To assess the association between PD-L1 expression and driver gene mutations in patients with non-small-cell lung cancer (NSCLC).We performed a meta-analysis of 26 studies (7541 patients) which were published from 2015 to 2017. Pooled odds ratios (ORs) with 95% confidence intervals (CI) were calculated to describe the correlation. Subgroup analysis was performed based on population characteristics, types of PD-L1 antibodies and quality of individual studies.p = 0.014). A negative correlation was also found at 1%  and 50%  cutoff for PD-L1 positive, elderly age group , female dominant group  and smoker dominant group . No significant differences in PD-L1 expression were observed among patients with different ALK, BRAF, HER2, PIK3CA status and MET expression level. Higher level of PD-L1 was found in tumors with KRAS mutation . PD-L1 expression level was not significantly different between triple (EGFR/ALK/KRAS) wild type NSCLCs and those with EGFR/ALK/KRAS mutation.A lower frequency of PD-L1 positivity was observed in NSCLCs harboring EGFR mutation (OR: 0.64, 95% CI, 0.45–0.91, PD-L1 expression in EGFR mutated NSCLCs were lower than those in EGFR wild type NSCLCs, while tumors with KRAS mutation showed higher levels of PD-L1. Lung cancer is the most common cancer and the leading cause of cancer-related mortality around the world. In 2015, a total of 733,300 patients  were diagnosed with lung cancer in China [Programmed cell death 1 ligand 1 , which belongs to B7 family, is an inhibitory cell surface molecule. PD-L1 expressed on NSCLC cells is shown to inhibit T cell proliferation and activation by combining with programmed cell death 1 (PD-1) receptor. Blockade of PD-1/PD-L1 represents a novel target for immunotherapy . In a reALK rearrangements containing exon 20 coding for the tyrosine domain occur in 3.0% to 11.5% of patients with NSCLC [Driver gene alternations including epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and Kirsten rat sarcoma viral oncogene homolog (KRAS) have been verified in a large proportion of NSCLCs. For example, 45% of never smokers and 7% of patients with tobacco-associated lung cancer harbor EGFR mutations. At least 30% of East Asian patients with NSCLC have EGFR mutation as compared to <10% of Caucasian patients . ALK reath NSCLC . KRAS muth NSCLC .et al. reported an objective response rate (ORR) of 3.6% in EGFR mutation or ALK rearrangement cohort treated with pembrolizumab, while the ORR in EGFR/ALK wild type cohort was 23.3%. Similarly, in Checkmate 057 and Keynote-010, neither nivolumab nor pembrolizumab showed an obvious superiority over docetaxel with respect to overall survival (OS) in the EGFR mutation cohorts [Gainor  cohorts . In contin vitro [Results of preclinical studies indicate a positive correlation between EGFR, ALK and KRAS mutations and higher PD-L1 levels, although the underlying mechanisms are not clear. EGFR mutation may upregulate PD-L1 expression through PI3K, MAPK, STAT3 and NF-κB signaling pathway , 17. Simin vitro , 21. Howin vitro . This meThe MOOSE guidelines were followed for this meta-analysis of observational studies . Flow diQ = 93.67, I2= 75.4%, p = 0.000). Using a random effect model, we found a statistically significant negative correlation between PD-L1 expression and EGFR mutation in NSCLCs   were inconsistent  Figure . In orde) Figure . Furthert Figure  and 5B. Q = 6.13, I2= 0.0%, p = 0.804). Using a fixed effect model, no statistically significant difference of PD-L1 expression was found among NSCLCs with different ALK status    Figure  and 5D iQ = 13.50, I2= 0.0%, p = 0.566). Pooled analysis using a fixed effects model revealed a statistically significant positive correlation between PD-L1 expression and KRAS mutation in NSCLCs    Figure . Statist) Figure  and 5F.p = 0.475) .To further explore the difference in PD-L1 expression between NSCLCs with and without EGFR/ALK/KRAS mutation, we performed a comparison of specimens with at least 1 EGFR/ALK/KRAS mutation and those with triple wild type. Four studies with 840 clinical specimens were included. As compared to triple wild type NSCLCs, those with EGFR/ALK/KRAS mutation did not show any significant differences of PD-L1 expression level with random effect model  Figure . A high p = 0.913), HER2 , MET expression , and PIK3CA  status with PD-L1 expression  and lower PD-L1 expression in the tumor microenvironment (TME) .et al. and Zhang et al. obviously influenced the pooled OR. However, pooled ORs were not statistically significant even after elimination of these 2 studies from the analysis.At cellular level, PD-L1 expression appeared to be associated with strong activation of driver gene (EGFR/ALK) pathways. Up-regulation of PD-L1 level was observed in cell lines with EGFR mutation or ALK rearrangement; further, treatment of EGFR/ALK-TKIs also induced down-regulation of PD-L1 expression . HoweverPrior to introduction of PD1/PD-L1 blockade agents, therapeutic options for NSCLCs with KRAS mutation were extremely limited because the molecular diversity of KRAS-mutant tumors is much greater than that of tumors initiated by other driver genes . The molThis study has a number of limitations. First, whether IHC is an ideal method to assess PD-L1 expression level is debatable. Partial PD 1/PD-L1 blocking response was seen in patient samples who were judged as “PD-L1 negative” by IHC , 29. SecIn conclusion, we found that PD-L1 expression levels in NSCLCs with EGFR mutation were lower than those with EGFR wild type. As compared to the KRAS wild type tumors, those with KRAS mutation tend to express higher levels of PD-L1. ALK, BRAF, HER2, PIK3CA and MET expression status were not significantly associated with PD-L1 expression. As compared to triple negative NSCLCs, driver gene (EGFR/ALK/KRAS) mutations did not alter the level of PD-L1.A search for relevant studies was conducted on PubMed, Web of Science and Embase databases. Studies published up