapy ,19,24. OKRAS is one of the most frequently mutated proto-oncogenes [KRAS-mutant NSCLC [KRAS-mutated NSCLC has not yet been established. Given the unfavorable prognosis in KRAS-mutant NSCLC patients [KRAS-mutant NSCLC.Among oncogenic drivers for NSCLC, ncogenes . Becausent NSCLC ,31 and tpatients ,33, the In this review, we address the clinical data of immunotherapy and preclinical evidence concerning immunologic characteristics in oncogene-driven NSCLC and discuss future directions for immunotherapy in patients with oncogene-driven NSCLC.PD-L1, a logical biomarker to predict the response to anti-PD-1/PD-L1 blockade, has been extensively evaluated in clinical studies. PD-L1 was regarded as a predictive biomarker in a first-in-human study of nivolumab, and a subsequent larger-scale phase I study of NSCLC further confirmed this finding; at a cut-off of 5% PD-L1-positive tumor cells, the overall response rate (ORR) of PD-L1-positive patients was 36%, whereas the ORR of PD-L1-negative patients was 0% . In a phThe central mechanism of anti-tumor immune responses is that activated cytotoxic T lymphocytes recognize tumor antigens specifically and eliminate cancer cells; therefore, TILs are a reasonable candidate to predict the efficacy of ICIs. Accumulating evidence has confirmed the prognostic impact of TILs in NSCLC patients, clarifying that increased levels of CD3 and CD8+ TILs are associated with better outcomes in NSCLC ,38,39. ROther factors along with TILs may be important. Kinoshita et al. focused on the relationship between TILs, histological type, and smoking habit, showing that a low number of CD8+ T cells in non-adenocarcinoma, a high FOXP3/CD4 ratio in smokers with adenocarcinoma, and a low number of CD20+ B cells in non-smokers with adenocarcinoma were identified as independent prognostic factors in resected NSCLC . It was Mutations, genetic rearrangements, insertions, and deletions can generate neoantigens that are specific to the tumor relative to normal somatic cells. Tumors with a greater mutational burden could possess more neoantigens; thus, the patient may possess a larger repertoire of extant tumor-specific T cells . RecentlSome unsolved issues persist concerning the clinical use of TMB as a biomarker for ICIs. First, the ranges of TMB were highly overlapped in responders and non-responders and the numerical cut-off of TMB that discriminates responders from non-responders has not been determined ,48. SecoThe loss of MMR genes is associated with a marked increase in TMB. The MMR-deficient genotype was identified through the detection of microsatellite instability (MSI). The relationship between this genetic subtype and the clinical activity of ICIs has been rigorously investigated mainly in colorectal cancer. The first hint of the predictive relevance of the MSI status arose from the finding that a durable response to anti-PD-1 treatment was observed in a patient with colorectal cancer, which is one of the cancers resistant to ICIs; this patient exhibited the MSI-high phenotype . ConsequEGFR mutations exhibit less efficacy of anti-PD-1/PD-L1 treatment than those with wild-type EGFR, whereas KRAS mutations are likely to be predictors of favorable outcomes. Few studies have addressed the relationship between ALK rearrangement and clinical benefit from ICIs due to the small number of recruited patients with ALK rearrangement. The efficacy of ICIs in oncogenic-driven NSCLC is summarized in Randomized phase III trials have indicated that patients carrying EGFR-mutant populations [EGFR-mutated patients tended to be inferior compared to that in EGFR-wild type patients [EGFR-mutant patients [EGFR T790M-positivity was associated with unfavorable clinical outcome in patients treated with nivolumab following EGFR-TKI treatment [EGFR-mutant patients who had progressive disease with EGFR-TKI treatment, even in patients with ≥50% PD-L1 expression [EGFR-mutant NSCLC compared to chemotherapy [EGFR-mutant NSCLC. Meanwhile, the ATLANTIC trial reported contradictory findings that durvalumab exhibited clinical activity regardless of the status of EGFR/ALK alterations in heavily treated NSCLC with higher expression of PD-L1 [EGFR/ALK-positive NSCLC in larger-scale cohorts. In this respect, it is noteworthy that a randomized phase II trial (WJOG8515L) is currently ongoing to compare nivolumab with carboplatin plus pemetrexed chemotherapy in EGFR-mutant NSCLC patients who have developed resistance to EGFR-TKI due to mechanisms other than T790M [Although several clinical trials have demonstrated the survival benefit of anti-PD-1/PD-L1 inhibitors over docetaxel in pretreated NSCLC patients, this has not been proven in ulations ,21,24. Fpatients . Consistpatients . A retroreatment . Anotherpression . In addiotherapy  or EGFR-otherapy . These fof PD-L1 . Furtheran T790M .EGFR or ALK genetic alteration status [EGFR exon19 deletion or L858R mutation compared to BCP in the subgroup analysis [EGFR-mutated NSCLC patients. Further studies of large-scale population will help to elucidate the therapeutic role of ICIs combined with bevacizumab plus chemotherapy in oncogenic-driven NSCLC after acquired resistance to TKIs.Recently, the IMpower150 trial demonstrated that the addition of atezolizumab to bevacizumab plus carboplatin plus paclitaxel (ABCP) significantly improved PFS and OS compared to bevacizumab plus carboplatin plus paclitaxel (BCP) in advanced non-Sq NSCLC patients including those who had received TKIs, irrespective of n status . Of noteanalysis , showingKRAS mutations, OS in the nivolumab group was significantly longer than that in the docetaxel group [KRAS mutations but not in those with KRAS wild-type [KRAS mutations may predict the response or outcomes in NSCLC patients treated with anti-PD-1/PD-L1 blockade, its clinical relevance has not been fully proven due to the retrospective nature and insufficient examination of the genetic status.Among NSCLC patients with el group , whereasel group . A recenild-type . CollectEGFR, ALK fusions, KRAS, MYC, PTEN, and p53; (2) exogenous inflammatory cytokines, such as interferon-γ; (3) PD-L1 amplification; and (4) disruption of the 3′-untranslated region of the PD-L1 gene [An association between PD-L1 expression and oncogenic drivers has been extensively explored in prospective studies and clinical practice. Recent experimental studies have elucidated the regulatory mechanisms of PD-L1 expression. Briefly, tumor cells can activate PD-L1 expression via the following mechanisms: (1) alterations of genes, including -L1 gene . PreviouEGFR-mutant NSCLC cell lines [Several preclinical studies have suggested that oncogenic EGFR signaling pathways upregulate PD-L1 expression. This issue was first addressed by Akbay et al., who demonstrated that forced expression of mutant EGFR in bronchial epithelial cells induced PD-L1 and that PD-L1 expression was reduced by EGFR inhibitors in ll lines . Other sll lines  or NF-κBll lines  is involEGFR mutations and PD-L1 overexpression has also been observed in NSCLC tumors. Azuma et al. showed that the presence of EGFR mutations was associated with high expression of PD-L1, independent of other characteristics [EGFR mutations was linked to a PD-L1-positive status [EGFR mutation status and PD-L1 expression [EGFR-mutant LUADs than in those with wild-type EGFR [EGFR mutations were observed in only 9% of all EGFR-mutant LUADs [EGFR mutations were associated with decreased expression of PD-L1 [EGFR mutation status, it is likely that anti-PD-1/PD-L1 therapy has limited activity for EGFR-mutant NSCLCs.The positive correlation between eristics . D’Incece status . In contpression ,82,83. Pype EGFR , and PD-nt LUADs . Concordof PD-L1 ,87,88. TEGFR-mutant NSCLCs. NSCLC patients with EGFR mutations showed a shrinking proportion of PD-L1+/CD8+ TILs and decreased TMB [EGFR-mutant NSCLC tumors has been observed [EGFR mutations.Recent studies have provided some implications for immunological features other than PD-L1 expression that could confer the limited efficacy of ICIs for ased TMB . Additioobserved ,89, possALK rearrangement to upregulate PD-L1 expression was first documented in ALK-translocated T-cell lymphoma [EML4-ALK-positive NSCLC cells was attenuated by the ALK inhibitor alectinib and by siRNA-mediated ALK knockdown [EML4-ALK-induced upregulation of PD-L1 [The oncogenic capacity of lymphoma . In NSCLnockdown . Hong etnockdown . Furtherof PD-L1 ,92,93.ALK rearrangement and PD-L1 overexpression. A previous study investigated the PD-L1 expression by IHC in resected NSCLC tumors, revealing that the ALK-rearranged tumors exhibited higher PD-L1 expression than those with wild-type ALK [ALK translocation was correlated with PD-L1 and PD-L2 expression in the resected LUADs [ALK-rearranged LUADs compared to those with EGFR mutation or those with wild-type EGFR/KRAS and non-ALK rearrangement [ALK rearrangement and PD-L1 expression [Clinicopathological studies have indicated a relationship between type ALK . Anothered LUADs . Furtherangement . By contpression ,88. KRAS mutations induce PD-L1 overexpression through activation of its downstream pathways in NSCLC [KRAS or ERK2 and the MEK inhibitor decreased PD-L1 expression in KRAS-mutant NSCLC with PD-L1 overexpression [KRAS siRNAs and the ERK inhibitor [KRAS mutations and PD-L1 overexpression [CD274 encoding PD-L1 varied greatly among KRAS-mutated NSCLC cell lines, implying that other mechanisms modulate PD-L1 expression in different KRAS-mutant NSCLCs.Previous studies have shown that in NSCLC ,96,97. Spression . ConsistKRAS mutations and PD-L1 expression in NSCLC [KRAS-mutated NSCLC tumors exhibited a higher positivity of PD-L1 expression [KRAS-mutated tumors were significantly associated with higher levels of PD-L1 expression [KRAS-mutant NSCLC. Calles et al. suggested that smoking is likely to be associated with increased PD-L1 expression; PD-L1 expression is detected in 44% of current smokers, 20% of former smokers, and 13% of never smokers, and a higher intensity of PD-L1 expression was frequently observed in smokers with more pack-years [Clinicopathological studies have indicated the positive correlation between in NSCLC ,99,100. pression . Severalpression ,88,99. Ick-years .KRAS-mutant NSCLC. Co-occurring genetic alterations in STK11/LKB1 (KL subgroup) and TP53 (KP subgroup), as well as CDKN2A/B inactivation coupled with low expression of TTF1 transcription factor (KC subgroup), define three major subgroups of KRAS-mutant LUAD with distinct biology and therapeutic vulnerabilities [TP53 and KRAS mutations in LUAD and provide some notable findings: (1) co-occurring mutation in TP53 and KRAS showed the highest PD-L1 expression, the highest proportion of concurrent PD-L1 expression and CD8+ T-cell infiltration, decreased expression of some other non-PD-L1 immune inhibitory checkpoints, such as lymphocyte activating 3 (LAG-3), and higher mutational load; (2) KRAS mutation manifested as various defects in DNA repair, including MMR-related genes; (3) TP53 mutations increased the mutation frequencies of POLE, which is associated with the disruption of exonuclease activity required for DNA proofreading and leads to high TMB; and (4) the patients with KRAS or TP53 mutations, especially those with co-occurring TP53/KRAS mutations, showed a marked clinical benefit to PD-1 inhibitors [KRAS-mutant LUAD specimens exhibited higher expression of PD-L1 in malignant cells and of B7-H3, T-cell immunoglobulin mucin family member 3 (TIM3), and indoleamine 2, 3-dioxygenase-1 (IDO-1) in stromal tumor-associated inflammatory cells than wild-type [Several experimental and translational studies have addressed distinct immunological features in bilities . In thishibitors ,104,105.hibitors . The mosild-type .BRAF is mutated in 2% to 4% of LUAD [BRAF-mutated NSCLCs have non-V600E mutations [BRAF-mutant NSCLC only target the V600E mutation, treatment strategies for non-V600E BRAF-mutated NSCLC should be urgently developed, and immunotherapy may contribute to establishing the strategies.The proto-oncogene  of LUAD , and apputations ,109. GivBRAF mutation have been observed in colon cancer [BRAF-mutant NSCLC, little is known about the immune response and immunological characteristics in such populations. A retrospective analysis in LUAD patients showed that BRAF mutation was significantly associated with a low density of intratumoral CD8+ T cells and high neutrophils density [BRAF V600E mutation (group A) and those with BRAF non-V600E mutation (group B) [BRAF mutation is associated with a high level of PD-L1 expression, low/intermediate TMB, and a microsatellite-stable status. This study also described that the ORRs with ICIs were 25% and 33% in groups A and B, respectively, and the median PFS rates with ICIs were 3.7 and 4.1 months in groups A and B, respectively, indicating that ICIs may have favorable anti-tumor activity for both BRAF V600E and BRAF non-V600E mutant NSCLC.Significant associations between PD-L1 positivity and n cancer ,111,112 n cancer . Owing t density . Recentlgroup B) . This stKRAS mutations or BRAF non-V600E mutations remains challenging, immunotherapy may play a pivotal role in patients carrying these genotypes. Thus, further investigation into the relationship between oncogenic drivers and immunological backgrounds should be required. Accumulating evidence has speculated that a single biomarker may not fully predict the clinical activity of immunotherapy. Thus, exploring the combination of biomarkers for patient selection to maximize the therapeutic benefits or minimize medical and economic loss would be a cornerstone of the development of cancer immunotherapy in the future.Although molecular targeted therapies have been established in some subsets of patients with oncogene-driven NSCLC, the role of immunotherapy remains unclear. Previous studies have suggested that the status of driver oncogenes in NSCLC tumors seems predictive for the efficacy of immunotherapy, but the predictive roles have not been determined. Given that targeting NSCLCs harboring


DNA methylation of promoter regions is often associated with epigenetic silencing of gene expression, and DNA methyltransferase (DNMTs) has been used to suppress gene expression. In order to explore the synergistic roles of two methyltransferase members Dnmt3a and Dnmt1, we constructed expression plasmid that could express a recombinant DNMTs consisting of the C‐terminal domains of both Dnmt3a and Dnmt1 fused to a zinc finger domain which binds to the PD‐L1 promoter of human prostate cancer cells (DU145). Programmed death ligand 1  is a transmembrane protein widely expressed on antigen‐presenting and other immune cells. The interaction of PD‐L1 with its receptor PD‐1 is considered an ‘immune checkpoint' for possible cancer therapy. DU145 cells treated with the Dnmt3aC‐1C plasmid showed significantly reduced expression of PD‐L1 as compared to Dnmt3aC or Dnmt1C alone. Our results show that the fusion of Dnmt1 improves the methylation activity of Dnmt3a and enhances its biological functions. This combinatorial strategy can be used to better control PD‐L1 expression to support cytotoxic T lymphocytes (CTL) response against tumors. CTLcytotoxic T lymphocytesDNMTsDNA methyltransferaseMSPmethylation‐specific PCRProgrammed cell death ligand 1  is a transmembrane protein that is widely expressed on antigen‐presenting and other immune cells, and is unregulated on tumor cells from a broad range of cancer types. Binding of PD‐L1 with its receptor PD‐1 on T cells delivers a signal that inhibits TCR‐mediated activation of IL‐2 production and T‐cell proliferation. PD‐L1 binding to PD‐1 also contributes to ligand‐induced TCR downregulation during antigen presentation to naive T cells Using a PD‐L1 inhibitor that blocks the interaction of PD‐L1 with the PD‐1 receptor can prevent cancer from evading host immune system. So, blocking the PD‐1/PD‐L1 interaction can suppress tumor immune escape, thereby improving the ability of initial T‐cell activation and CTL killing activity DNA methylation of promoter regions is often associated with epigenetic silencing de novo methyltransferases The DNA methyltransferase (DNMTs) comprises a family of DNA‐modifying enzymes that have a central role in epigenetic gene regulation. Global cytosine methylation patterns in mammals appear to be established by a complex interplay of at least three independently encoded DNMTs: DNMT1, DNMT3A, and DNMT3B DNA methyltransferases have been used to repress target gene through specific DNA methylation, with guidance to the promoter region of a target gene by fusing it to a specific zinc finger domain http://www.urogene.org/methprimer) was used to analyze the core promoter sequences of potential methylation site (CpG island), and zinc finger protein binding site on PD‐L1 gene promoter was identified by the software (http://www.scripps.edu/barbas/zfdesign/register.php?query). Putative methylation and zinc finger protein binding sites on PD‐L1 promoter are shown in Fig. We used Eukaryotic Gene Promoter Database to analyze PD‐L1 gene promoter sequence. CpG Island Analysis Software  by Neuron Biotech, Shanghai, China, and abbreviated as Ad‐ZF‐Dnmt3aC‐1C.NM_175629.1) and Dnmt1  were generated from pcDNA3.1/Flag‐Dnmt3a and pcDNA3.1/Flag‐Dnmt1 by PCR using primers listed in Table 2 to generate pZF‐Dnmt3aC‐1C and this to the C terminus of zinc finger (ZF). These recombinant Dnmt constructs were commercially packed into replication‐defective adenoviral vector pDOV‐mCMV‐GFP‐3FLAG (Neuron Biotech) to generate Ad‐ZF‐Dnmt3aC‐1C. The pDOV‐mCMV‐GFP‐3FLAG was considered as negative control (Ad‐control). The resulting vectors are schematically represented in Fig. The C terminus of Dnmt3a  per cell.Human prostate cancer cells (DU145) were obtained from the American Type Culture Collection . Cells were cultured in standard DMEM with 10% fetal bovine serum, 1% penicillin/streptomycin at 37 °C, and 5% COTotal proteins were collected from the infected cells after 24, 48, and 72 h, run on 10% SDS/PAGE, and detected with 3000× diluted anti‐PD‐L1 monoclonal antibody  overnight. The antibody was removed, and the poly(vinylidene difluoride) membrane was washed three times for 5 min each in TBST. Anti‐rabbit secondary antibody (Abcam) was used at a dilution of 1 : 5000 in TBST and incubated for 1 h at room temperature. The antigen–antibody complexes were detected using the G:BOX F3 gel doc system .flowjo software .Cells were cultured in 6‐well plates in regular growth media until they reached 70–80% confluency. Cells were fixed with 4% PFA and washed. PD‐L1 was detected using PE‐conjugated anti‐PD‐L1 antibody  on Calibur flow cytometer (BD Bioscience), and data were analyzed using Total RNA from treated and control cells were isolated after 24, 48, and 72 h using Trizol reagent  according to the manufacturer's instructions. For each sample, 2 μg of total RNA was reversely transcribed using the QuantiTect Reverse Transcription Kit . Gene expression was determined using the DyNAmo Flash SYBR Green qPCR Kit  on the Stratagene Thermocycler (Mx3000). The primers used are listed in Table methprimer software  for methylated DNA .To check the degree of methylation of PD‐L1 promoter, methylation‐specific PCR (MSP) was performed as described earlier spss software . The data are reported as means ± SD. Different experimental groups were compared by Student's t‐test. A value of P < 0.05 was considered statistically significant.Statistical tests were performed with the Analysis with Eukaryotic Gene Promoter Database showed that PD‐L1 gene promoter sequence is highly methylated in normal cells and is hypomethylated in tumor cells. Using Tools Ver3.0 design software, we identified PD‐L1 gene promoter and GC box including downstream GTCCGCCTGCAGGTAGGGAGC zinc finger binding site. The 100‐bp‐long fragment contained 15 potential methylation sites, of which three were located in GC box  were transduced with Ad‐ZF‐Dnmt3aC, Ad‐ZF‐Dnmt3aC‐1C, or Ad‐control, and the expression of PD‐L1 was checked at 24, 48, and 72 h. Western blot analysis showed that ZF‐Dnmt3aC‐1C suppressed the expression of PD‐L1 more efficiently as compared to ZF‐Dnmt3aC alone and the expression level was reduced to less than half after 72 h as shown in Fig.  and control group (18.7%) than the experimental groups. The Ad‐ZF‐Dnmt3aC showed 25.7%, Ad‐ZF‐Dnmt1C showed 35.3%, and Ad‐ZF‐Dnmt3aC‐1C group showed highest methylation reaching 84%. Targeting the DNA methylation‐based gene‐silencing machinery has assumed great significance in the field of cancer epigenomics et al. reported that there is an inverse correlation between PD‐L1 promoter methylation and protein expression in prostate cancer. They suggest that PD‐L1 is inversely correlated with miR as cellular component, which modifies the downstream processing of PD‐L1 mRNA. Differential expression of miR may therefore potentially interfere with the linear translation of PD‐L1 mRNA into PD‐L1 protein et al. also report on the inverse correlation between promoter methylation and respective mRNA expression Gevensleben de novo and maintenance functions have recently been re‐examined. Although single knockout of either Dnmt1 or Dnmt3b gene had minimal effects on DNA demethylation in colon cancer cells, Further we evaluated the cooperative role of different Dnmts with respect to DNA methylation and subsequent PD‐L1 expression. The specific roles of these Dnmts in The authors declare no conflict of interest.NZ, JH, XL, and ZW conceived and designed the project. XL and ZW performed the experimental work. XL wrote the manuscript, researched the data, reviewed and edited the manuscript. JX, HL, SZ, HY, LZ, BH, LY, and LL contributed to the discussion. Funding acquisition by NZ.


