We discovered an inherent trade-off in this mechanism between transcriptional redundancy, which maintains the robustness of TDP-43 metabolism, and vulnerability to specific interfering factors. These factors include an increased tendency of TDP-43 to aggregate, impaired nuclear-cytoplasmic TDP-43 transport, and a decreased efficiency of degrading abnormal proteins, all of which are functional abnormalities related to the gene that causes familial ALS/FTD. When these conditions continue at a certain intensity, the vulnerability of the autoregulatory machinery becomes apparent over time, and transcriptional redundancy enters a vicious cycle that ultimately results in TDP-43 pathology. The results obtained using this in silico model reveal the difference in TDP-43 metabolism between normal and disease states. Furthermore, using this model, we simulated the effect of a decrease in TDP-43 transcription and found that this decrease improved TDP-43 pathology and suppressed the abnormal propagation of TDP-43. Therefore, we propose a potential therapeutic strategy to suppress transcriptional redundancy, which is the driving force of the pathological condition caused by the specific factors described above, in patients with ALS presenting with TDP-43 pathology. An ALS animal model exhibiting TDP-43 pathology without overexpression of exogenous TDP-43 should be developed to investigate the effect of alleviating the transcriptional redundancy of TARDBP.Abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm and its disappearance from the nucleus are pathological features of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) and are directly involved in the pathogenesis of these conditions. TDP-43 is an essential nuclear protein that readily aggregates in a concentration-dependent manner. Therefore, cells must strictly maintain an appropriate amount of nuclear TDP-43. In one relevant maintenance mechanism, TDP-43 binds to its pre-mRNA and promotes alternative splicing, resulting in mRNA degradation via nonsense-mediated mRNA decay. The level of nuclear TDP-43 is tightly regulated by these mechanisms, which control the amount of mRNA that may be translated. Based on the results of previous experiments, we developed an  TARDBP gene, which encodes TDP-43, is present in 1–5% of patients with familial ALS, and these patients exhibit TDP-43 pathology similar to individuals with sporadic ALS. The same TDP-43 pathology has also been identified in patients with mutations in many ALS-causative genes, including hexanucleotide repeat expansions in C9ORF72, a major genetic cause of ALS/FTD  is a devastating neurological disease characterized by the degeneration of upper and lower motor neurons. ALS leads to death within 2–5 years as a result of muscle weakness, including respiratory dysfunction. Up to 50% of patients with ALS develop cognitive and behavioral abnormalities, and approximately 13% of patients with ALS present with concomitant behaviorally variant frontotemporal dementia (FTD) , a structured diagram editor used to draw gene-regulatory and biochemical networks  cells expressing human TDP-43 cDNA under the control of the endogenous mouse TARDBP promoter, the amount of TDP-43 protein increases approximately 3-fold in the absence of the TARDBP 3′-UTR, which is essential for autoregulation . The g Figure . A decreTARDBP pre-mRNA that undergoes alternative splicing in vivo. The percentage of alternatively spliced isoforms among all isoforms of the TARDBP mRNA was examined using the Droplet Digital PCR method  using NucleoSpin RNA II  to confirm the percentage of the d Figure . Since Dy Figure . For mosy Figure . The perA Figure ; Y axis 8 Figure ; R axis.8 Figure .TARDBP RNA is degraded by nonsense-mediated mRNA decay. This transcriptional redundancy may be influenced by several conditions, including conditions within the tissue or cell  model. Therefore, in a state in which no disturbance was applied, the levels of the TARDBP mRNA, nuclear TDP-43, cytoplasmic TDP-43, aggregates, and fragments were equal between these two models. We recorded the amount of each element that stabilized when each parameter was varied from half to twice its initial level.We subsequently examined the fragility of this autoregulatory mechanism by investigating how each element behaves when each parameter was varied. For this analysis, we compared a model with autoregulation  to a model without autoregulation [NAR (–)]. In the NAR (–) model, only the level of transcription was adjusted, which indicates that the initial expression level of the TARDBP heterozygous knockout mouse model  model, the level of nuclear TDP-43 decreased as its transcription level decreased, reaching approximately half of the initial value under conditions that mimic the l Figure . When thl Figure . The levl Figure . Howeverl Figure . Similarl Figure , 3A. ThuTARDBP mRNA  model, changes in the degree of nuclear import altered the nuclear TDP-43 level; however, these changes did not increase the fragmentation of TDP-43. In the NAR (+) model, the amount of nuclear TDP-43 remained robust until nuclear import was reduced to approximately three-quarters of the initial setting Figure . When nuA Figure . In the A Figure . As the A Figure . SimilarA Figure , 3B.TARDBP mRNA and subsequently exacerbate TDP-43-related pathology  are related to disturbances in the factors that lead to the vicious cycle of the autoregulatory system. Therefore, examinations of the TDP-43 metabolism and specifically the expression of TARDBP mRNA in model animals with mutations in these causative genes are necessary.At this stage, we propose that TDP-43 pathology involves a vicious cycle in which excessive TDP-43 is continuously produced as a result of an autoregulatory mechanism. As shown in a previous study, motor neurons from subjects with ALS displaying abnormal TDP-43 accumulation exhibit an increase in the amount of TARDBP mRNA was suppressed (Figure TARDBP mRNA levels under the pathological condition of persistent TARDBP mRNA expression driven by transcriptional redundancy. An animal model that maintains the autoregulatory mechanism that controls TDP-43 levels is necessary to provide supporting evidence for this treatment strategy. In this type of animal model, the endogenous TARDBP mRNA, but not exogenous TARDBP, should be overexpressed, as observed in motor neurons from patients with ALS (Koyama et al., TARDBP mRNA in ALS. Specifically, a strategy that employs an antisense oligonucleotide or CRISPR-dCas9 system to decrease the TARDBP mRNA level would conceivably be successful. A decrease in TDP-43 levels induces a loss in TDP-43 function, which may be detrimental to cell survival and function. However, because TARDBP expression is transcriptionally redundant, a 50% reduction in its transcription does not affect protein levels (Kraemer et al., TARDBP heterozygous knockout mice have normal survival times (Ricketts et al., TARDBP mRNA levels might be effective.Using this model, TDP-43 pathology was reversed and abnormal TDP-43 propagation was suppressed when the transcription of the d Figure . Similard Figure . Based oThe pathophysiology of neurodegenerative diseases is particularly difficult to understand due to the complexity, multifactorial, and dynamic aspects of the central nervous system. Approaches using systems biology may be useful for integrating all aspects of a given phenomenon and may constitute promising strategies. However, models of any scale have advantages and limitations (Vujasinovic et al., TARDBP transcription that is intended to maintain nuclear TDP-43 levels becomes the driving force that leads to TDP-43 pathology. This hypothesis answers several questions: why do ALS motor neurons enter a pathological state from which they cannot escape; why do many different ALS causative genes result in TDP-43 pathology; and why does TDP-43 pathology propagate between cells, thereby resulting in progression? Furthermore, based on our model, therapies for ALS targeting specific molecules are a possibility. In the future, an ideal animal model of this disease should be established to explore this hypothesis.Why does TDP-43 accumulate abnormally in the cytoplasm in patients with ALS/FTD when a mechanism that precisely maintains its expression exists? Studies aiming to resolve this issue are important to enhance our understanding of the pathology of sporadic ALS, which accounts for most ALS cases, and to promote the development of methods to treat this disease. Therefore, in the present study, we modeled and simulated TDP-43 dynamics within the cell. The trade-off relationship between the vulnerability and robustness of the mechanism that maintains nuclear TDP-43 levels might underlie TDP-43 pathology. Once this vulnerability is triggered, the redundancy of AS and OO designed the study. AS, TKA, and AK performed the biological experiments using the mouse cerebrum and investigated the results of previous experiments to create our model. AS analyzed the results. YK, SK, TKO, and TI contributed to the interpretation of the findings. AS and OO drafted the manuscript. All authors critically revised the draft and approved the final version.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.