to April 26, 2017 were eligible for inclusion. The combinations of key words used are listed in Table Eligible studies were required to meet the following criteria to be included: 1) Patients were diagnosed with NSCLC by histopathological methods; 2) Expression of PD-L1 in tumor tissues was evaluated by immunohistochemistry and the specific cutoff for PD-L1 positive expression was reported; 3) At least one driver gene was involved in the study; 4) Distribution of driver gene in participants was reported; 5) Sufficient data to form an 2 × 2 table was reported: number of PD-L1 positive specimens with or without driver genes mutation, and number of PD-L1 negative specimens with or without driver genes mutation.Exclusion criteria were as follows: 1) Studies not related to NSCLC; 2) Studies on patients with metastatic lung cancer; 3) PD-L1 expression was not evaluated in tumor tissues; 4) Other methods were used for evaluation of expression of PD-L1 such as ELISA, gene copy number or RNA expression level; 5) Studies that included patients <18 years of age or those that included patients with more than one cancer; 6) Non-English publication; 7) Overlapping studies; 8) Editorials, letters, reviews, case reports, duplicate publications, and expert opinions.In order to assess the quality of each included study, three investigators  scored the individual studies according to the Strengthening the Reporting of Observational studies in Epidemiology-Molecular Epidemiology (STROBE-ME) statement .supplem, qualityData from the included studies was extracted independently by two investigators (Bo Lan and Chengxi Ma). A third investigator (Chengyan Zhang) was responsible for resolving any disagreement between the first two investigators. The following information were collected: last name of first author, year of publication, ethnicity, median age, gender, stage, smoking status, histological type, primary PD-L1 antibody used for IHC assessment, cutoff level for PD-L1 positive, status of driver genes and PD-L1 expression . Pooled odds ratios (OR) of PD-L1 positive with 95% CI for driver gene status were calculated by fixed or random effect model and forest plots generated. ogeneity . A rando


HPV L1 protein is a corner stone in HPV structure, it's involved in the formation of the viral capsid; widely used as a systematicmaterial and considered as the main component in vaccines development and production. The present study aims to characterizegenetic variation of L1 gene of HPV 16 specimens and to evaluate in silico the impact of major variants on the epitope changeaffecting its conformational structure. A fragment of L1 gene from 35 HPV 16 confirmed specimens were amplified by PCR andsequenced. Overall, five amino acids residues changes were reported: T390P in 16 specimens, M425I and M431I in 2 cases, insertionof Serine at 460 and aspartic acid deletion at position 477 in all analyzed cases. The 3D generated model showed that T389P amino acidsubstitution is located in the H-I loop; the two substitutions M424I and M430I are both located in the H2 helice. The Serine insertionand aspartic acid deletion are located in the H4 helice and B-C loop, respectively. Superimposition of sequences' structures showedthat they share a very similar conformation highlighting that the reported amino acids variations don't affect the structure of the L1protein. However T389P, located in the H-I loop identified as an immunogenetic region of L1 capsid, was reported in 51.4% of casescould interact with vaccines induced monoclonal antibodies suggesting a potential impact on the efficacy of available anti-HPVvaccines. Worldwide, Cervical cancer is the fourth most common cancerin women, with an estimated 528,000 new cases in 2012, andmore than 85% of the global burden occurs in developingcountries, where it accounts for 13% of all female cancer . The intOn the surface of the pentamers, specific loops structures of theL1 protein contain type specific epitopes  and the Previous studies have reported that variations in L1 gene canaffect the viral assembly affecting the protein structure orconformation and leading to altered biological functions withclinical significance, including the immunological recognitionby the host ,22. On tDNAs from 35HPV16 positive cervical cancer samples wereavailable from our laboratory DNA bank ,18. OverA fragment of 450 bp of L1 gene was amplified usingMY09/MY11 consensus primers . PCR amplification wasperformed in a 25 μl volume containing 1,5mM MgCl2, 100 μMeach dNTP, 0,2 μM forward and reverse primers, 100 nggenomic DNA and 0,25 U gold Taq DNA polymerase  in 1x PCR buffer. The amplification mixtureswere first denatured at 94°C for 7 min. Then, thirty-five cyclesof PCR were performed with denaturation at 94°C for 1 min,primer annealing for 1 min at 55°C and primer extension for 1min at 72°C. At the end of the last cycle, the mixtures wereincubated at 72°C for 7 min. For every reaction, a positivecontrol, using DNA extracted from SiHa, an HPV16 positivecell line, and a negative control, without template DNA, wasincluded. PCR products were tested on an ethidium bromidestained 2% agarose gel.The ExoSaP ITR clean up system  was used to purifypositive PCR products. Sequencing of purified PCR productswas performed with BigDye Terminator v3.1 Cycle SequencingKit (Applied Biosystems). Sequencing reaction was performedin a final volume of 10μl containing 1μl of Big Dye v.3.1, 10pmol of forward primer and 2μl of purified PCR product. Themixture was incubated at 96°C for 1min and 25 cycles wereperformed: denaturation at 96°C for 10s, primer annealing at50°C for 5s and extension at 60°C for 4 min. The reaction wasset to 30μl. To eliminate the excess of labeled ddNTPs,sequencing reaction products were purified using sephadex G-50 gel-exclusion chromatography . Direct sequencing of amplified PCR products wasperformed on an ABI 3130xL Genetic Analyzer (AppliedBiosystems).The compiled