Biguanide forms bidentate metal complexes through the two imino nitrogens. Here, it is discussed that phenformin (phenylethyl biguanide) is a model for recruitment of endogenous Zn+2 to inhibit CysHis/CysHis(X) peptidolysis. Phenformin is a Zn+2-interactive, anti-proteolytic agent in bioassay of living tissue. Benzoyl-l-arginine amide (BAA) is a classical substrate of papain-like proteases; the amide bond is scissile. In this review, the structures of BAA and the phenformin-Zn+2 complex were compared in silico. Their chemistry and dimensions are discussed in light of the active sites of papain-like proteases. The phenyl moieties of both structures bind to the “S2” substrate-binding site that is typical of many proteases. When the phenyl moiety of BAA binds to S2, then the scissile amide bond is directed to the position of the thiolate-imidazolium ion pair, and is then hydrolyzed. However, when the phenyl moiety of phenformin binds to S2, then the coordinated Zn+2 is directed to the identical position; and catalysis is inhibited. Phenformin stabilizes a “Zn+2 sandwich” between the drug and protease active site. Hundreds of biguanide derivatives have been synthesized at the 1 and 5 nitrogen positions; many more are conceivable. Various substituent moieties can register with various arrays of substrate-binding sites so as to align coordinated Zn+2 with catalytic partners of diverse proteases. Biguanide is identified here as a modifiable pharmacophore for synthesis of therapeutic CysCt inhibitors with a wide range of potencies and specificities.Excessive activities of cysteinyl cathepsins (CysCts) contribute to the progress of many diseases; however, therapeutic inhibition has been problematic. Zn2+ ComplexPhenformin-Zn The human genome encodes 11 cysteinyl cathepsins (CysCts) and multiple peptide inhibitors . Deletions of various CysCt genes can decrease the progress of diverse pathogenic processes. Conversely, loss of endogenous protease inhibitors can increase these pathogenic processes. Optimal health and longevity require an unexplained balance between CysCt activities vs. Nature’s anti-proteolytic agents. Pharmacological opposition of one or more CysCt activity(s) might have great therapeutic benefits , where X is one of several amino acids, e.g. asparagine. Site-directed mutagenesis reveals that the essential catalytic partners are CysHis. The third amino acid of triads can be replaced with a non-catalytic amino acid without loss of activity; although this changes reaction kinetics  protease assays with a reaction buffer consisting of metal chelator, sulfhydryl reducing agent and optimal pH. Standard assay conditions can buffer a constant maximal reaction rate. However, Nature varies the in vivo reactions of CysCts over a wide range by changing the composition of their endogenous reaction buffer. Machinery regulating metals, redox and protons at the cellular and compartmental levels has been identified. Metals, redox and protons are now recognized as simultaneous variables that modulate the endogenous CysHis/CysHis(X) reaction mechanism Lockwood .  +2 is a natural inhibitory modulator of their reaction mechanism. Zn+2 forms multi-dentate associations with the thiolate anion of cysteine neighboring the imidazole ring of histidine in many Zn+2 finger proteins, e.g. Cys2 -Zn+2-His2  neighboring 1 His(imidazole) in catalytic sites is emerging as a major component of Nature’s reaction buffer. Recent evidence suggests that cell Zn+2 homeostasis is interactive with multiple pathways of cell protein degradation (see below section).An exploitable feature of CysCts has been overlooked. All proteases with the CysHis/CysHis(X) catalytic partners share something in common: ZnMost of the cell content of CysCts consists of inactive zymogens. Excessive ongoing proteolysis can lead to decompensated activation of pro-proteases, positive feedback, and necroptosis or apoptosis (see below section). Lesser excesses of intracellular or extracellular activities can be gradually injurious over many years. For some uses, complete inhibition of one or more CysCts might be required, e.g. inhibition of viral or parasite proteases. Importantly, excessive inhibition of various proteolytic pathways can be pathogenic under prolonged administration   Fig.  forms cons Figs. , 5. Certl-arginine amide (BAA) is a well-known artificial substrate of diverse proteases. Comparison of the active site of papain-like proteases with the structures of BAA and the phenformin-Zn+2 complex explains why one is a substrate for proteases; and the other is an inhibitor. The Zn+2 complex of phenformin is a prototype for design or discovery of other anti-proteolytic biguanide derivatives with a wide range of specificities and potencies.In silico chemistry now permits precise comparison of small molecules. Phenformin is a derivative of biguanide  the interaction of CysHis/CysHis(X) catalytic partners with the coordinated metal cation and (b) the interaction of substrate-binding sites with derivative moieties of the biguanide pharmacophore  excessive activity of one or more CysCts, and (c) elevated inflammatory/autoimmune processes seems likely. Such a relationship provides multiple points of therapeutic intervention.It has been reviewed that mammalian inflammation and autoimmunity are increased by ZnNature buffers the intracellular activities of CysHis/CysHis(X) cathepsins at submaximal rates by regulating components of their catalytic environment. Positive and negative controls can be superimposed upon the buffered reaction. Redox regulation and metal regulation are virtually inseparable in the buffering of CysHis/CysHis(X) peptidolysis; and pH regulation can influence both. Many redox interactions can influence the status of protease sulfur, e.g. GSSG/GSH ratio or oxygenations (Lockwood +2 can preemptively decrease the CysHis/CysHis(X) peptidolytic mechanism under otherwise optimal reaction conditions.Lockwood . A dedicLockwood . In addiLockwood . Zn2+ caThe CysHis peptidolytic mechanism responds to the range of endogenous cell Zn2+regulation. Intracellular Zn+2 is 98% sequestered by protein binding. The biological concentration of truly “free” Zn+2 is very low, but not known with certainty , has been suggested to be in the range of 1–100 picomolar. However, 1-3 micromolar cytoplasmic Zn+2 is neither free nor completely sequestered. Investigators have used various terms to refer to this intermediate form of Zn+2, e.g. “mobile”, “active”, “loosely buffered”, “exchangeable”, “chelatable” etc. These many forms of Zn+2 can be collectively referred to as “interactive” Zn+2. This Zn+2 exists in a dynamic exchange among myriad biomolecules with diverse Zn+2 affinities, e.g. citrate, phosphate, certain amino acids, peptides, and many more. Since Zn+2 has six ligand-binding sites . Importantly, the lysosomal vacuole is a major participant in cell Zn+2 regulation as well as protein degradation.Long ago we studied the rate of bulk intracellular protein degradation in a viable perfused tissue bioassay, i.e. minute-to-minute release of incorporated 3H-leucine from cell proteins. Endogenous extracellular Zn+2 with the Cys(thiolate)-His(imidazole) catalytic partners is influenced by additional variables. CysHis/CysHis(X) peptidolysis can be inactivated by oxidation of protease sulfhydryl to diverse products . Histidine can oxidize to 2-oxohistidine; however, the significance is uncertain. Many of the variables that interact with CysHis catalytic partners also interact with each other, independent of the protease. For example, glutathione interacts with CysHis proteolysis in several ways. The reducing action of GSH maintains reductive activation of protease sulfur. Oxidized GSSG inhibits cysteinyl proteases, thereby imparting sulfhydryl/disulfide redox buffering of the reaction   and multidrug and toxicant transporters (MATEs)  Lysosomes of some species can serve as Zn+2 storage depots for use in times of Zn+2 insufficiency  is not precisely known; however, it is probably near 10 or 11 (i.e. Kd = 10−11). EDTA completely reverses the inhibitory action of Zn+2 and Zn+2-interactive biguanides. The Kd of the hexadentate Zn+2-EDTA complex is 10−16.5, corresponding to 105–106 times greater affinity than biguanides. Stabilities of biguanide complexes with various metals, and “on–off” kinetics can vary. The intracellular and extracellular distribution of biguanide among complexed endogenous metal species is unknown. The most effective anti-proteolytic metal complex need not be the complex that is formed in greatest amount.The biguanide-ZnIn the late 1800s it was known that the papaya plant contains high amounts of a proteolytic activity  proteases; although Fe+3 is much less potent than Zn+2 . Chlorhexidine is an antibacterial agent that is widely used in dental practice. “Gingipains” are secreted bacterial cysteinyl proteases that contribute to periodontal disease. Chlorhexidine is a Zn+2 -interactive inhibitor of these proteases. Zn+2 increases chlorhexidine inhibition of gingipains