Idiopathic pulmonary fibrosis (IPF) is characterized by the complex interaction of cells involved in chronic inflammation and fibrosis. Global gene expression of a homogenous cell population will identify novel candidate genes.p-values < 0.05. CCL8 mRNA and protein levels were quantified using real-time PCR and ELISA. CCL8 localization was evaluated by immunofluorescence staining.Gene expression of fibroblasts derived from lung tissues (8 IPF and 4 controls) was profiled, and ontology and functional pathway were analyzed in the genes exhibiting >2 absolute fold changes with CCL8 expression was 22.8-fold higher in IPF fibroblasts. The levels of CCL8 mRNA and protein were 3 and 9-fold higher in 14 IPF fibroblasts than those in 10 control fibroblasts by real-time PCR and ELISA . The CCL8 concentrations in BAL fluid was significantly higher in 86 patients with IPF than those in 41 controls, and other interstitial lung diseases including non-specific interstitial pneumonia (n = 22), hypersensitivity pneumonitis (n = 20) and sarcoidosis (n = 19) . Cut-off values of 2.29 pg/mL and 0.43 pg/mL possessed 80.2 and 70.7% accuracy for the discrimination of IPF from NC and the other lung diseases, respectively. IPF subjects with CCL8 levels >28.61 pg/mL showed shorter survival compared to those with lower levels (p = 0.012). CCL8 was expressed by α-SMA-positive cells in the interstitium of IPF.One hundred seventy eight genes differentially expressed and 15 genes exhibited >10-fold change. Among them, 13 were novel in relation with IPF. CCL8 is a candidate molecule for the differential diagnosis and prediction of survival.Transcriptome analysis identified several novel IPF-related genes. Among them, The online version of this article (doi:10.1186/s12931-016-0493-6) contains supplementary material, which is available to authorized users. Idiopathic pulmonary fibrosis (IPF) is characterized by alveolar epithelial cell hyperplasia and increased myofibroblast with the interstitial deposition of extracellular matrix (ECM) . The disThe use of whole-lung tissues, however, may be a limitation of transcriptomic studies because transcriptomic changes are cell-type-specific . The patPlasma, BAL fluids, and lung tissues of the subjects with diffuse interstitial lung disease were obtained from a biobank in Soonchunhyang University Bucheon Hospital  after the study protocol was approved by institutional review board (IRB) in Korea National Institute for Bioethics Policy . Control BAL fluids were obtained from general population and hospital personnel, and blood samples were obtained from spouses, general population and hospital personnel after approval by the hospital ethics committee . An informed written consent to participate was obtained from each subject. The diagnostic criteria for IPF, NSIP, HP and sarcoidosis were based on the international consensus statement –14. All HP was diagnosed by the presence of compatible clinical manifestations with a non-necrotizing granulomatous interstitial bronchiolocentric pneumonitis . The dia2 cell culture flasks with tissue culture media (TCM) consisting of DMEM , 10% fetal bovine serum , 2mmol/L glutamine, and 1% penicillin-streptomycin-amphotericin  immunohistochemical stain  until the fourth passage. The cells were then stored at −170 °C. Fifth-passage fibroblasts (2.5 × 106) were seeded in 1mL TCM in 10cm2 dishes. After reaching 90% confluence, the fibroblasts were washed twice with PBS (Thermo Fisher Scientific Inc.) and used for RNA. Total RNA was extracted using TRI reagent . The cell pellets were prepared in RIPA buffer for immunoblot analysis, and protein concentrations were measured using a BCA kit (Thermo Fisher Scientific Inc.).Lung fibroblasts were cultured from the surgical specimens of 14 patients with IPF and normal lungs of 10 subjects who underwent surgery to remove stage I or II lung cancer as described in the previous publication . Brieflyp-values <0.01 were discarded to reduce the number of false positives, and the remaining 15,020 genes were analyzed. Fold change of gene expression was calculated as follows: a mean of expression levels of IPF-fibroblasts divided by that of control fibroblasts if the levels were higher in the IPF than the controls. In cases of countertrend, fold change was determined by dividing the mean value of control group by that of the IPF group and presented as minus value. The microarray data were analyzed using ScoreGenes software package (http://compbio.cs.huji.ac.il/scoregenes/). The general approach to analysis has been previously described , those in the NSIP , those in the HP  and those in the sarcoidosis patients   , while no correlations with macrophage, lymphocytes and eosinophil numbers in BAL fluid and physiological parameters  in the GDS1252 dataset, as well as in 6 sarcoidosis lung compared with 6 normal lungs (p = 0.016) in the GDS3580 dataset of NCBI GEO DataSet Browser (https://www.ncbi.nlm.nih.gov/geo/). CCL8 was included in the ontology categories of extracellular region, receptor binding, heparin binding, G-protein-coupled receptor binding, chemokine activity, carbohydrate derivative binding, and glycosaminoglycan binding, which are essential pathways in the pathogenesis of IPF. In validation, CCL8 levels in BAL fluids appeared useful as a candidate marker for the differential diagnosis from NC and other chronic interstitial lung diseases. Thus, we identified several novel genes, and demonstrated the clinical relevance of CCL8 as a candidate marker for the diagnosis and prognosis of IPF patients for the first time to the best of our knowledge.In our study, 178 genes were found to be differentially expressed by the fibroblasts derived from IPF lungs. Among the top 15 genes of them, only CCL8 is secreted into the peripheral circulation. However, no correlation between CCL8 protein levels in BAL fluids and peripheral bloods was observed. This may be due to sources of CCL8 other than fibroblasts or individual variance in the diffusion of CCL8 from IPF-fibroblasts into the bloodstream. Among the CC chemokine ligand family, CCL26 expression was increased in IPF patients compared with controls  in our study. In other studies of IPF-patients, CCL2 are present in metaplastic epithelial cells and vascular endothelial cells [CCL3, CCL4, and CCL7 expressions are elevated in BAL fluids [CCL2, CCL3, CCL4, and CCL7 in the lung.al cells  and CCL3L fluids , 30. TheCCL8 activates various immune cells, including mast cells, eosinophils, basophils, monocytes, T cells, and NK cells [CCL8 have been discovered in infection, immunity, and allergic inflammation. CCL8 recruits gamma/delta T cells, which preferentially express IL-17F and synergistically enhance neutrophil chemotaxis in the presence of IL-8 [CCL8 is induced by fibroblasts and endothelial cells when co-stimulated with IL1-β and interferon (IFN)-γ. IFN-γ has also a synergistic effect with activation of TLR2, TLR3 or TLR4. The application of both IFN-γ and dsRNA via TLRs resulted in the synergistic induction of CCL8 expression [CCL8 appears to be related with innate immune response in the development of IPF, but the exact role of CCL8 remains to be solved in near future.NK cells . Recentl of IL-8 . CCL8 ispression . All of CCL8 was positively correlated with those of IL-8, IL-13RA2, and CCL2, ADAMTS1, ADAMTS8, MMP10, MMP2, MMP3, TIMP2, ECM1, TGFBI, and CLEC3B, and inversely correlated with FN1   in our study were included in the fibroblast growth factor production and EMT categories. Among them, PTGS2 is known to be involved in the development of IPF [In the ontology analysis of the 178 genes, regulation of fibroblast growth factor production, extracellular region, positive regulation of the EMT, and positive regulation of cell morphogenesis were the most relevant groups. The fibroblasts used in our study expresses markers of smooth muscle differentiation, such as α-SMA . Lindahlem, FGF7 , POSTN [7 [POSTN , TSKU [3TN [TSKU , and TFPKU [TFPI  have beet of IPF . These rt-test and nonparametric TNoM scoring method to compare the differences in gene expression between NC and IPF groups because of no gene passed of less than 5% correction for multiple testing [P-values may be less problematic than omitting informative genes in studies aimed at identifying target genes responsible for biological mechanisms [n = 744, 10 controls and 3 IPF subjects) [n = 1813, 4 controls and 10 IPF subjects; GSE44723), those with Ronzani C’s study using  and those with Emblom-Callahan’s study using uncultured fibroblast  [Our study had the following limitation: the small number of lung tissue samples available for microarray analysis. Therefor we used the  testing . The usechanisms . In addiubjects) , those wTIMPs, β-FGF, and PDGF expression  [ALDH3A2, CDC42EP3, IGFBP2, MOXD1, NBEAL2, PITX1, POSTN, TMEM51, and UBE2K, were overlapped. This may be due to biological differences between the cultured fibroblasts used in our study and the uncultured ones from IPF-lungs. However, we validated the CCL8 concentration in BAL fluid in 86 patients with IPF and those in 41 controls and CCL8 protein expression using immunohistochemical stain with antibodies to CCL8 and α-SMA-positive cells in the lung tissues of IPF. Because the BAL fluids and lung tissues were obtained in un-cultured condition, so CCL8 protein may be generated per se, not solely due to a phenotypic shift of the fibroblasts. However, replication of the result is mandatory for useful biomarkers. Another limitation was the use of control fibroblasts from the resected cancer specimens. It cannot be excluded that the gene expression in fibroblasts derived from the lungs in which cancer has developed may be different form fibroblasts derived from the normal lungs.Fibroblasts derived from IPF lungs have distinct biological characteristics: a high percentage of apoptotic cells, and increased collagen, fibronectin, gelatinase B, enotype) , and a renotype) . Interesenotype) . When thEpigenetic changes, such as CpG methylation and miRNA, are widespread throughout the genome in IPF-lung tissues and may regulate the expression of important genes –46. AlthCCL8 was validated as a candidate in BAL fluids and lung tissues samples. A total of 178 genes showed differential expression; among 15 genes showing ≥ 10-fold changes, 13 were newly identified in relation with IPF. The CCL8 protein concentrations in BAL fluids were significantly higher in patients with IPF, and a cut-off value of 2.29pg/mL showed a high degree of accuracy for diagnosis. The levels were also higher compared to those in other interstitial lung diseases including NSIP, HP and sarcoidosis with a cut-off value (0.43 pg/mL) possessing a high degree of accuracy for the discrimination from the other interstitial lung diseases. The subjects with IPF with CCL8 levels >28.61 pg/mL showed a reduced survival rate. In conclusion, our transcriptome analysis identified new genes that may be involved in the pathogenesis of IPF. Among them, CCL8 may be useful as a candidate molecule for the differential diagnosis of IPF and prediction of survival.Global gene expression profiling of fibroblasts from lung tissues from IPF patients was performed to identify novel candidate genes.