nucleotide sequences were aligned usingClustalW2 software [http://www.ebi.ac.uk/clustalw/] and theprotein sequences were aligned using MUSCLE stands(MUltiple Sequence Comparison by Log-Expectation) .SeqLogoThe alignment was carried out using MUSCL and ClustalX2.Phylogenetic analysis was performed with the partial L1sequences, which were constructed by the Maximumlikelihood method and the Kimura 2-Parameter model byMEGA6, the Bootsrap proportions were calculated with 1000replicates. The phylogenic tree has been evaluated byShimodaira-Hasegawa-test and K-test. The thirty-five partialsequences of HPV16 variants were aligned with the prototype(GenBank: K02718.1) and other variants, As, Af-1, AA (GenBank: AF402678), and AA1 (GenBank: HQ644247) and E (GenBenk:NC_001526.2).The secondary structure of HPV16 L1 protein sequences waspredicted using Swiss Model Server(http://swissmodel.expasy.org). Predicted 3D structures ofHPV16 L1 were obtained using PHYRE2 Server , thealiResults from multiple sequence alignment of the 35 sampleswith the L1 reference sequence (ID: K02718.1) are reported inSequence analysis of a partial L1HPV nucleotide sequences'alignment has shown that 37.1% of the HPV16 variants identifiedwere in the African 1, European and Asia lineage and 51.5% HPV16 variant were in the African 2 lineage, while this tree shownone group of 11.4% variant which represent a low degree ofevolutionary change compared with other variants (X lineage).The thirty-five sequences were aligned using the MUltipleSequence Comparison by Log- Expectation software (MUSCLE).SeqLogo uses this alignment to generate conserved sequences,the sequence conservation is indicated by the height of residuelogos (bits). Overall, all sequences are presented with highresidue logos level which means that all our sequences areconserved, except in two regions, H-I loop and B-C loop. In thefirst region the proline and threonine residues at position 390, arepresented in equal proportion in our samples while in the secondregion all the specimens are characterized by the absence ofaspartic acid at position 477. In Helice 2 region, the twomutations Met/Ile at position 425 and Met/Ile at position 431 arenot significant as compared to the reference sequence. However,the insertion of serine at posThe crystal structure of the HPV16 L1 protein was obtainedfrom the RCSB Protein Data Bank (PDB id: 1dzl) and was usedas a template to model the 4 HPV16 L1 patterns, including thereference sequence of L1 HPV protein (K02718.1). The qualityof generated prototype model was evaluated usingPROCHECK statistics and the obtained results are reported inthe Ramachandran plot. Overall, 276 amino acids (AA) (71.2%)are located in the "Most favored regions" , 103 AA(26.5%) are located in the "Additional allowed regions" , 9 AA (2.3%) are located in "Generously allowed regions" and no AA (0.0%) is located in the "Disallowedregions".The 3D L1 protein generated by PyMOL, representing anindividual L1 protein molecule resulted from the expression ofHPV16 L1-ORF, is reported in The 3D carton structure of the L1 prototype and the threeobtained structures are illustrated in HPV16 and 18 L1 virus-like particles (VLP) are largely employedto develop prophylactic vaccines for the preventionof HPV infection. Currently, two prophylactic vaccines, Cervarix(GSK) and Gardasil (Merck), have been widely implemented inmany countries. The efficacy of these vaccines, targeting highriskHPV-16 and -18 alone, or additional HPV types, is stilldiscussed, depending on the VLPs used to produce the vaccineand the HPV variants present in the targeted populations.Indeed, it's widely accepted that HPV variants have been shownto differ in geographic origins affecting the lesion progressionand the vaccine protection ,35,36.In Morocco, prophylactic HPV vaccines were introduced in 2012and are still with limited utilization. Thus, it's of a great interestto assess HPV16 and 18 L1 genetic diversities, at the introductionof vaccination, as a baseline, for a potential follow-up of thevaccination efficacy in the future. Previous studies conducted inMorocco have highlighted a high genetic diversity of HPV16 andthe presence of both European (E), African (Af) and North-American (NA-1) phylogenetic clusters ,18. HoweOverall, 12 silent mutations were found in our specimens andmost of them were already reported in many studies fromIndia  NederlanPartial sequence of L1 gene was used to have a phylogeneticanalysis of HPV16 circulating in Morocco. Two differentgroups belonging to the African 1 and 2 lineages prevail, with37.1% and 51.5% respectively. These results are in agreementwith our previously reported data on the same isolates usingE6 and E7 genes. Indeed, DNA sequencing of E6 and E7 geneshighlighted that the predominance of HPV16 African variantsand the majority of isolates belong to the African 1 and 2lineages .et al. have shown that in human immune response, antibodies bind to regions DE, EF, FGand H-I loop, making these regions of a high interest for HPVVLP recognition [The computer modeling of L1 protein was generated for eachpattern to assess the impact on the obtained non-synonymousmutations on the structure of the major capsid protein andtherefore its potential interaction to available vaccines. In theL1 protein, the external loop regions: DE loop (AA 110-153),FG loop (AA 262-291), EF loop (AA 160-189), DC loop (AA 50-69) and the H-I loop AA 348-396) have been characterized asbeing antigenic [6 have beognition . Moreoveognition .Of particular interest, fine-mapping of the epitope footprintshowed that the five non-synonymous changes of amino acidsresidues, including the ATC insertion and GAT deletion, arelocalized in the H2 and H4 helices and H-I and B-C loopregions T389P amino acid substitution is located in the H-1 loop,identified as an immunogenetic region of L1 capsid and believedto contribute towards cross-neutralising antibody . M424IaThere's evidence that