by 3- to 30-fold , correspond to multiple substrate amino acid residues (“P”) in a linear relationship. The substrate amino acid sequence was proposed to bind within the groove between domains, i.e. like “beads on a string” .Metformin indicates that a phenyl ring at the P2 position is unnecessary for Zn+2-interactive inhibition of CysCts; however, the S2P2 interaction is a major determinant of potency. Both metformin and phenformin are Zn+2-interactive inhibitors of cell protein degradation as observed in viable tissue bioassay . There is no necessary “S-P” correspondence when the complexed metal is aligned with the catalytic pair. The two methyl groups of metformin impart a partial hydrophobic character to complexed Zn+2 insertion between thiolate and imidazole stops peptidase, esterase and non-peptide amidase reaction mechanisms. In the absence of metals the CysHis/CysHis(X) peptidolytic reaction involves formation of thiolate anion and imidazolium cation by transfer of the proton  does not require experimental addition of extracellular Zn+2 for the most rapid time course of its inhibitory action. Due to its great potency, phenformin can acquire sufficient intracellular Zn+2 for maximal anti-proteolytic action at 0.01 µM drug concentration. In contrast, the anti-proteolytic action of therapeutic metformin concentration (10 µM) was delayed and submaximal in the absence of extracellular Zn+2 added to the perfusate. Exposure to physiological extracellular Zn+2 concentration and therapeutic metformin concentration were highly synergistic. These experimental results are expected of two agents with similar metal-interactive mechanisms of action, but a great difference in binding affinity for the CysCt active site.+2 toward protease assays as observed under standard assay conditions of 5 mM DTT at pH 5.5 . Therefore, therapeutic metformin concentration does cause Zn+2-interactive inhibition in protease assay; however, its potency is much less than phenformin.We reported that metformin adds little to the inhibitory action of Zn+2 naturally influences many cellular processes and enzyme reactions, e.g. some of the kinases. It is conceivable that biguanides might inhibit lysosomal proteolysis indirectly by interference in vacuolar/cellular regulation of metals or pH, or autophagic substrate accumulation. Moreover, an agent that modifies the interaction of metals with biomolecules might have multiple effects that are unrelated to protein degradation  inhibits the DPAP reaction toward other peptide derivatives  proteases or groups of them.Finally, cellular Zn


In the Men who are overweight or obese are generally recognized as a hard to involve, yet high-risk group for obesity-related chronic disease treatment . A varieCrowdsourcing is a strategic model used to draw insights from an interested group of individuals who are able to suggest solutions beyond those offered by traditional forms of research. In other words, the crowd “solves” the problem that has scientific professionals puzzled . Web-bas22.1reddit.com, which is a social networking and news website with user-submitted content. The notice on reddit.com was posted in a specific section focused on weight loss www.reddit.com/r/loseit where many users spend time reading and commenting on other users' posts, links and photos. Reddit.com was specifically chosen due to the interactive nature of the website, the high number of users , and the fact that 71% of Reddit users are men  All questions were presented to users randomly, each with an equal chance of appearing for the user to answer. Data were collected for a two-week cycle in December 2015. Similar to previous studies, no target sample size was established, as it is impossible to estimate the number of participants or the number of questions and answers collected . For thi2.4Correlations between question responses and BMI were calculated between self-reported BMI and responses to questions for all male respondents. Pearson correlations were calculated for Likert scale and numerical responses and Spearman's rho was calculated for yes/no responses. All questions that received fewer than 50 responses were excluded from analyses due to insufficient response numbers to appropriately assess correlations.Anonymity is one of the advantages of the crowdsourcing mechanism, however this introduces a level of reporting bias in that survey users may not accurately represent themselves in the study  After collecting all questions and responses, we encountered six obviously falsified answers , which were removed from all analyses.3Five hundred twenty-two visitors initiated the survey during the two-week survey period. Males comprised 57% of respondents, therefore the sample size for data analysis was 298, once we removed participants who reported an implausible BMI. Men with obesity (BMI > 30) comprised 43.3% (n = 129) of the sample, overweight (BMI 25.0–29.9) 33.6%, (n = 100) healthy weight (BMI 18.5–24.9) 23.1%,(n = 69) and underweight (BMI < 18.5) 0%.In addition to the five ‘seed’ questions supplied by the researchers, participants proposed 192 new questions, 188 of which were approved and added to the survey. In total, participants provided 21,846 responses to the 193 questions. Participants could only answer each question once. On average, each question was answered 126 times. Twenty-six questions were excluded due to fewer than 50 responses.p < .05), 21 of which were significant to p < .01. Out of the total 193 questions that were posted to the survey, 37 questions were significantly correlated with self-reported BMI  posed by men were correlated with higher BMI. While moderating the comment stream on  limited . Certain limited  and shouThe crowdsourcing survey brought forth many questions about physical activity. The correlation between physical activity and BMI is nothing new or unexpected, but it is interesting that many of the questions posed are related to the enjoyment of physical activity instead of strictly the practice . Research indicates that increasing exercise improves body image in men . BecauseMany other well-documented weight-related factors were not significantly associated with BMI in this study. Sleep has been negatively correlated with BMI in a variety of studies, but was not found to have any correlation in this investigation . Regular4.1While crowdsourcing is a novel approach, there are limitations to utilizing this methodology that could be addressed in future studies. It is important to consider that not all participants answered each question. Over 300 users answered the first six questions, but the last few questions only collected 2–3 answers. While we did offer a lottery-selected financial incentive to answer all questions posted on the site at the time of each participant's initial visit, a different incentive structure to motivate participants to return to the site as more questions are added could be beneficial for further data collection. It may also be beneficial to log how long participants spent on the site, as a measure of seriousness of the survey participants. Additionally, we did not collect demographic information from crowdsourcing participants. This makes it difficult to interpret the influence of some demographic factors, such as socioeconomic status, race and age on survey responses.We are particularly intrigued by the introduction of video games as a significant sedentary behavior with high correlation to BMI as well as the very small number of proposed questions relating to intimate personal relationships and their impact on weight. It is certainly understood in the literature that sedentary behavior is associated with higher weight, and perhaps further investigation of this population of men who participate in “gaming” is worth pursuing. Additionally, future intervention design could focus on the relationship between weight and relationship status/household roles  perhaps to better decipher the consistent external influences on weight management for men.Finally, while we hoped to gather ideas related to male motivation and engagement in weight loss studies, the nature of crowdsourcing does not allow us to dictate what information we will receive from the “crowd”. Crowdsourcing allows researchers to pose questions, and while we could have perhaps guided our research question a bit differently to better target interest and enthusiasm, it was also our goal to not guide the discussion in one way or another and instead see what the “crowd” came up with. We did not end up collecting many ideas of how to better target men for weight loss interventions, but we were able to collect valuable information surrounding why men are perhaps overweight in the first place. In order to appropriately design a successful weight loss program for men, it is important to better understand the background and the behaviors to change; such as less time spent playing/watching video games or more time spent participating in organized physical activity such as team sports or planned outdoor activities.