PDAC) is considered a non‐immunogenic tumor, and immune checkpoint inhibitor monotherapy lacks efficacy in this disease. Radiotherapy (RT) can stimulate the immune system. Here, we show that treatment of KPC and Pan02 murine PDAC cells with RT and gemcitabine upregulated PD‐L1 expression in a JAK/Stat1‐dependent manner. In vitro, PD‐L1 inhibition did not alter radio‐ and chemosensitivity. In vivo, addition of anti‐PD‐L1 to high  but not low  RT doses significantly improved tumor response in KPC and Pan02 allografts. Radiosensitization after PD‐L1 blockade was associated with reduced CD11b+Gr1+ myeloid cell infiltration and enhanced CD45+CD8+ T‐cell infiltration with concomitant upregulation of T‐cell activation markers including CD69, CD44, and FasL, and increased CD8:Treg ratio. Depletion of CD8+ T cells abrogated radiosensitization by anti‐PD‐L1. Blockade of PD‐L1 further augmented the effect of high RT doses (12 Gy) in preventing development of liver metastases. Exploring multiple mathematical models reveals a mechanism able to explain the observed synergy between RT and anti‐PD‐L1 therapy. Our findings provide a rationale for testing the use of immune checkpoint inhibitors with RT in PDAC.Pancreatic ductal adenocarcinoma ( Surgeryet al, et al, et al, et al, et al, et al, et al, et al, + T‐cell infiltration  receptor and its ligand PD‐L1  is an important mediator of PD‐L1 signaling , anti‐PD‐L1 alone, or their combination . Additionally, we assessed the activation status of the CD45+CD8+ T cells based on IFNγ expression and found increased activated CD8+ T cells following combination of high RT doses and PD‐L1 blockade  KPC cells, then treated with anti‐PD‐L1 every 3 days. After 14 days, livers were harvested and weighted. Compared to control, prior treatment with 12 Gy reduced metastatic tumor burden that was further enhanced by anti‐PD‐L1 treatment Fig A. Combinrols Fig . Hence, et al, et al, Finally, we used mathematical modeling  RT induces a large population of tumor cells to become susceptible to immune cell killing and (ii) anti‐PD‐L1 increases the recruitment and killing ability of CD8 T cells and, importantly, prevents the establishment of suppressive microenvironment factors .et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, PD‐L1 plays a central role in permitting cancer evasion, mainly by interfering with T‐cell functions, and inhibitors of the PD‐1/PD‐L1 axis have changed the paradigm in the management of melanoma patients  that made tumor rechallenge impossible as the experiment had to be terminated.Although our observations with higher RT doses are in agreement with the majority of reports showing strong radiosensitization with PD‐1/PD‐L1  was sufficient to delay tumor growth in colorectal and triple‐negative breast cancers  or inflammatory processes via IFNγ (adaptive resistance)  are known to suppress antigen‐specific T cells  PDAC cells and assessed the impact of systemic PD‐L1 blockade (i.p.) in the development of liver metastasis. Both RT alone and anti‐PD‐L1 alone reduced metastatic colonization compared to untreated controls, but addition of anti‐PD‐L1 significantly enhanced the anti‐metastatic effect of RT. Recruitment of immune cells to the liver is known to precede development of liver metastases  tumors. Murine Pan02 pancreatic adenocarcinoma cells were obtained from the NCI–DCTD Tumor Repository, Maryland, USA. Gemcitabine was purchased from Eli Lilly and Company Ltd, UK. Murine anti‐PD‐L1 antibody (Clone 10F.9G2) was purchased from BioXCell.The murine PDAC cell lines KPC and Pan02 were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. KPC cells were derived from KrasLSL. Anti‐PD‐L1 antibody was added to the culture 1 h before irradiation with single dose of 2, 4, and 6 Gy. Similarly, cells were also treated with gemcitabine . Gemcitabine was diluted in DMSO (vehicle), and hence, we used DMSO for the untreated control group. Twenty‐four hours after treatment, cells were washed with PBS and replaced with fresh medium. The clonogenic survival of cells was assessed as described previously . Tumor size was measured with caliper using the formula Volume = (a × b2)/2, in which a and b are the largest and the smallest perpendicular diameters, respectively. Mice were randomized into different groups when tumors reached 100 mm3 and treatment was initiated (day 0) as described previously , 3, 6, and 9. We assessed the time it took for the tumors to grow to 400 mm3, starting from the beginning of treatment (day 0) to ensure that our experiments were according to the UK Home Office guidelines for animal welfare.C57BL/6 mice were injected subcutaneously (s.c.) with KPC and Pan02 cells (5 × 105 cells/100 μl in PBS) were injected intrasplenically into C57BL/6 mice that are either naïve or pre‐irradiated with 12 Gy 1 h before intrasplenic injection, as previously described  or long term  after completion of treatment with gemcitabine. Tumors were harvested and analyzed for PD‐L1 expression by flow cytometry as described below.The KPC transgenic mouse model has been described previously . All protocols were approved by the Committee on the Ethics of Animal Experiments of the University of Oxford.KPC and Pan02 cells were seeded in 6‐well plate. After 24 h, cells were treated with either 6, 4 × 2 Gy fractionated radiation given daily using cesium‐137 irradiators, or 20 nM gemcitabine. Gemcitabine was diluted in DMSO (vehicle), and hence, we used DMSO for the untreated control group and the radiation groups as well. Cells were harvested 24 h post‐treatment and washed with cold PBS. Cells were stained with mouse PE‐PD‐L1 and APC‐IFNγ. All the antibodies were purchased from eBiosciences.We also examined expression of PD‐L1 following blockade of either Stat1 or Stat3 using siRNA. For that purpose, cells were transfected with 50 nM negative control siRNA , 50 and 100 nM Stat1 , or 50 nM Stat3 siRNA  using Lipofectamine 2000 (Invitrogen) reagent. After 48 h, proteins were isolated and knockdown was confirmed by Western blotting. For flow cytometry analysis of PD‐L1 and IFNγ, siRNA‐transfected cells were either irradiated with 6 Gy or treated with gemcitabine and PD‐L1 expression examined as above. PD‐L1 expression was also assessed following addition of recombinant mouse IL‐6  for 1 h and subsequent treatments with 6 Gy and gemcitabine for 24 h.For flow cytometry analysis of mouse tissue samples, tumor, liver, and spleen were cut into small pieces and only liver samples were digested with 0.05% collagenase/dispase (Roche) for 15 min at 37°C in the presence of 0.01% trypsin inhibitor. Bone marrow was collected by flushing both femur and tibia with PBS using a 25‐G syringe. All cells were washed with cold PBS. Cells were then filtered through 70‐μm nylon strainer followed by red blood cell (RBC) lysis for 3 min at room temperature. Single‐cell suspensions were then stained with the following antibodies: FITC‐CD45, FITC‐CD8, PeCy7‐CD4, PeCy7‐CD11b, PE‐Gr1, PE‐Ly6G and APC‐IFNγ, PE‐CD25, APC‐FOXP3, CD69‐PE, APC‐CD44, and PeCy7‐FasL.g for 20 min at RT to isolate leukocyte population. The cells were washed with cold PBS and stained with FITC‐CD8, PeCy7‐CD4, and PE‐PD‐1. Flow cytometry was performed using a FACSCalibur (Becton Dickinson) and analyzed using FlowJo software . All the antibodies were purchased from eBiosciences, UK.Blood samples were collected in 40 mM EDTA and mixed with equal volume of PBS. Diluted blood was layered onto an equal volume of Histopaque (Sigma) and centrifuged at 400 Cells were first treated with 20 μM of JAK kinase inhibitor  1 h before RT (6 Gy) or addition of 20 nM gemcitabine. Following 24 h post‐RT and gemcitabine, cell lysates were prepared in RIPA buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). In total, 30 μg of proteins were separated on SDS–PAGE (Invitrogen) and transferred to Hybond‐C membranes (Amersham Biosciences). Standard procedures were applied for Western blotting. The following antibodies were used: Stat1, phospho‐Stat1 (Y701), Stat3, mouse PD‐L1 , actin .et al, Serum was obtained from the KPC allograft‐bearing mice 5 days after treatment with either vehicle (control), 12 Gy, anti‐PD‐L1, or 12 Gy + anti‐PD‐L1. Serum was assessed for expression of cytokines using a standard Cytokine Profiler kit and SDF‐1 ELISA kit (both from R&D Systems) according to the manufacturer instructions. Array spot intensities were quantified using a Matlab software as reported : ε1 = 0.02, φ = −0.2, T1 day 0 = 0.4, T2 day 0 = 99.6; radiation + anti‐PD‐L1 (day 6): ε1 day 0 = 0.002, ε1 day 6 = 0.02, φ day 0 = 3, φ day 6 = −0.2, T1 day 0 = 0.4, T2 day 0 = 99.6. The ODEs were integrated in Matlab  using ode45.t‐test and one‐way ANOVA (Bonferroni test) with the GraphPad Prism version 5 . P‐values lower than 0.05 were considered statistically significant. Error bars represent standard deviation.Differences between groups were determined using Student's paired in vivo studies. SL helped with the manuscript preparation. All authors discussed and approved the manuscript.AA, SYL, and EF designed the experimental strategy. AA, SYL, ZD'C, and EF organized the experiments and collected and analyzed the data. AD and SYL provided assistance with the experimental methodology. AA, SYL, and EF prepared the protocols for mouse radiotherapy. OJS, WGM, and RJM provided experimental advice. PK and OD generated the mathematical modeling. AA, SYL, and EF wrote the manuscript with advice from OJS, WGM, OD, and RJM. KJ assisted with the Part of this work was presented at the ESTRO 35 and the NCRI 2015 conference. The authors declare that they have no conflict of interest.Pancreatic ductal adenocarcinoma is characterized by a dismal prognosis and it is refractory to conventional treatment, such as radiotherapy and chemotherapy, and immune checkpoint inhibitor monotherapy. In contrast to the notion that all patients with PDAC die of metastatic disease, at least 20% suffer and die of local recurrence. Hence, there is a need to improve radiotherapy efficacy to enhance local control and clinical outcome. Evidence indicates potentially complementary roles for immunotherapy and radiotherapy, but this combination has not been explored in PDAC.in vitro and in vivo. Inhibition of PD‐L1 strongly enhanced tumor response to high  but not low  RT doses, and to gemcitabine‐based chemoradiotherapy in different mouse models of PDAC. The increased tumor response to radiation observed upon PD‐L1 blockade correlated with increased intratumoral T‐cell numbers and activation and reduced myeloid cell infiltration, whereas depletion of CD8+ T cells abrogated the radiosensitization by anti‐PD‐L1. Additionally, inhibition of PD‐L1 enhanced the impact of radiotherapy in preventing development of liver metastases. We generated a novel mathematical model that explains the synergistic effect of radiation and PD‐L1.Here, we show that radiation and gemcitabine chemotherapy have substantial immunomodulatory effects, including upregulation of PD‐L1 In the present work, we provide a strong rationale for testing the use of PD‐L1 immune checkpoint inhibition with radiotherapy in PDAC in the clinical setting that could lead to improved outcome in patients with this disease.https://www.ncbi.nlm.nih.gov/pubmed/26433823https://www.ncbi.nlm.nih.gov/pubmed/26270858AppendixClick here for additional data file.Source Data for AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 4Click here for additional data file.