among the 5 non-synonymous mutationsobtained in this study, 4 don't affect the immunogenic site of L1and therefore don't interfere with the binding of monoclonalantibodies targeting HPV 16 and only the T389P amino acidsubstitution present in 51.4% of cases was associated withpotential interaction to monoclonal antibodies induced vaccines.On the other hand, generated 3D carton structure of HPV16 L1homology models, harboring the 5 non-synonymous mutations,clearly showed that the L1 structure rests unchanged suggestingthat these mutations don't affect the overall structure of L1protein.The present study is very informative and give for the first timedata on genetic diversity on HPV16 L1 gene in a Moroccanpopulation and adress a prediction of potential reaction toavailable anti-HPV vaccines. However, the main limitation wasthe small number of HPV 16 DNA, which may not reflect the realsituation in Morocco and give an exhaustive picture on L1 genediversity and the mutational status highlighting mutations thatcould modify the L1 protein structure and consequently affectthe neutralization epitopes.The present study gives evidence on the genetic diversity ofHPV 16 L1 gene without any impact on the structure of the L1protein. The study highlights the presence of T390P mutation,located in the H-I loop and could interact with vaccinesinduced monoclonal antibodies suggesting a potential impactof this mutation on the efficacy of available anti-HPV vaccines.Other studies are needed on large samples and by sequencingthe entire HPV 16 L1 gene to predict the efficacy of HPV16 targeted vaccines and the success of the vaccination strategy inMorocco.


Helicobacter pylori (Hp) and Epstein-Barr virus (EBV) infection status in a GC cohort (n=340), then analyzed the relationship between the expression of PD-1, PD-L1/L2 and GC prognosis. We found that PD-1, PD-L1, and PD-L2 mRNA levels were up-regulated in GC tissues, and were positively correlated with one another . PD-1 protein expression in TIICs was observed in 22.6% of GC patients. The PD-L1 and PD-L2 positivity rates were 40.3% and 53.8% in TCs, respectively, and 60.0% and 60.9% in TIICs, respectively. PD-L1 was up-regulated in EBV-infected GC patients in both TCs (P=0.009) and TIICs (P=0.003). Hp status was not associated with PD-1 or PD-L1/PD-L2 expression. In TIICs, PD-L1 expression was independently associated with better GC prognosis . Co-expression of PD-1 and PD-L1, but not PD-L2, was a favorable prognostic marker that indicated a dose effect on the mortality risk of GC patients . Comprehensive evaluation of PD-1 and PD-L1 in TCs and TIICs could help predict the prognosis of gastric cancers, as well as reveal patients who might benefit from targeted treatment.While the prognosis of gastric cancer (GC) remains poor, PD-1 and PD-L1/L2 are promising prognostic biomarkers. We evaluated PD-1 and PD-L1/L2 expression in tumor cells (TCs) and tumor-infiltrating immune cells (TIICs). We determined the  Gastric cancer (GC) is a major cancer-related threat to global health , 2. In 2PD-1 and its ligand PD-L1/PD-L2 are a group of negative co-stimulatory molecules that can suppress T cell proliferation in carcinoma –8. The cThe stroma of gastric cancer is infiltrated with numerous T lymphocytes . This imHelicobacter pylori  and Epstein-Barr virus (EBV) infections are the most common infection-related risk factors of gastric cancer [H. pylori infection is positively associated with early inflammatory, precancerous lesions of GC [H. pylori, EBV can stimulate the oncogenic process of GC by promoting chronic inflammation and increased tissue damage [Hp and EBV infections promote the formation of the tumor immune microenvironment [Infection promotes the susceptibility to, and development of, gastric cancer. c cancer , 18. H. ns of GC . Similare damage . Hp and ironment , 22.H. pylori-infected GC and EBV-positive GC may have different states of immunity compared with other types of GC and may lead to an increased PD-1 and PD-L1/L2 expression. In a Western cohort, abundant PD-L1 expression was found in EBV-infected gastric cancers. In a large Asian cohort, PD-1 and PD-L1/L2 expression could not be definitively correlated with H. pylori-infected or EBV-positive GC.A comprehensive molecular classification of gastric adenocarcinomas was reported by The Cancer Genome Atlas (TCGA), and the EBV-positive GC (EBVaGC) accounted for 5.0-17.9% of gastric cancers , 24. EBVHelicobacter pylori (Hp) and Epstein-Barr virus (EBV) infection status in a large GC cohort, then analyzed the relationship between the expression of PD-1, PD-L1/L2 and GC prognosis.In the current study, we evaluated PD-1 and PD-L1/L2 expression in tumor cells (TCs) and tumor-infiltrating immune cells (TIICs). We determined the In a total of 357 cases, 6 patients died of postoperative complications within 30 days, and 11 patients were lost to follow-up at the first time point. These 17 patients were excluded from our study, and the remaining 340 gastric cancer patients who underwent surgical resection were included in the final analysis. The median follow-up time was 48 months (ranging from 1 to 111 months). During follow-up, 169 (50.0%) patients died of GC, 2 (0.5%) patients died from other causes, 134 (39.0%) patients were still alive, and 35 (10.0%) patients were lost to follow-up  gastric cancer patients, especially in the lymph follicles Figure . PD-L1 aH. pylori infection status data were available for 102 patients, and Hp infection was found in 62 (60.8%) cases. There were no associations between the expression of PD-1, PD-L1, or PD-L2 and Hp status (Table P<0.01). EBV-infected GC patients also had greater PD-L1 expression in the TIICs (P<0.01). We found no association between the expression of PD-1 and PD-L2 with EBV status , and it was more prevalent in patients with smaller tumors (diameter<5 cm) than in those with larger tumors (P=0.024) (Table P<0.05). PD-L1 expression