Indium nitride (InN) luminescence is substantially enhanced by the introduction of a multilayer graphene interlayer, mitigating the lattice mismatch between the InN epilayer and the Gallium nitride (GaN) template on a sapphire substrate via weak van der Waals interaction between graphene and nitride layers. The InN epilayers are deposited by radio-frequency plasma-assisted molecular beam epitaxy (MBE), and are characterized by spatially-resolved photoluminescence spectroscopy using confocal microscopy. A small blue shift of the emission band from the band gap evidences a low density of equilibrium carriers, and a high quality of InN on multilayer graphene. A deposition temperature of ~375 °C is determined as optimal. The granularity, which is observed for the InN epilayers deposited on multilayer graphene, is shown to be eliminated, and the emission intensity is further enhanced by the introduction of an aluminum nitride (AlN) buffer layer between graphene and InN. Indium nitride (InN) is a promising material for near-infrared optoelectronic devices owing to its narrow band gap (~0.65 eV), small electron effective mass, and high peak electron velocity . HoweverIn principle, the low emission efficiency caused by the low crystal quality can be circumvented by controlling energy transfer between radiating dipole and localized plasmons . HoweverThis approach opens a new route to alleviate one of the most critical problems in III-nitride growth, i.e., the substrate compatibility. In future perspective, combining distinctive band structure and physical properties of graphene with III-nitrides may lead to novel optoelectronic devices.It is already demonstrated that the introduction of graphene improves the crystal quality and luminescence intensity of GaN ,9, AlN , and IndThe InN deposition has been accomplished by radio-frequency plasma-assisted molecular beam epitaxy (rf-MBE), which is known to provide a better structural quality of InN epilayers than that achievable by metalorganic chemical vapor deposition (MOCVD). The deposition temperatures were varied, and the optimal growth temperature was estimated. Additionally, a sample with an AlN interlayer, introduced between ML graphene and InN to affect the coalescence processes in the InN layer deposition, was prepared and studied.Exfoliation of graphite was used to prepare multilayer (ML) graphene flakes which were then transferred onto gallium nitride (GaN)-on-sapphire templates. Indium nitride (InN) layers were deposited using radio-frequency plasma-assisted molecular beam epitaxy (rf-MBE), setup . We used droplet elimination by radical ion beam (DERI) process  for the The surface topography was analyzed with a 10-nm lateral resolution, using atomic force microscopy (AFM) in tapping mode . The crystal structure of InN layers was studied by the electron backscatter diffraction (EBSD) technique. The emission properties were studied by photoluminescence (PL) spectroscopy using a confocal microscope . A CW laser diode emitting at 660 nm (Integrated Optics) was used as the excitation source. The PL spectra were recorded using a spectrometer  equipped with an Indium gallium arsenide (InGaAs) detector array . The spatial resolution of 1.3 µm was achieved using a high numerical aperture (NA = 0.55) microscope objective. To eliminate the noise from the confocal microscopy InGaAs images, PL spectra were approximated by a Gaussian function at every pixel of the image before extracting PL parameters. All the measurements were performed at room temperature.Typical spatially-integrated PL spectra from InN deposited on ML graphene flakes  and InN layer outside ML graphene  are shown in The most long-wavelength position of the PL band of MBE-grown InN was observed at 0.63 eV  and 0.64To maximize the positive effect of the ML graphene interlayer, the InN deposition temperature was varied. The effect of growth temperature on PL parameters is demonstrated in Typical images of the spatial distribution of PL peak intensity and band peak position of the samples studied are shown in To compare the spatial inhomogeneity of PL parameters with surface morphology, the PL images obtained using the confocal microscope were matched with atomic force microscopy (AFM) images. A combined image for sample S375 is shown in Meanwhile, the average lateral size of InN grains on ML graphene increases from 125 nm in InN deposited at 325 °C to 256 nm at 375 °C, and decreases to 192 and 186 nm as the temperature is further increased to 425 °C and 435 °C, respectively. This grain size variation might be explained by a low migration barrier of group-III metals on ML graphene due to weak van der Waals interaction between InN and graphene, and by the dissociation of InN reducing the grain size at elevated temperatures [We attempted to improve the homogeneity of InN epilayers on ML graphene by introducing additional interlayers. The lack of dangling bonds on the surface of pristine graphene obstructs the direct growth of III-nitrides, as there are no sites to promote bonding with foreign atoms. To overcome this issue, the graphene surface can be functionalized. Buffer layers of AlN ,19 and ZMoreover, the average PL peak position is slightly blueshifted by the introduction of an AlN interlayer. This is an indication of a higher density of donor states.The smallest observed diameter of the bright spots in PL peak intensity images is limited to ~1.3 µm by the spatial resolution of the confocal microscope. Nevertheless, the comparison of the images with the AFM images leads to a conclusion that the overall PL efficiency is higher in the areas containing larger InN grains. The overall PL efficiency, considered here as the ratio of the numbers of photons emitted per photons incident, depends on (i) excitation conditions, (ii) internal quantum efficiency, and (iii) light extraction efficiency. A dedicated study is necessary to reveal the relative contributions of these three efficiencies.A high density of unintentional donors and, consequently, strong filling of the conduction band resulting in a high-energy shift of the optical band gap is an inherent property of InN. Therefore, we studied the correlation of PL intensity and band peak position. In each sample, we selected regions of 0.6 × 0.6 µm² in size at the center of several bright spots, and extracted the PL intensity and the band peak position spatially-integrated within the regions. The pairs of the PL intensity/peak position values for each region in the five samples studied are represented as points in The large PL intensity and band width variations might be explained by the mixing of hexagonal and cubic crystal phases on ML graphene flakes. The contribution of cubic and hexagonal phases was previously suggested to explain double-peaked PL spectra of GaN on graphene . In InN,In conclusion, InN epilayers were grown by rf-MBE on exfoliated ML graphene transferred onto GaN-on-sapphire templates. Substantial enhancement of PL intensity of InN by introduction of ML graphene interlayer is observed. The introduction of an ML graphene interlayer also results in a smaller blue shift of the PL band in respect to the band gap of InN, which indicates a lower density of donors providing free electrons into the conduction band. The optimal growth temperature for the deposition of InN on ML graphene was found to be in the vicinity of 375 °C. It is shown that the InN epilayers on ML graphene are inhomogeneous and consist of grains ranging from 0.2 to 1.2 µm in size. An AlN buffer layer was found to improve the homogeneity of the InN layer and to further enhance the spatially-integrated PL intensity. The comparison of the spatially-resolved PL measurements with the surface morphology characterized by AFM shows that the enhancement is caused by surface filling rather than by the enhancement of PL efficiency. The results demonstrate that the ML graphene interlayers are beneficial for growing thin epitaxial layers of InN with high overall emission efficiency. Further optimization of growth conditions to enhance the overall emission efficiency by improving the surface coverage, light extraction conditions and internal quantum efficiency are feasible.


Therapy of children with post‐transplantation lymphoproliferative disorder (PTLD) after hematopoietic stem cell (HSCT) and solid organ transplantation (SOT) can be challenging. In this retrospective study, we investigated PD‐L1 and PD1 expression in all PTLD categories of childhood and adolescence to see whether checkpoint inhibition with PD‐L1/PD1 inhibitors may serve as a therapy option. We included 21 patients aged 19 years or younger (at date of transplant) with PTLD following SOT or HSCT having adequate tumor samples available (n = 29). Using immunohistochemistry, we evaluated PD‐L1/PD1 expression on both tumor cells and cells of the microenvironment in all samples. Availability of consecutively matched tumor samples during 6 of 21 patients' disease courses also allowed an intra‐individual assessment of PD‐L1/PD1 expression. We observed lower PD‐L1 and higher PD1 expression in non‐destructive lesions, and higher PD‐L1 and lower PD1 expression in polymorphic and, in particular, in monomorphic PTLD, mostly diffuse large B‐cell lymphomas . The amount of PD‐L1‐ and PD1‐positive cells changed in the opposite way in sequential biopsies of the same individual correlating well with the PTLD category. This is the first comprehensive pediatric study assessing PD‐L1 and PD1 expression on tumor cells and in the microenvironment of PTLD including not only monomorphic, but also non‐destructive early lesions. PD‐L1 expression of the tumor cells inversely correlated with PD1 expression in surrounding tissues, with the highest expression in DLBCL. Since PTLD can be therapeutically challenging, our results indicate a potential efficacy of checkpoint inhibitors if standard immune‐ and/or chemotherapy fail or are impossible. We therefore recommend routine staining of PD‐L1 and PD1 in all PTLD categories. Among them, LMP1 and EBNA2 have been extensively studied.Post‐transplantation immunosuppression in an EBV‐positive patient reduces the activity of the patient's EBV‐specific cytotoxic T‐cell surveillance, which increases the chances of uncontrolled proliferation of EBV‐infected B‐cells and subsequent progression to PTLD.Immune checkpoints regulate T‐cell responses to maintain self‐tolerance. They deliver co‐stimulatory and co‐inhibitory signals to T‐lymphocytes.In this study, we investigated the expression of PD‐L1 and PD1 in the different categories of PTLD in 21 pediatric SOT and HSCT patients, allowing not only an inter‐individual comparison of the results, but also the analysis of the course of expression on an intra‐individual level in patients with consecutively collected matched tumor samples. Based on our results, we propose checkpoint inhibition as a potential treatment option for children and adolescents with PTLD if standard immune‐ and chemotherapy is not successful or impossible.22.1Twenty‐seven consecutive cases of pediatric and adolescent PTLD were retrieved from the patient's databases of the St. Anna Children's Hospital, the Department of Pediatrics and Adolescent Medicine, and the Department of Pathology of the Medical University of Vienna, Austria. Twenty‐one/27 (78%) cases  according to the manufacturer's instructions. EBV status was assessed by in situ hybridization  using the automated Leica Bond III Immunostainer (Leica Biosystems).All hematoxylin and eosin stained slides of each patient were reviewed and diagnoses were established according to the 2016 revised WHO  Classification of Hematopoietic and Lymphoid Tissues by two authoring hematopathologists .2.3Immunohistochemical analyses were performed on 2 µm slides of FFPE tissue using the automated Leica Bond III Immunostainer. The following primary antibodies were used: for PD‐L1: DAKO, monoclonal mouse anti‐human antibody, clone 22C3, Glostrup, Denmark, and for PD1 detection: CellMarque, monoclonal mouse anti‐human antibody, clone NAT 105, Darmstadt, Germany. Heat induced epitope retrieval was done with Bond Epitope Retrieval Solution (Solution 2 for PD‐L1 and Solution 1 for PD1) for 20 minutes. Primary antibodies were incubated for 30 minutes. Staining was done using the Bond Polymere Refine Detection Kit DS9800 according to the manufacturer's guidelines. Tissue sections were counterstained with hematoxylin.Double immunohistochemical staining for PAX5  und PD‐L1 were done in selected cases. Stains were performed in a sequential manner using the Leica Bond III Immunostainer platform with the protocol described above except for counterstaining, which was performed only once at the end. PAX5 antibody was used as the first agent and developed with brown color followed by a red color staining for PD‐L1 using the BOND Polymer Refine Red Detection Kit DS9390. Immunohistochemistry was assessed by two hematopathologists together on a multi‐headed microscope. Several high‐power fields  were examined and hot spots were defined. A distinct membranous staining on cells with medium or high intensity was rated positive. Lymphocytes in germinal centers served as internal positive controls for PD1. The amount of immunolabeled cells with respect to total cellularity (for PD1 and PD‐L1) and of the stained tumor cells (for PD‐L1) was estimated in each case by the two hematopathologists. Analyses were done on hot spots. The cut off level for PD1 positivity was set at ≥ 20% immunolabeled cells. According to previous publications, different cut‐off levels for PD‐L1 positivity were applied.33.1Patient's