In the last four decades, tumor immunology has shed light on identity and functions of cells and molecules involved in tumor rejection through the involvement of the immune system . SeveralIn response to the aim of the special issue, four original research papers and one review article are presented below.The study reported by B. Cerbelli et al. from Sapienza University of Rome, Italy, showed that immunohistochemical PD-L1 expression in ≥25% of triple negative breast cancer (TNBC) chemo-naïve cells, derived from core biopsies, is an independent predictor for pathological complete response (pCR) after neoadjuvant chemotherapy, thus discussing potentials and limits of PD-L1 future applications as a predictive biomarker for neoadjuvant treatment response in this subset of patients affected by such a clinically aggressive disease.M. Moschetta et al., at Sarah Cannon Research Institute of London, UK, presented a study assessing the impact of “neutrophil-to-lymphocyte ratio” (NLR) in predicting PFS among 55 advanced patients enrolled into PD-1/PD-L1 inhibitors phase 1 clinical trials. Results showed a significant longer PFS in patients with a reduction of NLR after two treatment cycles compared to the median baseline NLR, thus advancing the hypothesis that NLR may be a helpful predicting tool in cancer patients treated with anti-PD-1/PD-L1 agents.An international collaboration between USA  and Japan , coordinated by K. Suda et al., obtained evidence concerning molecular mechanism behind the low expression of PD-1/PD-L1 in NSCLC, associated with reduced efficacy of checkpoint inhibitors (CI) treatments. The study highlighted that EGFR-mutated lung cancer cell lines do not show high PD-L1 expression and, furthermore, after acquisition of resistance to EGFR-TKIs, EGFR phosphorylation affects PD-L1 expression, thus identifying a molecular event able to influence the expression of biomarkers, which regulate patients' access to CI agents.Apart from its immunomodulatory function, F. Zheng et al., from Huazhong University of Science and Technology, China, identify PD-L1 molecule as a potential biomarker of melanoma cancer stem-like cells, since blocking PD-L1 in melanoma cell lines expressing PD-L1 and ALDH1 impaired tumorsphere formation and induced the apoptosis of tumorsphere cells. These findings raise the need to elucidate the relationship between tumor response to checkpoint inhibitors and clonal evolution of cancer stem cells in the future.Finally, the review paper by M. Wei et al., Southeast University of China, discusses the role of gastric cancer patients' T cells immunity and disease prognosis, providing a critical synthesis of recent evidence on this still controversial topic.In conclusion, we find this special issue to be a good opportunity for improving knowledge in the field of cancer immunobiology and immunotherapy, which is a pivotal step to respond adequately to the questions of our time in the battle against cancer.Ilary RuscitoElena Ioana BraicuMaria Luisa GasparriIlaria Grazia Zizzari


Sarcoidosis is a multisystem disease characterised by the formation of granulomas within various organs, mainly the lungs. Several studies from different countries have been undertaken to investigate sarcoidosis with extrapulmonary involvement except from China. The objective of this study is to investigate a comparative clinical analysis in patients with pulmonary sarcoidosis with and without extrapulmonary involvement from China.Data from inpatients diagnosed with sarcoidosis at Shanghai Pulmonary Hospital  between January 2009 and December 2014 were retrospectively collected and analysed. Six hundred and thirty-six patients with biopsy-proven sarcoidosis were included in the study, including 378 isolated pulmonary sarcoidosis and 258 pulmonary sarcoidosis plus extrapulmonary involvement.Two hundred and fifty-eight (40.6%) patients with pulmonary sarcoidosis had extrapulmonary involvement. Extrapulmonary localisations were detected mostly in extrathoracic lymph nodes (n=147) and skin (n=86). Statistically significant differences were demonstrated between patients with pulmonary sarcoidosis plus extrapulmonary involvement and patients with isolated pulmonary sarcoidosis for fatigue , serum ACE (SACE) levels , and high-resolution CT (HRCT) findings .Extrapulmonary involvement is common in patients with pulmonary sarcoidosis, with the most common sites being extrathoracic lymph nodes and skin. Patients with sarcoidosis with extrapulmonary involvement are more symptomatic (fatigue), have higher SACE levels and more deteriorating HRCT findings, to which clinicians should pay attention. The present study provides a large number of patients with pulmonary sarcoidosis with and without extrapulmonary involvement from China.The patients with sarcoidosis had a long-term follow-up (24–42 months) on chest high-resolution CT.There is a risk of selection bias due to the retrospective design.Sarcoidosis is a granulomatous disease of unknown aetiology. It is characterised by the formation of immune granulomas in many involved organs.Sarcoidosis is a multisystem disease. Although the lungs are most commonly involved, pulmonary sarcoidosis is often accompanied by the involvement of extrapulmonary organs,8It is well known that the features of sarcoidosis have varied widely among various populations around the world. To date, several studies from different countries have been undertaken to investigate sarcoidosis with extrapulmonary involvement. However, there is limited information in Chinese patients with sarcoidosis. Therefore, the purpose of this study is to compare the clinical characteristics and prognosis of patients with pulmonary sarcoidosis with and without extrapulmonary involvement from China.Data were retrospectively collected from inpatients diagnosed with sarcoidosis at the Shanghai Pulmonary Hospital  between January 2009 and December 2014. Patients who had not received a chest radiograph or chest high-resolution CT (HRCT) scan examination or who had not received tissue biopsy were excluded. All aspects of the study were performed in accordance with relevant guidelines and regulations.Diagnosis of sarcoidosis is based on standardised criteria,3Organ involvement was determined and classified in each patient in accordance with the criteria proposed in the ACCESS formulation,The following variables were obtained from the medical records: general and anthropometric information, symptoms, chest radiographs or CT films, serological indicators and pulmonary function parameters. The chest radiographs or CT films were read at each site by two investigators; the findings were evaluated and staged according to the modified Scadding criteria.2 test. A logistic regression model was used to determine the factors that were independently associated with deteriorating chest HRCT findings. The variables that presented statistically significant differences (P<0.05) in the bivariate analysis and were of clinical interest were included as the independent variables in the model. The OR and 95% CI were calculated for the independent variables. Statistical analysis was performed using SPSS V.22.0 package software with a value of P<0.05 considered statistically significant, and graphs were drawn using GraphPad Prism V.5 software.Quantitative variables were expressed as the mean±SD, and qualitative variables were expressed as absolute numbers and percentages. The Kolmogorov-Smirnov test was used to analyse the distribution of variables. In the bivariate analysis, the t-test for independent variables was used to analyse normally distributed variables, and the Mann-Whitney U-test was used to analyse non-normally distributed variables. Qualitative variables were compared using the χA total of 636 patients with biopsy-proven sarcoidosis (378 isolated pulmonary sarcoidosis and 258 pulmonary sarcoidosis with extrapulmonary involvement) were included in the study. Among these 636 patients, 298 responded fully to the follow-up study on HRCT (182 isolated pulmonary sarcoidosis and 116 pulmonary sarcoidosis with extrapulmonary involvement), the median follow-up time after discharge was 30 months (ranging from 24 to 42 months). Thirty-nine patients had deteriorating chest HRCT findings after their discharge from the hospital.The organ involvement and baseline characteristics of the subjects are shown in The differential characteristics of patients with or without deteriorating chest HRCT findings were shown in The OR values and 95% CIs of variables related to deteriorating HRCT findings in all patients were illustrated in The current study aimed to retrospectively compare the clinical characteristics of patients with pulmonary sarcoidosis with and without extrapulmonary involvement, as well as their prognosis on HRCT. So far, several studies conducted by different countries have been undertaken to investigate extrapulmonary sarcoidosis except for China. Since clinical features of sarcoidosis have varied among various populations around the world, our study would provide valuable information and, therefore, may help facilitate the prognosis and management for Chinese patients with sarcoidosis.The prevalence of extrapulmonary sarcoidosis involvement varies among populations. Based on our study, we found that 40.6% patients with pulmonary sarcoidosis had extrapulmonary involvement, which was consistent with previous study in which 30%–50% of patients showed extrapulmonary disease localisations.The most common symptom of mediastinal–pulmonary manifestations of sarcoidosis is persistent cough. Dyspnoea is rare in the early stages, but more frequent later on.Of note, in our study we found patients with pulmonary sarcoidosis with extrapulmonary involvement had higher level of SACE than patients with isolated pulmonary sarcoidosis . ACE is a carboxypeptidase enzyme that converts angiotensin I into angiotensin IIet al found that change in radiographic extent was more applicable to routine monitoring in sarcoidosis than change in radiographic stage.Using chest radiograph or CT to view the internal structure is the most common imaging biomarker for evaluating sarcoidosis. The sarcoidosis granulomas can be detected with plain radiographic or CT radiographic techniques.There are several limitations in this study. First, there is a risk of selection bias due to the retrospective design. Therefore, we conducted a cross-sectional and observational study which might reduce the risk of selection bias. Second, we conducted this study more from a pulmonary perspective. Since sarcoidosis is a multisystem disease, a more comprehensive study could be performed to investigate other parameters related to sarcoidosis. Third, to date, no reliable prognostic biomarkers have been identified. Based on the previous research,In summary, our results suggest that extrapulmonary involvement is common in patients with pulmonary sarcoidosis, with the most common sites being extrathoracic lymph nodes and skin. Patients with sarcoidosis with extrapulmonary involvement have more complaint of fatigue, higher SACE levels and more deteriorating HRCT findings than those with isolated pulmonary sarcoidosis, to which clinicians should pay attention.