in TIICs was positively correlated with males, tumor type of tubular adenocarcinoma, larger tumor size, tumor with neural invasion, and lower TNM stages (P<0.05)  than in the older patients (4) Table . In TCs,5 than in5) Table .P=0.027). Patients without lymph metastasis or any distant metastasis lived longer than patients with lymph metastasis or any distant metastasis (P<0.001 and P=0.003). Overall, patients with TNM stages I-II had better survival than those with TNM stages III-IV (P<0.001).Tumor size ≥5 cm, positive vascular invasion, and positive neural invasion were associated with poor GC prognosis . PatientP=0.024). The overexpression of PD-L1 in the TIICs appeared to be associated with better overall survival, although it did not reach statistical significance (log-rank P=0.063). PD-L2 expression in TCs and TIICs was not associated with overall survival (log-rank P=0.919 and P=0.452). Tumors were defined as overall positive for PD-L1 if PD-L1 was expressed in either TCs or TIICs. The same rule was applied to PD-L2. Patients with PD-1/PD-L1 co-expression had better prognosis (log-rank P=0.019). Patients with PD-1/PD-L2 co-expression also had better overall survival, although this did not reach statistical significance (log-rank P=0.065) . PD-L1 expression in TIICs was independently associated with better GC prognosis . The co-expression of PD-1 and PD-L1 was independently associated with better prognosis of GC, and indicated a dose-effect on the mortality risk of cancer patients . Compared to the group with no PD-1/PD-L1 expression, the HR of the group with either PD-1 or PD-L1 expression was 0.70 . In the PD-1/PD-L1 co-expression group, the HR was 0.48 . PD-L2 was not independently associated with GC prognosis, in either TCs or TIICs (P>0.05). The presence of at least one of the PD-1/PD-L1 or PD-1/PD-L2 combinations was positively connected and almost correlated with better survival, but without statistical significance . Additionally, a higher TNM stage was always independently associated with worse prognosis of GC .Multivariate Cox regression showed that PD-1 expression in TIICs seemed to be correlated with better survival, but this association did not reach statistical significance  GC samples  GC samples . PD-L2 expression also increased as PD-L1 expression increased  and positively correlated with one other  and ‘EBV negative’ (14 cases) groups to determine s Figure . PD-L1 as Figure  and 4C, d Figure  to 4F. Tne other .PD-L1 mRNA levels did not display significant differences between the EBV-positive and EBV-negative patients (P=0.582). To better understand this, we next analyzed the correlation between PD-L1 mRNA and protein levels. We found that PD-L1 mRNA expression in the PD-L1 protein-positive group was not different from that in the PD-L1 protein-negative group  patients , 26. ConPD-L1 mRNA is always done simultaneously on TCs and TIICs. However, the expression of PD-L1 protein in the TCs and TIICs are evaluated separately and combined as simply positive expression or negative expression. Another possible reason could be that there is some post-transcriptional regulation taking place. Similar to the mRNA expression, biomarkers that expressed in tumor cells or immune cells could not be distinguished by western blot. Western blot also presented a trend that aligned with the IHC results, especially in the patients with positive expression of PD-L1 both in TCs and TIICs  and CagA protein in H. pylori-infected gastric cancer cell lines [H. pylori positive and H. pylori negative GC. Using the data from the TCGA, we found that there was no association between the level of PD-L1 mRNA and H. pylori-infected gastric cancer  from 2007 to 2014. A total of 357 patients who were newly diagnosed with gastric cancer and had undergone a physical tumorectomy for adenocarcinoma of the stomach qualified for the study. Before surgery, none of the patients had received chemotherapy, radiotherapy, or anti-PD-1/PD-L1 treatment. Two independent pathologists confirmed each patient's diagnosis. The principal clinical characteristics included gender, age, WHO classification of the primary tumor, tumor sizes, TNM stage according to AJCC/UICC, 2010 classifications, and chemotherapy status.All patients underwent follow-up after the tumorectomy in the third month, sixth month, and every year until death or the last scheduled follow-up. The duration from the date of surgery to the date of death or the last successful interview date was defined as the survival time. Patients who died due to complications of the surgical procedure during the perioperative period or were lost at the first time of interview were excluded in the survival analysis. Before enrollment, all participants signed informed consent forms. All of the analyses in our study were performed after surgery. Our study was approved by the Institutional Review Board of the First Hospital of Jilin University.Helicobacter pylori IgG enzyme-linked immunosorbent assay (ELISA) test  was used to diagnose H. pylori infection in the patients’ sera. A value of 30 EIU or more indicated H. pylori infection. The tumors were labelled by DNA in situ hybridization  for EBV infection using tissue microarrays . Nuclei stained brown indicated EBV infection.The 2O2. The sections were pre-treated with 10% normal goat serum  before the primary antibodies were applied for 90 min at room temperature. The sections were further incubated with horseradish peroxidase-labeled secondary antibody  for 15 min at room temperature. The signals were visualized with 3, 3-diaminobenzidine (DAB), and then the slides were counterstained with Mayer's hematoxylin.Histological sections (4 μm) of 10% formalin-fixed paraffin-embedded tumor specimens of GC patients were used for immunohistochemical (IHC) staining. We have tested different dilutions of antibodies against PD-1, PD-L1/L2 in patients during the preliminary experiment and the optimal IHC