characteristics are presented in Table Among the eight patients with non‐m‐PTLD, four patients (Pt. #15‐18) were primarily diagnosed as p‐PTLD. Two showed regression of their PTLD category at subsequent biopsies with the morphology of a non‐destructive florid follicular hyperplasia (FFH) PTLD (Pt. #17‐18), one patient progressed to DLBCL (Pt. #15) and patient #16 did not have further sampling. Of note, one patient developed p‐PTLD as a progressive disease in a cervical lymph node 8 months after the diagnosis of non‐destructive infectious mononucleosis (IM) PTLD in the tonsil (Pt. #19).Among the eight patients with non‐m‐PTLD, another four patients  were primarily diagnosed as non‐destructive PTLD, including 1 plasma cell hyperplasia (PH) PTLD prior to the diagnosis of PBL (Pt. #11), 1 IM PTLD prior to the diagnosis of p‐PTLD (Pt. #19), 1 without further sampling  and 1 case (Pt. #21) which showed a persisting PTLD category at two subsequent biopsies 3 and 7 months later, with all specimens showing the morphology of both FFH and PH. Of note, two patients showed non‐destructive PTLD (FFH) at later time points of the disease course  after having had p‐PTLD as the primary disorder (Pt. #17‐18).c‐MYC translocation was detected in 1 patient each with BL (Pt. #13) and PBL (Pt. #12). Ten of 11 DLBCL samples investigated were negative for a c‐MYC translocation (Table c‐MYC rearrangement (Pt. #4).All samples analyzed in the present study except for two cases of DLBCL (Pt. #5‐6) and one patient with PBL Pt. #12) were EBV‐positive . Thus, overall, we examined 29 specimens: DLBCL, n = 11; BL, n = 1; PBL, n = 1; MALT lymphoma, n = 1; p‐PTLD, n = 7; non‐destructive early lesions, n = 8.The results of PD‐L1 immunohistochemical analyses are provided in Table A PD‐L1‐positive microenvironment was detected in 4 of 11 DLBCL samples , 6 of 7 p‐PTLD samples Figures E,F,H,I aIn summary, PD‐L1 expression regardless of cell type was observed in 9 of 11 DLBCL samples Figures B and 4, PD‐L1 expression regardless of cell type was found in 6 of 7 p‐PTLD specimens with a percentage of positive cells ranging from 20%‐30% , non‐destructive lesions did not reveal PD‐L1 positivity Figure . In 6 of3.3In the 29 samples of PTLD analyzed, PD1 expression was only detected on lymphocytes but not on tumor cells Table . The den3.4In individual patients, the density of PD‐L1 expression changed in the course of disease and according to the PTLD category  vs PD1 expression in 1 of 11 (9%) cases] and in p‐PTLD [6 of 7 (86%) vs PD1 expression in 2 of 7 (29%) cases], and the lowest expression in non‐destructive lesions [1 of 8 (12.5%) vs PD1 expression in 6 of 8 (75%) cases]. We also clearly demonstrated for 6 patients with matched tumor samples available not only a maintained inverse correlation of PD‐L1 and PD1 expression, but also very well‐correlating dynamics of PD‐L1 and PD1 expression with progression, persistence, and regression of the WHO PTLD category.Depending on the cut‐off levels used for positivity, previous studies on adult DLBCL indicated a wide range of 26%‐75% of patients to be PD‐L1‐positive.PD1‐positive lymphocytes were, with one exception, only sparsely detected in our 11 cases of DLBCL. Kiyasu et alAs for p‐PTLD, PD‐L1 positivity in 6 of 7 cases within a range of 20%‐30% was below the level detected in our DLBCL samples (20%‐80%). Intermingled Hodgkin‐ and Reed‐Sternberg‐like cells, which were present in two p‐PTLD samples, displayed PD‐L1 positivity in one of them. A strong PD‐L1 expression in the majority of the malignant cells is characteristic and usually observed in 70%‐87% of cHL samples.To the best of our knowledge, there are no studies on pediatric patients and only a single report dealing with PD‐L1 und PD1 expression in p‐PTLD. Kinch et al investigated the expression patterns of PD1, PD‐L1, and PD‐L2 in 81 PTLD patients after SOT and found positivity for PD‐L1 in two and PD1 positivity in three of five p‐PTLD samples, respectively.With only one sample with a PD‐L1‐positive microenvironment, non‐destructive PTLD exhibited the lowest rate of PD‐L1 expression, but the highest PD1 expression. The PD‐L1‐positive microenvironment was observed in 1 case of IM PTLD where a clustering of PD‐L1‐positive macrophages with EBV‐positive cells was evident, pointing out the pathogenetic contribution of EBV Figure G,H. FlorInterestingly, among the 9 PD‐L1‐positive DLBCL cases, 5 had PD‐L1‐positive tumor cells only, another 3 had PD‐L1‐positive macrophages only, and just 1 case showed both types of cells to be PD‐L1‐positive. Among the 6 PD‐L1‐positive p‐PTLD cases, all had PD‐L1‐positive macrophages including 3 which also showed PD‐L1‐positive tumor cells. In the single PD‐L1‐positive case of non‐destructive PTLD only the macrophages were PD‐L1‐positive. This observation shows that although the rate of PD‐L1 expression is similar between m‐PTLD and p‐PTLD, the predominant type of PD‐L1‐positive cell is different with the macrophages almost always being involved in the less aggressive category of PTLD. Intriguingly, recent research has implied a significant role of non‐malignant tumor‐associated PD‐L1‐positive macrophages in, that is, Hodgkin's disease, with respect to prognosis, suggesting that with a higher amount of PD‐L1‐positive macrophages response to chemotherapy becomes poorer.Numerous studies have tried to identify genetic predictors attributable to PD‐L1 expression in tumor cells.We acknowledge that our small series does not allow strong conclusions, but the inter‐ and intra‐individual correlation of PD‐L1 and PD1 expression with the PTLD category is conspicuous. The amount of PD‐L1‐ and PD1‐positive cells changed in the opposite way in sequential biopsies of the same individual correlating well with progression, persistence and regression of the WHO PTLD category. Accordingly, we observed less PD‐L1 expression in non‐destructive lesions and higher expression in p‐ and m‐PTLD. Since PTLD in childhood represents a therapeutic challenge when reduction of immunosuppression and standard immune and chemotherapy fail or are not possible, our results indicate a potential efficacy of checkpoint inhibitors for this group of patients, at least for p‐ and m‐PTLD. Therefore, co‐staining of PD‐L1 and PD‐1 is advisable in this group of patients and should be routinely implemented for immunohistochemical analysis of PTLD of childhood and adolescence. Nevertheless, as the use of checkpoint inhibitors in this very peculiar group of transplanted patients could be associated with the risk of auto‐/allo‐immune reactions, graft‐versus‐host disease, graft rejection, among others, a phase 1 and  2 trial in refractory PTLD or PTLD not tolerating standard therapies should be considered in order to assess safety and efficacy in a controlled setting.The authors declare no competing financial interests.AA, AIS, ES, and ISK designed and planned the study; AIS, ES, CH, and AA wrote the manuscript. AIS, ES, and AF collected and analyzed the data. AIS and ISK were in charge of the histopathological analyses. All the other authors  as well as AA, ES, and CH recruited, treated, and identified the patients. All authors read and approved the final version of the manuscript. Click here for additional data file.


In this paper, we propose a simple and efficient method for designing a broadband terahertz (THz) absorber based on singular graphene patches metasurface and metal-backed dielectric layer. An accurate circuit model of graphene patches is used for obtaining analytical expressions for the input impedance of the proposed absorber. The input impedance is designed to be closely matched to the free space in a wide frequency range. Numerical simulation and analytical circuit model results consistently show that graphene metasurface-based THz absorber with an absorption value above 90% in a relative bandwidth of 100% has been achieved. Terahertz 0.1–10 THz) absorbers have attracted great interest for their extensive applications in sensing –10 THz a, detectiGraphene, a material consisting of one monolayer of carbon atoms, has been widely investigated as a promising platform for designing broadband tunable MAs in THz regime due to its exotic properties, such as optical transparency, high absorption, high electron mobility ,15. In aIn this paper, a simple and efficient method for designing broadband THz MAs based on a periodical monolayered graphene patch array has been proposed. This method is based on the equivalent circuit model, in which an analytical surface admittance is employed to describe the properties of the graphene metasurface. Moreover, Conditions for broadband absorption have been analyzed. A set of closed-form relations for material properties and geometrical parameters of the absorber have been derived. As a consequence, we can successfully design a broadband THz absorber for a given central frequency based on the formulas derived from this approach. To validate the approach, the results have been compared with those obtained by the finite-difference time-domain  simulations, excellent agreements have been achieved.This paper is organised as follows. The equivalent circuit model of the proposed device is derived in x and y directions and embedded into the dielectric material. This layer is used to induce high absorption. The dielectric spacer layer has a refractive index of To realize electromagnetic wave absorbers at THz frequency, we can take a simple structure. A schematic of the proposed absorber is shown in e is the electron charge, ℏ is the reduced Planck’s constant, T is the temperature, As we know, the surface conductivity of graphene is composed of interband and intraband contributions derived using the Kubo formula . In the ude form ,27:(1)σg=10−13 s .x direction) is illustrated in Here, we use an equivalent circuit model to design the broadband graphene-based absorber. Based on the transmission line theory, the whole structure of the absorber can be considered as circuit elements . The equa is the weighted coefficient, D is the period of the patch array, g is the gap between the graphene patches ω0 at 1 TEquation  as μc=0.Equation , we obtaWith the aid of the above analytical equations, a perfect absorber for a given central frequency is designed. It can be seen that the absorption decreases rapidly with the corresponding frequency away from the perfect matching point in To further verify the inpact of real and imaginary part of the input admittance upon the absorption of the absorber, We test the absorption spectra of a perfect absorber with the central frequency at 1.5 THz a. The baThe additional condition leads to a small and slowly varying imaginary part in a wide range of frequencies near the central frequency . With βdSimilarly, the admittance of the graphene metasurface for frequencies around R, is then obtained from Equation (Differentiating Equations  and 16)16) at ω=Equation :(17)R=4τMoreover, in order to fulfill the requirement of a broadband absorption , , , and 1919. Now lEquation ), we setEquation , one obtThe absorption of this structure is shown in Considering the actual manufacturing errors, effect of the variations of Fermi energy and gap of graphene patches on the absorber performance are examined. As shown in R should be large than It should be pointed that the design algorithm has some limitations. Firstly, to achieve high absorption, be valid .A novel approach for graphene metasurface-based broadband THz absorber design has been proposed. This approach significantly simplified the design of broadband THz absorbers. Based on the equivalent circuit model, the imaginary part of the absorber input admittance and its derivative are designed to be zero at a given center frequency. Then, the real part of the device input admittance is tuned to match the free-space admittance around the central frequency. As a result, a relative absorption bandwidth of


P < 0.0001; OS 5.3 vs 4.1 months, P < 0.0001) were moderate.Gastric cancer is the fifth most frequently occurring cancer worldwide. China has the largest gastric cancer population and nearly half of the patients are diagnosed as advanced-stage . The proSeveral biomarkers were found for PD-1 antibodies in AGC such as microsatellite instability-high (MSI-H) and programmed cell death-ligand 1 (PD-L1) expression , 5. AccoP = 0.038). This study is the first to identify TMB as a predictive biomarker for ICI use in gastrointestinal (GI) tract cancers.Tumor mutational burden (TMB) has been recently described as a new biomarker for PD-(L)1 antibody treatment. However, its predictive effect in AGC had not yet been demonstrated. In a study recently published in Annals of Oncology, Wang et al.  have ideP = 0.027). Only one patient who was PD-L1 negative and was in the TMB-Low group responded to toripalimab. This result suggests that TMB should be further evaluated to identify AGC patients who may respond to PD-1 antibody monotherapy besides PD-L1 testing. For the use in daily clinical practice, a smaller and standardized panel should be developed and needs to be validated in clinical trials.It is also noticeable that TMB-High and PD-L1 positive populations were mostly not overlapping in their study (3.9%). TMB-High and PD-L1 positive patients showed significantly higher ORR (33.3% vs 3.0%) and OS (12.1 vs 4.0 months, HR = 0.47, Further findings in the trial  with torIn summary, TMB may be another good predictive biomarker for PD-1 antibody monotherapy in AGC. A combination of the two biomarkers, TMB-High and PD-L1+, could have the potential to identify a wider range of AGC population who may benefit from ICIs.