PD‐L1 expression combined with CD8+TILs density in patients with resected NSCLC and correlations with clinicopathological features. We retrospectively enrolled 178 patients with resected NSCLC from 2011 to 2015. All surgical primary and 58 matched metastatic lymph node specimens were tested for PD‐L1, CD8+TILs, and oncogenic alterations. PD‐L1+ was detected in 71 (39.9%) and CD8highTILs in 74 (41.6%) cases. Smoking, SqCC, and EGFR− were associated with both PD‐L1+ and CD8highTILs. Patients with CD8highTILs had longer OS (P = 0.012). PD‐L1− was significantly associated with longer OS in patients with oncogenic alterations (P = 0.047). By multivariate analysis, CD8highTILs , rather than PD‐L1, was the independent predictive factor for OS. The longest and shortest OS were achieved in patients with PD‐L1+/CD8high and PD‐L1+/CD8low, respectively (P = 0.025). Inconsistent PD‐L1 expression levels were observed in 23 of 58 (39.7%) patients with primary and matched metastatic lymph node specimens. Of them, CD8highTILs was significantly associated with longer OS in patients with metastatic lymph nodes and/or consistent PD‐L1 expression . The combination of PD‐L1 and CD8+TILs density, instead of PD‐L1 alone, suggested impressive prognostic values in NSCLC patients. Less than half of patients with resected NSCLC experienced inconsistent PD‐L1 expression between primary and metastatic lesions. The level of PD‐L1 expression in advanced NSCLC needs to be evaluated more comprehensively.To investigate the prognostic value of  To date, few studies focusing on this issue have been published, and the case number of those studies is small + TILs. Their results concerning the correlation between PD‐L1 expression/CD8+ TILs and prognosis in NSCLC are inconsistent Programmed death receptor‐1 (PD‐1) is a protein receptor of the CD28 family expressed on the surface of T, B, and natural killer (NK) cells that regulates their activation and proliferation. Its ligand, PD‐L1, is frequently overexpressed in many kinds of human malignancies. The binding of PD‐L1 to PD‐1 induces apoptosis or exhaustion in activated T cells and limits the effector function of T cells in peripheral tissues during inflammatory responses + TILs density in Chinese patients with surgically resected NSCLC. We also analyzed the correlation between clinicopathological characteristics and PD‐L1 expression/CD8+ TILs density in NSCLC.Hence, we performed this study that aimed to investigate the prevalence and prognostic value of PD‐L1 expression and CD8Patients who were diagnosed with NSCLC and underwent surgical resection at Yancheng Third People's Hospital and Shanghai Pulmonary Hospital from 2011 to 2015 were evaluated. Patients with missing baseline clinicopathological characteristics and follow‐up data were excluded. Of the remaining patients, those who had formalin‐fixed and paraffin‐embedded (FFPE) specimens representing primary lesions containing adequate tumor cells and T cells were enrolled in this study. Patients who were staged as p‐TxN1‐3Mx without FFPE specimens representing metastatic lymph nodes were excluded. Finally, 178 total eligible patients were included. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee and Institutional Review Board (IRB) of Yancheng Third People's Hospital and Shanghai Pulmonary Hospital. A written informed consent was obtained from each participant in order to use the clinical data for research.−/+ was determined by cut‐off value of 5%, which was based on several previous clinical trials of anti‐PD‐1/PD‐L1 drugs in NSCLC; + cases, we defined PD‐L1 strong positivity (PD‐L1high) as PD‐L1 > 50% . To express ODM concisely, ODM+ refers to any one or more oncogenic driver mutations and ODM− refers to pan‐negative for the six above‐mentioned oncogenic diver mutations.All oncogenic driver mutation (ODM) analyses were performed at Thoracic Cancer Institute, Tongji University School of Medicine . + TILs density and patients' clinicopathological characteristics were performed with Chi‐square test for categorical variables and ANOVA and Tukey's multiple comparison tests for continuous variables. The Kaplan–Meier method was used for survival analyses, and the significance of differences between groups was evaluated by the log‐rank test. The Cox proportional hazards model was used for univariate and multivariate analyses to calculate the hazard ratios (HR) and 95% confidence intervals (95% CI). Overall survival (OS) was calculated from the date of diagnosis to death from any cause or was censored at the last follow‐up date. P values were considered statistically significant if <0.05 (two‐sided). All statistical analyses were performed by SPSS for Windows .Correlations between PD‐L1 expression/CD8EGFR, KRAS, HER2, RET, ALK, and ROS1 alterations were detected in 73, 16, five, four, three, and three cases, respectively . We found that PD‐L1high was more likely to be detected in patients with SqCC (P = 0.001), EGFR wild type (EGFR−) (P = 0.045), and ODM− (P = 0.046) . One‐hundred thirteen (63.5%) patients were male, and 103 (57.9%) patients had a history of smoking. The majority of histological types was adenocarcinoma , including five lepidic, 18 papillary, 81 acinar, 29 solid, three enteric and one minimally invasive ADC (IMA), which was followed by 41 squamous cell carcinomas (SqCCs). Of them, 85 (47.8%), 47 (26.4%), 39 , and seven (3.9%) patients had stages I–IV diseases at the time of diagnosis. With respect to ODM status, + was detected in 71 (39.9%) patients, and it was significantly associated with male gender (P = 0.005), current/former smokers (P = 0.002), SqCC (P = 0.002), and EGFR− (P = 0.002), but not with age (P = 0.782), p‐stage (P = 0.782), or KRAS mutation status (P = 0.053). We restricted our analyses to CD8+ TILs due to tissue availability, which is generally thought to be the population mainly benefitting from treatment with PD‐1/PD‐L1 antibodies high TILs were detected in 74 (41.6%) cases and were significantly associated with smoking (P = 0.027), SqCC (P = 0.031), p‐stage I (P = 0.020) and EGFR− (P = 0.010). In addition, it was observed that CD8high was associated with PD‐L1+/PD‐L1high, and CD8low was associated with PD‐L1− (P < 0.001) . Among the entire cohort, patients with CD8high TILs had longer OS (P = 0.012), but there was no statistical difference between PD‐L1−/+ and OS (P = 0.987). In patients with ADC and solid ADC, we obtained similar results in which CD8high TILs were associated with better OS , and PD‐L1−/+ showed no correlation with OS . Interestingly, in patients with non‐solid ADC and SqCC, PD‐L1+ was associated with more satisfactory OS than PD‐L1− , however, CD8+ TILs density did not correlate with OS . When we sub‐grouped PD‐L1low/high in PD‐L1+ cases for survival analyses, statistically significant difference was only achieved in patients with non‐solid ADC (P = 0.037) , not even both combined (P = 0.082)  and CD8high TILs  were independent and significant predictive factors for OS in NSCLC instead of PD‐L1 expression  . Statistical differences were achieved in patients with NSCLC, ADC and non‐solid ADC , but not in patients with solid ADC and SqCC . Among the whole cohort, the longest OS was achieved in patients with PD‐L1+/CD8high, and the shortest was in PD‐L1+/CD8low (median OS was not reached). In groups of ADC and non‐solid ADC, patients with PD‐L1+/CD8low experienced the worst OS , and patients with PD‐L1−/CD8high achieved the best OS (median OS was undefined in both groups)  Fig. .EGFR− or KRAS−, CD8high TILs were associated with better OS , but there was no statistically significant difference between PD‐L1 expression levels and OS. In patients with EGFR+ and KRAS+, PD‐L1 expression levels or CD8+ TILs density were not associated with OS. In patients with ODM+, it was observed that PD‐L1− was associated with longer OS (P = 0.047); in contrast, PD‐L1+ was associated with longer OS in patients with ODM− (P = 0.048), and there was no correlation between CD8+ TILs density and OS in both groups of patients . It is worth mentioning that whether statistically significant differences were reached or not, CD8high TILs showed a tendency for better OS than did CD8low TILs in each group of patients, and PD‐L1+/PD‐L1− showed a tendency for longer OS than did PD‐L1−/PD‐L1+ in patients with ODM−/ODM+, respectively  were noted, but no correlation between CD8+ TILs density and OS in patients with inconsistent PD‐L1 expression (P = 0.162). Due to tissue availability, we assessed the expression consistency of CD8+ TILs in 160 patients. Consistent expression was detected in 87 patients. Survival analyses indicated that CD8high TILs were associated with better OS in patients with either consistent or inconsistent expression , and just as seen in the results in patients with metastatic lymph nodes, there was no prognostic value of PD‐L1 expression in OS (P > 0.05)  were strong positive (PD‐L1high). PD‐L1high was also associated with SqCC and EGFR− (P = 0.001 & 0.045). The reported extents of PD‐L1 positive expression in NSCLC ranged from 7.4% to 72.7%, which could be explained by the differences in the choice of primary IHC antibody, ethnicity, cut‐off value of positivity, and histologic types + was common in male, smokers, SqCC, and EGFR−. Of note, there was a significant linear correlation between male, smokers, SqCC and EGFR− in our study. Therefore, PD‐L1+ may be actually associated with one of mentioned‐above parameters.PD‐L1high TILs occurred in 41.6% patients and was associated with smokers, SqCC, p‐stage I, and EGFR−. The similar clinicopathological features of CD8high TILs with those of PD‐L1+ may be explained by the positive correlation between CD8+ TILs density and PD‐L1 expression levels high TILs, instead of PD‐L1+, was significantly associated with longer OS. CD8+ TILs, which form the pivotal component of the cellular immune system, constitutes the effector arm of adaptive immunity against tumor cells. More recent studies found CD8+ TILs was associated with better survival in NSCLC although the correlation remains controversial + TILs and longer OS only in SqCC, particularly in early stage + TILs were associated with poor survival in non‐ADC high TILs and longer OS in all NSCLCs except non‐solid ADCs. On the contrary, there was a significant correlation between PD‐L1+ and longer OS in non‐solid ADCs and SqCCs. These findings could be explained in part by the possible impact of histological types on the correlation between CD8+ TILs/PD‐L1 expression and OS.Our study found CD8+ TILs because CD8+ TILs could produce IFN‐γ and induce PD‐L1 expression in different solid tumors. In all NSCLCs, the longest OS was achieved in PD‐L1+/CD8high group while the shortest OS was in PD‐L1+/CD8low. Tokito et al. found that the PD‐L1−/CD8low group had the shortest survival but the PD‐L1−/CD8high group had the longest survival in locally advanced NSCLC patients receiving concurrent chemoradiotherapy + TILs may be more useful to predict the prognosis of NSCLC and the efficacy of immunotherapy.We explored the prognostic effects of PD‐L1 combined with CD8+ was associated with EGFR−, but not with other oncogenic alternations mentioned in our study. We also observed that PD‐L1+ was associated with male, smokers, and SqCC. As we know, EGFR− was common in male patients with smoking history and SqCC histology. Moreover, CD8+ was also only associated with EGFR−. It may be attributed in part to the significant correlation between PD‐L1+ and CD8high TILs in our study. The number of studies evaluating the correlation of PD‐L1/CD8+ TILs with ODM was limited. Some studies reported a significant association between PD‐L1 expression with EGFR+ or KRAS+ in NSCLCs, but others did not EGFR+ and ALK+ in NSCLC with up to 72% and 78% of PD‐L1 positive rates in EGFR+ and ALK+ patients, respectively PD‐L1+ TILs and longer OS in patients with consistent PD‐L1 expressions between primary lesions and metastatic lymph nodes but not in patients with inconsistent PD‐L1 expression. The question is which specimens should we use to detect PD‐L1 expression in advanced NSCLC. The primary lesions or the metastatic lymph nodes? We considered that it may be better to evaluate PD‐L1 expression in both for advanced NSCLC patients. Patients with consistent positive PD‐L1 expressions may obtain better survival benefit from anti‐PD‐1 inhibitors.The last but not the least, our study showed inconsistent PD‐L1 expression levels between primary lesions and metastatic lymph nodes occurred in 23 of 58 (39.7%) patients. Moreover, there was a significant correlation between CD8Although many studies and clinical series reported the prevalence, clinicopathological and/or molecular features, and prognostic value of PD‐L1 expression in NSCLC, there were conflicting results on these aspects. The above‐described discrepant results could be explained by the differences in ethnicity, specimen types, PD‐L1 IHC protocols (including primary antibodies), scoring criteria, cut‐off values, targeted cell types (tumor cells or TILs), and locations (membranous or cytoplasmic). The absence of a universally accepted PD‐L1 IHC standard and its interpretation make it extremely difficult to determine the prognostic or predictive value of PD‐L1 expression in NSCLC. Besides, the significance of PD‐L1 expression from a single‐biopsy specimen in advanced NSCLC may be overestimated in clinical practice. Therefore, a large‐scale, prospective study is warranted to determine the prevalence and role of PD‐L1 expression, comparison on PD‐L1 expression between single‐biopsies and resected specimens, and the correlation with TILs in NSCLC population.The authors have declared no conflicts of interest.Figure S1. Representative IHC images for PD‐L1 and CD8+ TILs in NSCLC.Click here for additional data file.Figure S2. Representative images of positive and negative controls for PD‐L1 IHC staining.Click here for additional data file.