dilutions of each antibody had been well determined. From the instructions and related references about the antibodies above, there was no evidence that the antibodies had a neutralizing effect. Finally, the sections were stained with primary monoclonal antibodies against PD-1 , PD-L1 , and PD-L2 . After routine dewaxing, the slides were boiled for 2min (20 min for PD-L1) in a pressure cooker in citrate buffer (ethylenediamine tetraaceticacid for PD-L1 without high pressure) for antigen retrieval. Endogenous peroxidase activity was blocked using 3% HThe PD-1 staining was assessed in TIICs by two experienced pathologists blinded to the clinical data. A proportion of stained cells ≥5% in TIICs with a membranous staining was considered PD-1 positive. For PD-L1 and PD-L2, expression was predominantly observed in the cell membrane and cytoplasm. The IRS system was used to assess the staining intensity and percentages of the tumor cells. The intensity and expression prevalence was subdivided into four categories each: 0 , 1 , 2 , or 3 , and the percentages were subdivided into four grades: 0 (<5% expression), 1 (5 to 19% expression), 2 (20 to 49% expression) and 3 (≥50% expression). Adding the intensity and percentage scores resulted in IRS values ranging from 0 to 6. A total score of more than 2 was defined as positive expression of PD-L1 or PD-L2 in TCs. For the evaluation of the expression of PD-L1 and PD-L2 in TIICs, the cases were simply scored as negative (<5% expression) and positive (≥5% expression).glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous reference gene. The expression of PD-1, PD-L1, and PD-L2 was quantified using specific primers (Qiagen), and the mRNA levels of these three genes were analyzed with the 2−ΔΔCt method.Among the 340 GC patients for whom IHC staining was conducted, total mRNA was extracted from 21 patients’ tumor tissues and paired adjacent non-tumorous tissues (located > 3 cm away from the tumor and confirmed by the pathologists with H&E staining). After reverse transcription, cDNAs were amplified in the presence of primers, and The total protein of gastric tumor tissues and paired adjacent non-tumorous tissues was extracted using a mammalian protein extraction kit , and the concentration of various proteins was measured using a BCA kit . The levels of PD-1 , PD-L1 , PD-L2 , and GAPDH  were measured with ECL reagents (Thermo Fisher Scientific) using Molecular Imager Chemi Dox XRS+ imaging system .2 test. Continuous variables with normal distribution were represented as the mean ± standard deviation, and compared by Student's t-test. Continuous variables with non-normal distribution were represented by the median (Q1-Q3), and compared using Wilcoxon's rank-sum test. The log-rank test was used to compare Kaplan-Meier survival curves. Univariate and multivariate Cox regressions were performed to assess the hazard ratios (HRs) and 95% CIs of the possible prognostic factors. The correlation between PD-1, PD-L1, and PD-L2 mRNA levels were calculated with Spearman's rank correlation. P<0.05 was considered to be statistically significant. All of the analyses were conducted with the SPSS program  or GraphPad Prism 5.0.Categorical variables were represented as frequency (percentage) and compared using the χPD-1, PD-L1, and PD-L2 mRNA levels were up-regulated in GC tissues and positively correlated with one another. Co-expression of PD-1 and PD-L1, but not PD-L2, was a favorable prognostic marker in gastric cancer. The comprehensive evaluation of tumor cells and tumor-infiltrating immune cells could help in predicting the prognosis of gastric cancers and selecting patients who might benefit from targeted treatment.PD-L1 was over-expressed in EBV-infected GC. The


Sarcoidosis is a systemic inflammatory disease of unknown etiology. Osteopontin  is an extra cellular matrix glycoprotein and cytokine with a known role in granuloma formation and in autoimmune and inflammatory diseases.To determine whether plasma OPN levels are elevated in patients with sarcoidosis and compare the frequency of four single nucleotide polymorphism (SNPs) variants in the OPN gene in sarcoidosis patients compared to healthy controls.Demographic and clinical information, radiological studies and pulmonary function tests were evaluated in 113 patients with sarcoidosis and in 79 healthy controls. Blood samples were analyzed for SNPs of the OPN gene and for plasma OPN and CRP levels. Association between clinical features of disease and OPN levels as well as SNP frequencies was determined.Plasma OPN levels were higher in sarcoidosis patients than in healthy subjects, . Area under the curve for receiver operator curves (ROC) was 0.798 (0.686–0.909 95% CI.) No differences were observed between sarcoidosis patients and controls in the frequency of any of the SNPs evaluated. Presence of lung parenchymal involvement was associated with SNP distribution at rs1126772 (p = 0.02). We found no correlation between SNPs distribution and plasma OPN levels.Osteopontin protein levels are elevated in sarcoidosis. We found no evidence for an association between SNPs on the osteopontin gene and plasma OPN levels or the presence of sarcoidosis, however, an association between genotype and several phenotypic clinical parameters of disease was observed. Sarcoidosis is a systemic inflammatory disease of unknown etiology, characterised by non-caseating granuloma formation in various organs, with several recognized genetic and environmental risk factors. The prevalence of sarcoidosis varies from 4.7–64 per 100,000, with an estimated annual incidence of 1.0–35.5 in 100,000 , with a Insertion/Deletion polymorphism has been tested for association with the risk of sarcoidosis. Published results are unequivocal, however, it was found that genotyping for this I/D polymorphism improves the diagnostic value of serum ACE levels measurements [Genetic susceptibility to sarcoidosis has been found