N = 92). The last 10 min of each lecture session were used for reviewing the lecture content by either answering short-answer questions, multiple-choice questions, or reading summarizing statements about core lecture content. Three unannounced criterial tests measured the retention of learning content at different times . A positive testing effect emerged for short-answer questions that targeted information that participants could retrieve from memory. This effect was independent of the time of test. The results indicated no testing effect for multiple-choice testing. These results suggest that short-answer testing but not multiple-choice testing may benefit learning in higher education contexts.Review of learned material is crucial for the learning process. One approach that promises to increase the effectiveness of reviewing during learning is to answer questions about the learning content rather than restudying the material (testing effect). This effect is well established in lab experiments. However, existing research in educational contexts has often combined testing with additional didactical measures that hampers the interpretation of testing effects. We aimed to examine the testing effect in its pure form by implementing a minimal intervention design in a university lecture ( Learners tend to remember less learning content when reading or listening to it only once e.g., . StudentThe testing effect is a robust finding in laboratory settings e.g., , which hnet testing effect. The aim of the present study was to examine the net testing effect in the real-world educational context of a university lecture.Despite the promising results and recommendations, the generalizability to educational contexts and the conditions under which the effects occur remain an open question. Based on a review of key findings from lab experiments and a discussion of studies investigating the testing effect in real-world educational settings, we argue that many of the extant field studies suffer from limitations regarding the generalizability of the results. These limitations stem mostly from methodological problems such as a third variable that cofounds the comparison of testing vs. restudying. In this article, we refer to the pure (unconfounded) difference between testing and restudying as the d/Hedges’ g) ranging from 0.50 to 0.61. These meta-analyses also have identified moderators of the testing effect that are potentially relevant for applications in educational contexts.The testing effect has been a major focus of lab-based memory research for more than a century. Summarizing this research, recent meta-analyses by Two factors that reliably affect the testing effect are feedback  and retrConflicting results have been reported for different question formats used in the practice tests. In the meta-analysis by The robust testing effect found in laboratory experiments has spawned a growing body of research in educational contexts. One of the first studies of this kind was a study by d = 0.54 for studies that compared testing and no testing. g = 0.67 for classroom settings. Finally, d = 0.56 for studies in which psychological learning content was taught in the classroom.Since then, the testing effect has been demonstrated in different age groups  in criterial tests at three different times: before and after the final exam and half a year after the final exam. In the criterial tests, we also included questions that were not targeted in the testing conditions but contained related information as well as questions that targeted learning content not subject to testing or restudy, in order to control for differential effects of these question types on multiple-choice and short-answer testing.We expected a positive testing effect to occur. Furthermore, we examined as exploratory research questions whether the testing effect would depend on question format in the practice tests, the time of the criterial test, and retrievability. We reasoned that short-answer questions would be more suitable for prompting active retrieval of knowledge, leading to a stronger testing effect. Moreover, assuming that testing is a desirable learning difficulty, the benefits of testing vs. restudying might become visible, particularly at later criterial tests. Finally, retrievability might matter because the testing effect can only occur when retrieval is successful, especially when no feedback is given for responses in the practice tests.SD = 7.74).Participants were 137 undergraduate students in their first semester, most of them female (71%) and students of psychology (92%). They participated in at least one lecture session and one criterial test. All students gave their informed and written consent prior to participation. Participants’ age ranged between 18 and 74 with a mean age of 23.15 . Short-answer questions were created by asking for the key information of the information unit . Multiple-choice questions were created by adding four response options with only one correct answer to the short-answer question .For each of the seven lecture sessions, eight information units were randomly drawn from the 24 information units prepared for this session. Based on the selected information units, revision materials were prepared for each lecture session. The revision materials consisted of a one-page questionnaire asking for basic demographic information and two pages of revision items corresponding to the selected information units, consisting of either (a) eight summarizing statements (restudy condition), (b) eight short-answer questions , or (c) eight multiple-choice questions . In all three versions, information units were presented in the same order with four information units on each page.Three criterial tests  were constructed that consisted of questions based on the pool of 24 information units determined for each of the seven lecture sessions. The pool of questions was expanded by creating alternate versions of the questions used in the revision material. Alternate questions were created by asking for the key information in another way . For each information unit, an alternate short-answer question and an alternate multiple-choice question were created.Each of the three criterial tests consisted of three components: (a) questions corresponding to information units included in the revision materials, (b) questions corresponding to information units not included in the revision materials, and (c) alternate questions, corresponding to information units but not identical to questions included in the revision materials. Additionally, questions previously asked in criterial tests were also included in Criterial Tests 2 and 3. Table Each criterial test consisted of short-answer questions and multiple-choice questions in equal proportions. Two versions were created (Versions A and B) by altering the order of questions and the question format  of the same question between criterial test versions so that all multiple-choice questions in Version A were short-answer questions in Version B and vice versa. All study materials are made available upon request to interested researchers.Multiple-choice questions were scored with 1 when only the correct option was ticked (correct answer) vs. 0 when a distractor was ticked or no response was given (incorrect or missing response). Short-answer questions were scored with 1 (correct response) vs. 0 (incorrect or missing response). Two independent raters scored all responses to short-answer questions. Inter-rater reliability was high across all lectures and criterial tests  and thus scores from only one rater was included in the analyses. The performance scores based on both question types served as dependent variable.The study was conducted over a period of two semesters. In the first semester (October 2015–February 2016), a weekly introductory psychology lecture was taught that covered basic principles of cognitive psychology. In lecture Sessions 4–10, the manipulation of review condition (testing with multiple-choice or short-answer questions) took place. The three criterial tests, which assessed the learning of content taught in the seven lecture sessions, were administered unannounced to the students at scheduled times after the last lecture with learning content . Criterial Test 1 was administered 1 week after Session 10. Criterial Test 2 was administered in the first session of the second semester (April 2016–July 2016), 12 weeks after Session 10, and Criterial Test 3 was administered in the final session of the second semester, 23 weeks after Session 10.In each of the lecture Sessions 4–10, the last 10 min were reserved for the manipulation of the review condition. Participation was voluntary. Students were allowed to leave the lecture hall after the end of the regular lecture. Research assistants then administered the review materials, assigning participants randomly to one of the three review conditions . Participants first filled in basic demographic information. They were then given 4 min to complete each page of the two pages of revision items. This was the sole opportunity to review the learning content according to one of the three conditions. Finally, participants were thanked for their participation, and the materials were collected.All students present in the respective lecture sessions were allowed to take Criterial Test 1, 2, or 3, irrespective of previous participation in the study. In each of these sessions, the two versions of the criterial test were then administered in an alternating way so that participants sitting next to each other received different versions. Students were allowed 45 min to complete the test and could leave when they finished.The design was a 3 × 3 within-subjects design with the independent variables review condition  and time of test . Each participant received one of two versions of each criterial test, which differed in format (short-answer vs. multiple-choice question) and order of questions. The dependent variable was the performance (percent correct) on the multiple-choice and short-answer questions in the criterial tests.n = 32:33), 2 (n = 40:40), and 3 (n = 25:28). We assume that missing data is missing completely at random and thus inferences can proceed by analyzing the observed data only  with a logit-link function  with theFor comparisons between conditions and extracting mean performance scores for different experimental conditions the R package lsmeans was used . For allSeparate models were estimated to examine the testing effect based on short-answer questions and the testing effect based on multiple-choice questions. In each of the two models, the testing condition was compared to the restudy condition that involved reading the summarizing statements that provided the correct answer . We additionally tested whether the testing effect depended on the time of the criterial test by including two dummy-coded predictors for Criterial Test 2 and Criterial Test 3  and the interactions of these predictors with testing vs. restudying. In addition, the models included the retrievability of learned information in form of two dummy-coded predictors that contrasted items of medium retrievability and low retrievability with items of high retrievability as the reference condition. We examined whether higher retrievability rates were associated with a larger testing effect. To construct this predictor, we grouped the short-answer questions and the multiple-choice questions separately into three equally sized, ordered categories (tertiles) according to their difficulty in the practice tests. To avoid distortions from extreme values, we discarded the lowest and the highest 5% of the distribution before the grouping. Item difficulties to the multiple-choice questions were corrected for guessing. For each of the two item types (short-answer and multiple-choice questions), grouping resulted in three categories of items with high , medium , or low retrievability . Finally, the models included the interaction of retrievability with testing vs. restudying. All predictors and their interactions were entered simultaneously in the models.SE = 0.24, p = 0.033, one-tailed). However, the interaction of testing vs. restudying with the predictor comparing low to high retrievability was significant . Likewise, the interaction with the predictor comparing medium to high retrievability was significant . Planned contrasts revealed a testing effect only for items with high retrievability  but not for items