Raman and XPS analysis of a carbon colloidal performed at different irradiation stages indicate the formation of Graphene multilayers with an increasing number of layers: the amount of layers varies from a monolayer obtained in the first few seconds of the laser irradiation, up to two layers obtained after 10 s, and finally to Graphite and amorphous carbon obtained after 40 s of irradiation. The obtained colloidals are pure, without any presence of impurities or Graphene oxides, and can easily be deposited onto large surfaces (in the order of cm2) for being characterized or for being used in diverse applications.In this paper we introduce a laser-plasma driven method for the production of carbon based nanomaterials and in particular bi- and few-layers of Graphene. This is obtained by using laser-plasma exfoliation of amorphous Graphite in a liquid solution, employing a laser with energy in the order of 0.5 J/mm This simple 2-D material, composed of a single layer of carbon atoms, is characterized by high electron mobility and a field-generated band gap, therefore, it is defined as a zero-gap semiconductor4. Nevertheless, technological applications that take advantage of Graphene electronic transport properties require structurally coherent Graphene on a large scale , or large arrays of Graphene flakes positioned with a unique azimuthal orientation on a substrate.Graphene is one of the most promising materials for nanoscience applications4, which – being a low-budget technique - strongly contributed to arousing interest for Graphene. However, the produced Graphene flakes have irregular shapes and a random azimuthal orientation. Currently, Chemical Vapor Deposition (CVD) on transition metal substrates7 can produce the best quality Graphene. In particular, Graphene obtained with CDV deposited on copper foils forms large uniform layers. Nonetheless, the electrical properties of CVD Graphene cannot be tested on a metal substrate, since the testing requires a transfer process of the Graphene layer onto an appropriate insulating substrate. The transfer process often affects the Graphene integrity, its properties, and thus the overall performance: Wrinkle formation, impurities, Graphene tearing, and other structural defects can occur during the transfer. Moreover, CVD Graphene grown on substrates has its size limited by the reactor size, which restricts a large-scale production. Alternative methods for the production of Graphene are epitaxial growth on metal surfaces or graphitization of hexagonal SiC9. In all these cases, the quality of Graphene is strictly related to the substrate properties such as size, crystallinity, purity, since, similarly to the CVD, these production processes require a transfer.In the last decades, many preparation processes have been suggested in order to improve the quality of Graphene and its properties. The first method was the mechanical exfoliation from Graphite proposed in 200410. The most employed laser method is the Laser Ablation, which can be performed in vacuum, controlled gaseous atmosphere, or in liquid (Laser Ablation Synthesis in Solution - LASiS). Laser methods allow having a precise control in the dimensions and shapes of the produced nanomaterials, with an in-situ tuning of the material properties being possible by simply changing the laser and plasma characteristics. While these methods are efficiently used for the synthesis of nanoparticles and quantum dots12, the high temperatures and pressures reached on the generated plasma prevent their use in the Graphene synthesis. Higher temperatures can induce the amorphization of the carbon structures leading to a complete loss of the hexagonal lattice.In addition to these processes, recently also laser-driven methods for the synthesis of nanomaterials were proposed as alternative to chemical techniquesIn the following, we propose a simple method for the large-scale production of colloidal solution of Graphene flakes based on the interaction between a higher power laser beam (power of about 100 MW) and a material bulk. The technique , which however is requiring a high vacuum system, a Highly Oriented Pyrolytic Graphite base sample, and a substrate for the Graphene deposition. The method presented in refs.16 allows the production of Graphene oxide at a liquid interface, while in our case we obtain pure Graphene in a colloidal solution. The method proposed in ref.17 requires the use of liquid nitrogen and consequently the use of very low temperatures. This, since the nitrogen molecules are crucial for the exfoliation of the graphite. All the above-mentioned techniques are at the base of our proposed method, however, compared to them, we are able to relax some of the required stringent manufacturing characteristics (e.g. low temperature) making our technique more accessible and easier implementable.Our method is similar to other laser-based synthesis processes that have been explored previously (see later), although our synthesis process has the advantage of combining a much easier implementation with a much faster Graphene production, very suitable for large scale production, still enabling high quality optical and electrical properties. In first pioneering works (see refs.2. The laser fluence is about 0.5 J/pulse (with a pulselength of 7 ns). The laser source used for the LE reaches the target from the top surface of a cuvette. The colloidal solution, produced at different irradiation times, is transferred to a copper surface by drop-casting and heated in air up to 70 °C for solvent evaporation. Analytical characterization of each deposited drop was performed by morphological (AFM-SEM) and chemical (RAMAN and XPS) information. Differently from the classical Laser Ablation technique employed for the synthesis of nanoparticles, where the approach is typically bottom/up (i.e. atoms nucleate producing nanoparticles), the physical phenomena involved in the LE can be attributed to both, a top/down and a bottom/up approach: When the laser irradiates the carbon surface, it generates (similarly as for the generation of nanoparticles) different phenomena such as the detachment of single carbon atoms (which aggregate in amorphous carbon particles (bottom/up approach)), and the exfoliation of a single or multi-layer of Graphene (top/down approach). All these detached materials are distributed in the plasma plume and are dispersed in the colloidal solution. While the multi-layer of Graphene stays suspended in the colloidal solutions, the carbon atoms aggregate producing amorphous microparticles.Our Graphene growth method is based on the laser exfoliation (LE) of amorphous Graphite, realized by using an experimental method that is typically employed in the conventional Laser-method for nanomaterials synthesis, the “Laser Ablation Synthesis in Solution” (LASiS). The preparation of Graphene flakes using LE has the advantage of not requiring vacuum conditions , and as such allows achieving layers with higher purity produced in diverse solutions (permitting to choose the solution which is most suitable for the applications). In our experimental setup, a Graphite plate, with nominal purity of 99.99% and dimensions of about 1 cm × 1 cm, is placed on the bottom of a vessel cuvette containing an aqueous solution of acetone (90%). We chose as solvent acetone (oxygen-free ambient) for preventing the oxidation of nanoflakes during the synthesis process and the formation of Graphene oxide. The plate is ablated with the second harmonic (532 nm) of a pulsed YAG:LASER. The laser spot size on the target surface is of about 1 mmMorphological analysis of the colloidal drops deposited onto a silicon surface indicates the presence of Graphene bi- and multi-layer and of amorphous carbon aggregates generated by the aggregation of single atoms detached during the laser irradiation. Nevertheless, the amount of the Graphene multilayer or the amorphous aggregates changes with irradiation time, indicating that the two phenomena (particle synthesis and Graphene exfoliation) occur at different stages. The presence of amorphous carbon in the solution is higher after the first 10 s of irradiation, clearly indicating that the breaking of the interlayer bonding is the predominant phenomenon in a first phase of the laser irradiation while this effect (the breaking) becomes negligible respect to the amorphous aggregation in the second phase.The colloidal solution is finally deposited by drop cast on a conductive glass substrate and the film is heated in air to about 50 °C for facilitating the solvent evaporation. The deposited layer is analysed in order to check the possibility of producing very large substrates covered with our produced Graphene multilayers and to test the electrical and optical properties.AFM images are obtained with the ICON AFM microscope manufactured by the company Bruker working in the tapping mode. Each image is taken with a resolution of 512 × 512 and 1024 × 1024 pixels and a frequency of 1 Hz. Dimensions of the Graphene flakes are computed conducting a statistical analysis on about 100 flakes collected in several AFM images. For each sample, we scan several areas (about 50) in a window of 5 µm × 5 µm (resolution of 1024 × 1024 pixels). SEM analysis was conducted using a field emission SEM microscope (Quanta 200 F FEI/Philips) working with an energy of 20 keV.−9 torr. Non-monochromatic Mg-Kα X-rays (hν = 1253.64 eV) are used as excitation source. The XPS spectra are calibrated with the C1s peak of a pure carbon sample (energy position located at 284.6 eV). All XPS spectra are corrected for analyzer transmission and the background is subtracted using the straight-line subtraction mode. Moreover, the XPS data are fitted assuming a Gaussian distribution. Finally, the KLL Auger structure of carbon is analyzed in a Derivative mode for evaluating the D parameter. The Raman measurements are taken by a Raman Microscope (Thermo Fisher DXR), equipped with a 532 nm laser and a spot size with a resolution of 1 micron. The spectra are obtained with a 50 × objective . The spectral resolution, by using the 1800 grooves/mm grating, was estimated to be better than about 2 cm−1. Each micro-Raman spectrum was collected in 20 s and three accumulations.XPS measurements are conducted in a UHV chamber equipped for standard surface analysis with a pressure in the range of 10The optical transmittance of the Graphene multilayer are measured by depositing different colloidal quantities onto a conductive glass substrate, and irradiating the film with a halogen lamp under confocal microscope conditions (Olympus 900 by Horiba). The transmitted spectra are taken using a Triax 320 (Horiba–Jobyn–Yvon) spectrometer working in the 300–800 nm range.2).The electrical measurements are conducted on some of the films with best uniformity in coverage (deposited onto a conductive glass). The ohmic film resistance (R) is measured using a Keithley DC Current generator Model 2100 with four point probe, while conductivity (σ) is measured using the following formula:Chemical information on the produced carbon nanostructures is obtained studying the evolution of the G and 2D Raman structures of carbon with increasing irradiation time, while the final confirmation of the Graphene formation in the best colloidal solution is obtained by analyzing the D parameter of the KLL Auger line of carbon in the XPS data. Both measurements (G and 2D Raman and D parameter) are indicated in literature as fingerprints of the carbon allotropes.18. The systematic study in Graf et al. (ref.18 indicates as most relevant parameters for the individuation of Graphene (single or multilayer) the peak position of the 2D band and the ratio between the G and 2D band intensity. In detail, a Graphene monolayer shows a 2D peak position of 2678.8 cm−1 and a G/2D ratio around 0.25; for multilayers of Graphene (from 2 up to 5 layers) the 2D peak becomes larger, splits in different substructures, and the energy