to be independently related to both HLA Class I and HLA Class II groups such as HLA-DRB1 , HLA-DR5urements ,12.HSP70 can be used to discriminate it from idiopathic uveitis [Certain phenotypes in sarcoidosis have a genetic component. Early-onset sarcoidosis, together with familial Blau syndrome is associated with mutations of Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), also known as caspase recruitment domain-containing protein 15 (CARD15), that cause constitutive NF-kappa-B activation. NOD2 mutations are related to Crohn's disease as well . Sarcoid uveitis . Several uveitis .Osteopontin (OPN), also known as secreted phosphoprotein 1 (SPP1) and early T lymphocyte activation 1 (ETA1), is a secreted phosphoprotein, part of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family . It was The OPN gene resides on the long arm of chromosome 4. Crosby et al  showed tPatients: Patients were recruited from two large outpatient pulmonary clinics in Jerusalem, Israel between January 2011 and September 2015. All participants signed an Informed Consent Form and the study was approved by the Hadassah Medical Center Ethics Committee. Charts of patients with biopsy findings compatible with sarcoidosis as well as a compatible clinical picture as determined by symptoms, laboratory abnormalities and/or imaging and without another identified cause of granulomatous disease were included in the cohort.Demographic and clinical information including radiological and pulmonary function tests was gathered from patient questionnaires, hospital medical records, and outpatient medical records obtained from treating physicians’ records. All data and radiological studies were reevaluated by the pulmonary physicians and radiologists at the Hadassah Medical Center. Assessment of disease extent, severity, duration of symptoms prior to diagnosis, and extent and nature of organ involvement in the disease was evaluated. For this study, we used lung function tests (LFT) that were performed prior to and as close to blood drawing as was available.Controls: A cohort of healthy subjects with no significant medical background was recruited as a control group.A single sample of 15ml venous blood into commercially available EDTA-treated tubes for DNA analysis and plasma analysis of osteopontin and CRP levels was obtained from patients and control subjects.Plasma was separated from the blood samples and frozen at (-20)°C. OPN and CRP levels in the plasma were measured using the commercially available kits . The detection range for the OPN ELISA is 62.5pg-4000pg/ml, the detection range for CRP ELISA is 15.6-1000pg/ml. Normal range for CRP 1–3 mg/L. The samples were diluted when needed.Genomic DNA was extracted from anticoagulated whole blood collected in EDTA from patients and controls. Isolation of DNA was done by phenol chloroform extraction  or using® Predesigned SNP Genotyping Assays containing sequence-specific forward and reverse primers, and two MGB probes: VIC®-labeled probe and FAM™-labeled probe, together with TaqMan® Genotyping Master Mix according to protocol provided by the manufacturer (Applied Biosystems®). SNP genotyping was performed using StepOnePlus™ Real-Time PCR System. The results were obtained by TaqMan® Genotyper™ Software [Four different single nucleotide polymorphisms in the OPN gene were evaluated: rs4754, rs1126616, rs9138 and rs1126772 using commercially available primer/probe pairs: TaqManSoftware .Data was analyzed using SPSS version 20.0 Software . P-value of 0.05 was considered statistically significant.A total of 113 sarcoidosis patients and 79 control subjects were recruited for this study between January 2011 and September 2015. Tables Protein levels of OPN and CRP in plasma of sarcoidosis patients and controls are presented in We evaluated possible associations between plasma OPN levels and clinical and epidemiological parameters of disease in sarcoidosis patients. OPN levels were significantly elevated in smoking patients (p = 0.049), median levels of 247ng/ml in present and past smokers vs. 179 ng/ml in never-smokers. OPN levels were significantly decreased in patients whose presenting symptoms were rash or skin involvement .Serum OPN may serve as a diagnostic test for sarcoidosis. Using ROC curves the optimal cut-off value to differentiate sarcoidosis from healthy controls is 180ng/ml which gives a specificity of 89.5% and sensitivity of 64% .We found no differences between sarcoidosis patients and controls in the frequency of the three variations in any of the four SNPs we examined , Table 3All individual data are available in In this study, we found elevated levels of plasma osteopontin (OPN) in patients with sarcoidosis when compared to healthy controls. We found no correlation between the presence of four single nucleotide polymorphisms (SNPs) tested and plasma OPN levels or presence of disease. However, some of the clinical parameters were found to correlate with genotype.OPN is expressed in T- cells as an early response molecule in bacterial infections. It interacts with macrophages and induces a type-1 cytokine inflammatory response, potentiating production of IL-12 and IFN- γ. OPN has also been previously linked to “Th1-diseases” such as multiple sclerosis , rheumatWe considered whether plasma osteopontin could be of use as a biomarker to diagnose sarcoidosis or as a marker of disease activity and of response to treatment. As regards diagnosis, receiver operator curves for OPN show a diagnostic value (area under the curve–AUC) of 0.798, which is similar or slightly better than existing markers, which are being using routinely. For example, angiotensin converting enzyme (ACE), which is known to be non-specific and increased in neoplastic and chronic infectious conditions, was found to have AUC of 0.779  and sIL-2R had an AUC value of 0.667  . ChitotrOur findings do not support the use of OPN for disease monitoring. We found no correlation between