with medium  or low retrievability   compared to Criterial Test 1 .The interactions with time of tests were not significant, suggesting that the testing effect obtained for short-answer questions was independent of the time of test. However, there was a main effect of the predictor comparing Criterial Test 2 to Criterial Test 1. The probability of giving a correct response was higher at Criterial Test 2  compared to Criterial Test 1 .The model estimates for the effects of testing with multiple-choice questions can be found in Table In sum, the results indicated no testing effect for multiple-choice questions.The present study investigated the testing effect in a university education setting by implementing a minimal intervention in an existing university course. In contrast to many previous studies with a similar aim, we took care to avoid confounding factors and based our study on an experimental design. The main finding was a testing effect for practice tests based on short-answer questions, provided that participants in the testing condition were able to retrieve this content. No evidence was found for a testing effect for practice tests based on multiple-choice questions.Our study method shares many features with lab experiments investigating the net testing effect  influenced learning behavior during lectures and review conditions, when taking the criterial tests, or in the days and weeks between the lectures and the criterial tests is unknown. For example, the performance in the criterial tests increased steeply from the first to the second criterial test, which is likely caused by participants’ increased study activities in preparation for the upcoming exam. Participation in the study in each of the lectures was voluntary, which might have caused selection effects. However, it must be noted that these selection effects likely affected all experimental conditions to the same extent, because participants were unaware of the review condition that they would be assigned to when they made their decision to participate.Another limitation that our study shares with other studies on the testing effect is the potential confound of test properties for the practice and criterial tests. For example, multiple-choice questions not eliciting a testing effect might be due to the low demand on retrieval effort involved in answering multiple-choice questions e.g., . Thus, dTo conclude, this research contributes to the literature by demonstrating a testing effect for practice tests with short-answer questions in the real-world educational context of a university lecture. Previous research has examined the testing effect, normally combined with additional features or based on quasi-experimental designs, which has hindered interpretation of the testing effect reported in these studies. In contrast, the present study provides clear evidence for the claim that answering short-answer questions only once and without feedback, compared to restudying key points of the lecture, benefits retention of learning content even beyond the final exam. However, one important condition is that the difficulty of these questions must be at a level such that students are able to answer most of these questions correctly. To use the testing effect to foster learning, educational practitioners should identify the most important topics of their lecture, teach these thoroughly, and use short-answer testing to solidify the knowledge about these topics. Finally, presenting students with multiple-choice questions might be ineffective, compared to restudying key points of the lecture. Given these findings, we advise practitioners to use short-answer testing rather than multiple-choice testing to foster learning in university lectures.After publication, the data files underlying the analyses reported in this study will be made publicly available via Open Science Framework [www.osf.io].For the reported study, no ethics approval was required per the guidelines of the University of Kassel or national guidelines.TR: supervision of the project, design of the research, and revision of the article. SG: design of the research, organization of experiment conduction, data analysis and interpretation, and writing of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Graphene-based materials that can spontaneously response to external stimulations have triggered rapidly increasing research interest for developing smart devices due to their excellent electrical, mechanical and thermal properties. The specific behaviors as bending, curling, and swing are benefit for designing and fabricating the smart actuation system. In this minireview, we overview and summarize some of the recent advancements of stimuli-responsive actuators based on graphene materials. The external stimulus usually is as electrical, electrochemical, humid, photonic, and thermal. The advancement and industrialization of graphene preparation technology would push forward the rapid progress of graphene-based actuators and broaden their application including smart sensors, robots, artificial muscles, intelligent switch, and so on. Peng group reported the mechanically actuating fibers by carbon nanotube displaying a rapid response for using as the smart window and louvers . The value is −7 ppm KIn summary, the recent researches on graphene materials for the devices of actuators have brought up a great deal of government and social attention and support. Particularly, stimuli-sensitive actuators based on graphene materials which spontaneously respond to external stimuli will be given rise to a promising field for developing novel smart devices. The great progress has been made for stimuli-responsive actuation systems that could convert electrical, chemical, photonic, thermal and other types energy/signal to mechanical energy/action. Yet, there are several challenges to overcome like the problem of the graphene production, the unsatisfactory mechanical strength of graphene and the effective of actuation response and so on. Therefore, development of advanced technique and promotion of industrialization are demanded to realize the variety practical applications.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the evaluation of the well-known gold-standard combinations (Agilent 22C3 PharmDx on Dako Autostainer versus Roche's Ventana SP263 on BenchMark), the results confirmed the literature data and showed complete overlapping between the two methods. With regard to the performances by using open platforms, the combination of 22C3 with Dako Omnis or Benchmark obtained good results basically, while the 28,8 clone seemed to be associated with worse scores.An important harmonization effort was produced by the scientific community to standardize both the preanalytical and interpretative phases of programmed death-ligand 1 (PD-L1) immunohistochemical (IHC) testing in non-small-cell lung cancer (NSCLC). This analysis is crucial for the selection of patients with advanced-stage tumors eligible for treatment with pembrolizumab and potentially with other anti-PD-1/PD-L1 checkpoint inhibitors. This multicentric retrospective study evaluated the reproducibility of PD-L1 testing in the Italian scenario both for An important harmonization effort was produced by the scientific community to standardize both the preanalytical and interpretative phases of programmed death-ligand 1 (PD-L1) immunohistochemical (IHC) testing in non-small-cell lung cancer (NSCLC) . This an closed platforms to obtain the gold-standard scores . PD-L1 staining was evaluated by two lung-committed pathologists  in blind and then jointly for the final agreement. Secondly, further TMA blanks were stained using 7 alternative protocols for PD-L1 scoring on open platforms  histological samples from 18 lung surgical specimens with a NCSLC were retrospectively selected for this study. The series included adenocarcinomas and squamous cell carcinoma. The inclusion criteria were the following: adult patients (>18 years old) who underwent total or partial pneumonectomy in the period between 1 December 2016 and 31 January 2018 for NSCLC; no previous neoadjuvant chemoradiotherapy was administered. The original samples were recovered from the archive of the Pathology Department of University Milan Bicocca-ASST Monza, San Gerardo Hospital, Monza. The study was approved by the Ethical Committee of ASST Monza, under the approval #N.1311, dated 17/07/2018. To maximize the homogeneity in preanalytical variables, cases were selected from a unique institution with available trackable processing phases. For this study, fixation time was set at 24 hours following the surgical procedure, as previously described [latforms . The sli closed standard platforms was evaluated (Roche Ventana SP263 on Benchmark and Dako PharmDx 22C3 on Autostainer). In  gold-standard scores included 6 negative cases and 7 intermediate, and 5 strong expressors. While the conclusive results were equivalent, a certain degree of diversity was noticed in terms of intensity. SP263 produced stronger IHC reactivity, just easily perceptible at 4x; to assess correctly 22C3 staining, a greater magnification than 4x had to be used, in contrast to what happened with SP263 staining, where a low magnification was sufficient : exemplificative situations are listed in Figures Pathologist interpretative errors (P): typical examples are shown in Figures  gold-standard) and underestimated signal by the pathologists produced the error.Mixed errors (M): in these cases, a combination of low intensity due to technical reasons  were affected by the three error sources, globally; error rate (ER) of the single centers ranged from 25% (5/ 18) to 5% (1/18). Sensitivity ranged from 69% to 92% and specificity from 33% to 100%; the best performance was obtained by protocol n.2 using clone 22C3 on Dako Omnis  and protocols n.3,7  using clone 22C3 on Dako Omnis and Ventana Benchmark, respectively .Five out of 7 protocols assigned correctly all the negative cases . For 2 pIn the intermediate group a certain disagreement persisted; however technical (or mixed) errors seemed to be more relevant than in negative cases. Two out 10 errors were scored as strong expressors instead of intermediate; in the majority of the situations the score was underestimated .The positive group had a good diagnostic agreement for technical and interpretative variables . Case n. closed and open platforms. In the evaluation of the well-known gold-standard combinations (Agilent 22C3 PharmDx on Dako Autostainer versus Roche's Ventana SP263 on BenchMark), the results confirmed the literature data and showed complete overlapping between the two methods. As regards the intensity levels of the staining, the use of the Ventana platform produced more intense reactions in the face of a morphological more difficult distinction between the tumor cells and the immune cells  while the Dako platform provided fairly soft reactions but with morphological differentiation of the most obvious cell types. Secondly, the comparison between the gold-standard and the PD-L1 IHC staining obtained by using 7 alternative locally validated protocols on open platforms reflected some possible sources of errors in the routine practice. The study identified mainly two exemplificative situations: interpretative errors  produced equivocal results, due to a particularly challenging TMA core that included isolated (PD-L1 positive) tumor cells. For the technical errors, a significant proportion of them may be related to the limitations of a TMA-based study; the heterogeneity in the serial levels of the histological sections may in fact exclude focal PD-L1 positive foci from the analysis. Moreover, due to TMA intrinsic characteristics, some cores cannot be adequately examined . With re caveat. On the other hand, after several efforts in order to harmonize the read-out lecture of PD-L1 status among different antibody clones, assays, and platforms, pathologists have now focused experiences and adequate training to give more detailed and reproducible PD-L1 results to clinicians [This study was designed to stress the methodological challenges of the PD-L1 IHC testing and collected particularly difficult cases by a preliminary histological selection of NSCLC samples that did not reflect necessary a normal case-mix. By these limitations, we can conclude that oncologists should remember that the bioselection of NSCLC patients by the PD-L1 staining has still some technical and interpretativeinicians , 21.