position shifts of about 19 eV for 2 layers up to about 26 eV for 6 layers. The G/2D ratio varies from 0.4 (2 layers) up to 0.8 (6 layers).The effectiveness of the Raman diagnostic for the individuation of Graphene presence on a surface is well known in literature and generally used as a proof of Graphene formation and depositionet al., in ref.19 indicate in the D parameter, which calculates the distance between the positive maximum and negative minimum of the KLL line derivative, the key parameter for the identification of carbon allotropes. Their data indicate for D a value of about 15 eV for Graphene, 21 eV for Graphite and 13 eV for Diamond.In addition, the confirmation of Graphene formation can also be obtained by studying the changes in the carbon Auger emission. More precisely, Kaciulis 2 surface and for each deposited droplet we took a Raman spectrum, following the evolution of the G and 2D bands. As visible from the Raman spectra in Fig. 18 obtained for droplets collected after 10 seconds of irradiation, to Graphite values (>1)18 obtained after 30/40 seconds of laser irradiation. A detailed analysis of the Raman spectrum of a colloidal deposited after 10 s of irradiation 18 is strongly reduced after 10 s of irradiation, indicating the formation of states with less lattice imperfections and confirming the formation of Graphene bi-layers. Our assumption is reaffirmed by the fact that the 2D peak  for a bi-layer material. The analysis of the D parameter  onto a SiOion Fig.  indicateeak Fig.  is splitsee Fig. . The D vsee Fig. . Finallysee Fig.  shows a 10. The observed starting time for the aggregation (30 s) is identical to what observed for metal and semiconducting nanoparticles22, indicating a plume expansion and evolution similar to that observed for the synthesis of nanoparticles12. The main stages in the LASiS exfoliation process can be resumed as follows: the process starts with the absorption of the laser pulse energy by the Graphite target. This generates both the detachment of carbon atoms from the surface and the breaking of the Van Der Waals bonding between the carbon layers in the first micron of the Graphite surface. A plasma plume containing carbon atoms and the exfoliated mono- and bi-layers of carbon expands into the surrounding liquid. During the expansion, the plasma plume cools down and releases energy to the liquid solution. This phenomenon generates a cavitation bubble inside the bulk target, bubble that expands in the liquid and then collapses in a time scale in the order of hundreds of microseconds. The exfoliation of mono- and multilayer and the desorption of carbon atoms are estimated to occur in a timeframe ranging from 10−6 and 10−4 s after the impact of the laser pulse on the surface (the laser has a pulse duration of about 7 ns). These first phases are followed by a multilayer aggregation and a microparticle nucleation, which both form amorphous carbon structures and Graphite.Progressive aggregation of nanomaterials in the LASiS plume during the laser irradiation is a well-known phenomenon23.The final confirmation of Graphene bi-layer formation is obtained by morphological analysis of the small droplet 5 µL) of colloidal deposited onto copper after 10 s of irradiation. The 2D and 3D AFM images and SEM pictures in Fig.  µL of co2) obtained with different solution amounts. The area was completely covered with the 200 µL of the colloidal solution. AFM images  obtaining uniform films with high optical transmission, indicating that the flakes do not aggregate during deposition. Finally, electric measurements on the deposited films indicate, at atmospheric pressure, a conductivity in the order of 480 S/m, typical for Graphene films.In this paper, we introduce a method to synthesize bi-layers of Graphene using a Laser-Exfoliation based on the Laser Ablation techniques typically employed in the nanoparticle production. The obtained results indicate that the quality and the allotropes of the obtained carbon colloidals are strictly dependent on the laser irradiation time. Graphene bi-layer is obtained in the first 10 s of irradiation while for irradiation time greater than 30 seconds the colloidals are mainly composed by amorphous carbon and Graphite. The obtained bi-layers are characterized by Raman and XPS measurements, which indicate the formation of pure carbon layers with absence of oxygen impurities or Graphene oxide. Moreover the obtained micrometric flakes can be distributed on large areas (order of cm


Monoclonal antibodies targeting the PD-1/PD-L1 axis have gained increasing attention across many solid tumors and hematologic malignancies due to their efficacy and favorable toxicity profile. With more than 1 agent now FDA-approved in a wide variety of tumor types, and with others in clinical trials, it is becoming more common that patients present to clinic for potential treatment with a second PD-1/PD-L1 inhibitor.In this report, we present two patients with renal cell carcinoma and one with melanoma who received PD-1/PD-L1 inhibitors. Upon progression on their first-line PD-1/PD-L1 inhibitors, these patients received a different PD-1 inhibitor  and all had progressive disease as their best response to the subsequent PD-1 inhibitor. The reported clinical information focuses on the course of the disease and the responses to all treatment regimens.Clinicians should refrain from using multiple PD-1/PD-L1 inhibitors sequentially outside of clinical trials until there is sufficient data to support this practice routinely. Prospective studies that allow prior treatment with PD-1/PD-L1 are needed to validate the efficacy and safety of these drugs in the second line or later setting. Furthermore, ongoing efforts that aim to identify mechanisms of resistance to immunotherapy will be informative and may ultimately assist physicians in select the optimal treatment following progression on PD-1/PD-L1 inhibitor. The programmed cell death protein-1 (PD-1) and its ligands, PD-L1 and PD-L2, are part of a pathway that cancer cells utilize to evade immune surveillance . Five ofA 54-year-old man underwent a radical nephrectomy which revealed an 11.5 cm pT2bN0M0 clear cell renal cell carcinoma (ccRCC) on pathologic review. Four years later, he was diagnosed with metastatic disease to the lungs and hilar lymph nodes and began treatment with an anti-PD-L1-based combination. The patient had a best response of stable disease with tumor shrinkage and remained on treatment for 15 months. He discontinued therapy for progressive disease to the sacrum and the cerebellum and subsequently underwent stereotactic radiosurgery to the brain. Approximately 7 weeks after the last dose of the anti-PD-L1-based combination, he initiated treatment with 3 mg/kg nivolumab monotherapy every 2 weeks. After the patient received 4 doses, imaging showed progressive disease in the lung and hilar lymph node after 7 weeks.A 67-year-old male was diagnosed with pT2aN0M1 ccRCC with multiple subcentimeter metastases to the lungs. The patient initially underwent metastatectomy to remove a 0.6 cm tumor in the left upper lobe, but he experienced progression 1 year later. An anti-PD-L1-based combination was started and he had stable disease as best response with tumor shrinkage and remained on treatment for 8 months until he discontinued therapy for new liver metastases. He then progressed after 2 cycles of axitinib. The patient received 8 doses of 3 mg/kg nivolumab monotherapy every 2 weeks, 6 months after the last dose of the anti-PD-L1-based combination. He experienced disease progression in the lung, lymph nodes, and liver after 4 months.V600mutant cutaneous melanoma with metastases to the kidney, adrenal, and lymph node. The patient began treatment with a vemurafenib, a BRAF inhibitor, but discontinued after two months for progressive disease. He then progressed through treatment with cytotoxic chemotherapy, ipilimumab, and a combination of anti-BRAF/MEK (dabrafenib plus trametinib) combination therapy. He later received therapy with anti-PD-1 pembrolizumab for 5 months before being discontinued for treatment-related insulin-dependent diabetes mellitus, chronic pruritus, and joint pain. The patient had a complete response on treatment. Approximately 20 months after the patient’s last dose of pembrolizumab, he progressed and initiated nivolumab every 2 weeks for 3 doses. Although the patient continued on insulin for treatment of diabetes mellitus throughout his time on nivolumab, neither the pruritus nor the joint pain recurred after treatment with nivolumab. Treatment was discontinued after 7 weeks due to disease progression in the bilateral adrenal lesions, lymph nodes, and a new lesion in the liver.A 78-year-old gentleman was diagnosed with stage IVM1c BRAFGiven the approval of different PD-1 and PD-L1 inhibitors in several solid tumor and hematological malignancies as of May 2017, it is anticipated that clinical decisions facing patients and physicians as described above will become more common. For example, there are already two PD-1 inhibitors, pembrolizumab and nivolumab, approved for NSCLC and melanoma in both the first and second-line setting. The shifting treatment paradigms in NSCLC and other diseases such as melanoma and RCC are complicated by the paucity of data for patients who fail PD-1/PD-L1 inhibitors. This is partly because randomized trials in which these agents were investigated in the second-line setting or beyond, did not allow inclusion of patients who had prior treatment with a PD-1 or PD-L1 inhibitor , 15, 18.When successful, PD-1 blockade establishes a favorable equilibrium between the immune response to, and immune evasion by, cancer cells . In someAlthough many patients experience resistance after responding to PD-1 or PD-L1 inhibitors, there may be situations where re-treatment has plausible rationale. Re-treatment with PD-1 blockade may be especially effective in patients who discontinue the first PD-1 or PD-L1 inhibitor for reasons other than disease progression. In fact, successful re-introduction of anti-PD-1 therapy after delayed tumor recurrence has been reported in one patient with melanoma . Data haThere are numerous opportunities to address mechanisms of resistance to immunotherapy other than retreatment with PD-1/PD-L1 monotherapy. For example, anti-Vascular Endothelial Growth Factor (VEGF) therapy has been shown to increase T-cell infiltration  and precHowever, it is unlikely possible to provide clinical trials for all potential immunotherapy sequences. Therefore, databases that captures real-life clinical data on the safety and efficacy of anti-PD-1/PD-L1 therapy after treatment with another PD-1/PD-L1 inhibitor may potentially help to better characterize the outcomes of these patients in an unselected setting. For example, there is a United States-based registry for patients treated with high-dose IL-2, Proleukin® Observational Registry to Evaluate the Treatment Patterns and Clinical Response in Malignancy (PROCLAIM), which adds real-world experience for patients treated with IL-2 . SimilarCurrently, there is insufficient data to support treatment with a different PD-1/PD-L1 inhibitor after progression on PD-1 pathway blockade, and such practice should be discouraged. Ideally, prospective studies that investigate the efficacy and safety of a PD-1/PD-L1 inhibitor in the second-line setting or later, which allows prior treatment with one of these agents, are needed to validate the use of sequential PD-1 blockade. Combined strategy such as dual immune agents or combination of PD-1/PD-L1 non-immune therapy appears to be more supported by preclinical data and such trials represent a better opportunity to improve outcomes. Furthermore, ongoing efforts that aim to identify mechanisms of resistance to immunotherapy will be informative and may ultimately assist physicians in selecting a rational approach to the initial, and sequential, treatment options for these patients.


Although immune checkpoints inhibitors have exhibited promising activity in clinical trials in non-small cell lung cancer (NSCLC) patients, the current programmed cell death-ligand 1 (PD-L1) assays are inconsistent in terms of the staining analysis and scoring system used. To verify the interchangeability of the available PD-L1 assays, we performed immunohistochemistry using three antibody clones used in clinical trials  and the E1L3N clone as a laboratory developed test for 97 resected NSCLC specimens. Matched tissue microarray specimens were also stained. Staining with 22C3 yielded a greater proportion of stained tumor cells, whereas SP142 staining consistently labelled fewer tumor cells. However, when various cut-off criteria were applied, the positivity rates for PD-L1 were similar, with high concordance, under assay-specific cut-offs. Moreover, seven cases of discordant PD-L1 expression between the resected specimen and matched tissue microarray specimens were observed. In conclusion, despite of inter-assay variability of the PD-L1 status in NSCLC, the positivity rate appears to be similar under assay-specific criteria. Hence, an appropriate clinically defined algorithm or cut-off should be separately applied for each assay. Moreover, multiple biopsy specimens from different tumor areas should be obtained to reduce false results due to intratumoral heterogeneity in PD-L1 expression. Programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) checkpoint inhibitors for heavily pre-treated patients with advanced non-small cell lung cancer (NSCLC) represent major advances in immunotherapy . Recent Recently approved tests for anti-PD-1/PD-L1 therapy in NSCLC include the assessment of PD-L1 expression using immunohistochemistry (IHC) as a companion diagnostic test (22C3 for pembrolizumab) , 7 and 2Although companion/complementary PD-L1 assays are developed using a ‘one drug–one assay’ paradigm, it is impractical to run a different test for each drug, and most pathology laboratories currently use only one platform. Hence, it is important to verify the interchangeability of these assays.The intratumoral heterogeneity in PD-L1 expression is also important to consider . PD-L1 tIn the present study, we aimed to 1) compare the analytic results between 4 different PD-L1 IHC and scoring systems, and 2) evaluate the correlation of PD-L1 expression between tissue microarray (TMA) specimens and the corresponding resected specimen to better understand the frequency of discrepancies and the underlying characteristics.In the training set, PD-L1 expression in the tumor cells was observed in 40.0% 20/50), 38.0% (19/50), 18.0% (9/50), and 30.0% (15/50) of cases in the 22C3, SP263, SP142, and E1L3N assays, respectively. In the validation set, 30.0% (14/47), 30.0% (14/47), 14.9% (7/49), and 17.0% (8/47) of cases showed a tumor proportion score (TPS) >1% in the 22C3, SP263, SP142, and E1L3N assays, respectively. There was no difference in tumor cell PD-L1 expression between the sets. Among the total 97 cases, 38 showed PD-L1 tumor cell positivity in at least 1 assay; 22C3 showed the highest TPS, followed by SP263 and E1L3N, whereas SP142 showed the lowest TPS Figure . The 22C0/50, 38.In the training set, PD-L1 expression in immune cells was observed in 36% (18/50), 28% (14/50), 12% (6/50), and 18% (9/50) of cases in the 22C3, SP263, SP142, and E1L3N assays, respectively. In the validation set, PD-L1 expression was observed in 40% (19/47), 34% (16/47), 17% (8/47), and 32% (15/47) of cases, respectively. In all assays, the proportion of positive immune cells was significantly lower than that of the tumor cells Figure . FurtherNext, the PD-L1 status for the 97 NSCLC cases was compared after classifying the cases according to the combined pre-specified and selected cut-offs Table . The 22CTable 2The concordance of the PD-L1 positive rate improved with the assay-specific cut-off. Table We compared PD-L1 status which means above or below the specific cut-off threshold for each assay. Ten of the 97 cases (10%) were above the cut-offs used for all 4 assays, indicating that PD-L1 positivity would be concordant regardless of the assay used. Moreover, 79 of 97 (81%) cases were found to be below the cut-off value for PD-L1 negativity, regardless of the assay used. Eight cases (8%) showed variations in the classification, being classified either above or below the assay-specific cut-off. These data indicate that using an alternative validated assay and an assay-specific scoring algorithm to evaluate PD-L1 expression would yield different results in only approximately 8% of cases. However, the replacement of the validated cut-off for each assay with any other cut-off reduces the overall agreement in comparison with the reference standard.We used the 22C3 assay for assessing the TMA sections, and obtained PD-L1 IHC results for matched WTS and TMA specimens in 50 cases in the training set. With regard to the TPS, among the 50 cases, 29 (58%) cases were PD-L1 negative (TPS=0) and 14 (28%) were PD-L1-positive (TPS≥1%) in both the WTS and TMA specimens. Seven of 50 (14%) cases exhibited discordant PD-L1 expression, with negative findings in the TMA compared with those in the WTS (TPS=1–50%)  cases were PD-L1-negative and 11 (22%) were PD-L1-positive in both the WTS and TMA specimens. Although 21 of 50 (42%) cases showed discordant PD-L1 expression in the immune cells between the WTS and TMA, the immune cell-positive rate was low, ranging between 1–15%.In the present study, we demonstrated that 1) the PD-L1 status could be designated differently depending on the assay-specific cut-off, and 2) small biopsy/TMA specimens underestimated the PD-L1 status, as compared with resected specimens.Our results showed that the 22C3 and SP263 assays were similar in terms of the PD-L1 staining performance. The E1L3N assay, an LDT, exhibited a similar staining pattern, although the TPS was slightly lower than that of the above-mentioned 2 assays. The SP142 assay generally stained fewer tumor cells, consistent with the Blueprint Project Phase I results . All assA negative or positive PD-L1 status depends on the cut-off used and is crucial for subsequent treatment decisions. When various cut-offs, including assay-specific criteria, were used to designate the PD-L1 status in the present study, the 22C3 and SP263 assays displayed similar positive rates in the overall range, followed by the E1L3N assay, whereas the SP142 assay showed the lowest rate. Because of differences in the TPS for each assay, the application of the same cut-off to all assays resulted in a large positive rate discordance. When the assay-specific cut-offs were applied, the overall agreement rate was high, and only approximately 8% of cases exhibited variable PD-L1 status. Especially, only a few cases designated as PD-L1-negative showed values below the cut-off in the 22C3 or SP263 assays but were PD-L1-positive with the SP142 assay, owing to variations in the assay-specific cut-off. Therefore, it may be possible to infer the PD-L1 staining level results between assays, while using clinically defined assay-specific cut-offs for each drug.Further, we performed laboratory-developed PD-L1 tests using the E1L3N clone. The current companion/complimentary PD-L1 IHC assay was developed using a specific clinical program covering the staining systems and scoring/interpretation guidelines for a specific drug, and validated using the patient's clinical outcome. However, several LDT antibodies and IHC protocols have been validated using tissue samples. Herein, we compared the staining performance of these LDT antibodies with that of commercial assays. As a result, we found that LDTs are not inferior to commercial assays and can be used interchangeably when using the appropriate cut-offs. Thus, following strict validation of the analysis and clinical response, LDTs may be used as a screening method to validate the results of commercial assays.PD-L1 staining concordance between biopsy and resected specimens is important. In a recent study, significant discordance of PD-L1 expression using the E1L3N and SP142 clones between the TMA core and the corresponding WTS in 49 NSCLC cases was observed . SimilarTo our knowledge, this is the first comparative study of PD-L1 IHC assays, including LDT assays, in a NSCLC resection cohort. However, the lack of clinical response data for anti–PD-1/PD-L1 therapy is an important limitation of our analysis. Because the ultimate goal of determining PD-L1 IHC status is to predict the therapeutic response, the debate regarding the use of PD-L1 IHC assays may be settled using relevant clinical trial data for the drug being considered. Moreover, the threshold of the drug, rather than the assay, is a key parameter. Nevertheless, further studies are needed to determine the most appropriate cut-off and compare the sensitivity of the assays according to the therapeutic response.In conclusion, despite the different PD-L1 assay staining results, the PD-L1 status observed with assay-specific cut-offs was similar, suggesting that appropriate clinically defined algorithms or cut-offs should be applied to each assay. Furthermore, to consider LDTs as equivalent to the commercially available companion/complementary assays, rigorous validation is required. Hence, it is important to standardize the PD-L1 assays and enhance communications between clinicians, pathologists, and providers to establish both national and international guidelines for PD-L1 testing.We retrospectively collected formalin-fixed paraffin-embedded whole-tissue sections (WTS) from 97 resected NSCLC cases at Seoul National University Bundang Hospital. Fifty NSCLC WTS (32 adenocarcinomas and 18 squamous cell carcinomas) resected from 2010 to 2011 were assigned as the ‘training set’, and 47 NSCLC WTS  resected from 2015 to 2016 were assigned as the ‘validation set’. For the training set samples, a TMA was constructed using 2-mm-diameter cores derived from the representative tumor areas of formalin-fixed paraffin-embedded tissue blocks by SuperBioChips Laboratories , as previously described [Ninety-seven WTS slides were stained using the Dako  and Ventana  platforms and their PD-L1 IHC assays. The Dako pharmDx assay was stained with an anti-PD-L1 22C3 mouse monoclonal primary antibody with the EnVision FLEX visualization system on a Dako Autostainer Link 48 system, along with negative control reagents and cell line run controls, as per the manufacturer's instructions . For theTwo pathologists (HK and JHC) scored the 388 WTS IHC slides (97 cases × 4 assays) and 50 TMA IHC cores independently, and recorded 438 raw percentages of tumor cells and immune cells expressing PD-L1.PD-L1 expression was defined in tumor cells if membranous alone or membranous and cytoplasmic staining was present , 6, 7, 9We evaluated the PD-L1 expression using a scoring system with various cut-offs , including assay-specific cut-offs for the 22C3, SP263, and SP142 assays. The TPS was a common element of PD-L1 scoring in all assays. For 1 assay (SP142), the area of PD-L1-positive immune cells was integrated into the scoring system .Statistical analysis was conducted using SPSS , with Cohen's κ coefficient of agreement performed for comparing the dichotomized expression values between 2 assays.