OPN and CRP levels in sarcoidosis patients, despite both being elevated relative to levels found in the control group. CRP is generally considered a marker of disease activity in inflammatory diseases and OPN clearly does not simply reflect the information provided by CRP. Further studies using serial measurements pre and post treatment may clarify whether there is any correlation between OPN and disease activity.We chose to study four of OPN's SNPs based on previously published studies that found association between these specific SNPs and different autoimmune and Th1 related diseases, such as Systemic Lupus erythematosus, type I Diabetes Mellitus and rheumatoid arthritis, or Behcet`s Disease and Vogt-Koyanagi-Harada Disease, which are also characterized by aberrant Th1 and Th17 response. The alleles distribution of rs9138, rs7687316, together with allelesrs1126616T and rs1126772A have all been associated with SLE ,35. A siAs regards studies in sarcoidosis, in the Slovenian population rs4754 was found to have different genotype frequency in sarcoidosis patients, with CC being overrepresented. This was found significant, but not after correction for multiple testing. Haplotype TT for rs11730582-C/T, rs11728697-C/T and rs4754-C/T were significantly decreased and therefore considered protective . In contThe failure to identify correlation between genotype and the presence of sarcoidosis in our cohort was surprising and somewhat disappointing. Although unlikely, this may be due to a type II error related to the size of our patient cohort. Another possible explanation is that we did not check for haplotypes, but for isolated SNPs, and the only correlation previously found between the genotype of OPN and the presence of sarcoidosis, after correction for multiple testing, was for the whole haplotype. The lack of correlation could also be reflecting perhaps another, non-genetic, epigenetic or other mechanism in which OPN contributes to Th1 related diseases, in a way similar to previous studies in Behçet's disease and type-1 diabetes. Lastly, correlation studies regarding SNP's are invariably population related and therefore may be different in our cohort of Israeli patients, especially bearing in mind that our patients have heterogeneous ethnic origins.Some clinical parameters were found to correlate with the frequency of SNP's we have examined, with the presence of constitutional symptoms showing the strongest correlation. These findings could be explained in numerous ways. They could be reflecting a subset of sarcoidosis with genetic predisposition which is affected by the OPN genotype and which manifests with a prominent systemic reaction rather than as disease limited to the chest. In patients presenting with fever or weight loss, all four SNPs examined were found to have the same distribution, suggesting a similar haplotype for this presentation.From our study it is not clear whether OPN is involved in the pathogenesis of sarcoidosis or merely a marker of the disease. If OPN is found to be relevant to the pathogenesis, it may serve as a target for future therapy. There is increasing interest in OPN and its ligands as a therapeutic target in multiple sclerosis , and in In conclusion, our study demonstrates that there is an association between the protein osteopontin and sarcoidosis. Plasma osteopontin may be a suitable marker for the presence of sarcoidosis, but control groups with other lung diseases need to be studied. Surprisingly, no genetic association was found between osteopontin and the presence of sarcoidosis, however, a genetic association was found with some clinical subtypes of disease.S1 Table(XLSX)Click here for additional data file.


This asymmetry in answers is in line with work on negativity bias in other areas of linguistics and psychology. Building on these findings, this study investigated whether question polarity also affects the answers to VAA statements. In a field experiment  during the Dutch municipal elections we analysed the effects of polarity for 16 out of 30 VAA statements with a large variety of linguistic contrasts. Analyses show a significant effect of question wording for questions containing a wide range of implicit negations , as well as for questions with explicit negations . These effects of question polarity are found especially for VAA users with lower levels of political sophistication. As these citizens are an important target group for Voting Advice Applications, this stresses the need for VAA builders to be sensitive to wording choices when designing VAAs. This study is the first to show such consistent wording effects not only for political attitude questions with implicit negations in VAAs, but also for political questions containing explicit negations.Online Voting Advice Applications (VAAs) are survey-like instruments that help citizens to shape their political preferences and compare them with those of political parties. Especially in multi-party democracies, their increasing popularity indicates that VAAs play an important role in opinion formation for citizens, as well as in the public debate prior to elections. Hence, the objectivity and transparency of VAAs are crucial. In the design of VAAs, many choices have to be made. Extant research in survey methodology shows that the seemingly arbitrary choice to word questions positively (e.g.,  VAAs are online tools that help citizens to shape their political opinions and to figure out which parties or candidates are most in line with their own individual position, in order to make a vote choice. After reacting to a range of statements, the user’s answers are compared to the positions of all the political parties in a certain election. This way, VAAs offer a concise way to obtain information about the issues and party positions in a particular election in a time-efficient and user-friendly way (e.g. ).The popularity of VAAs has increased over recent years and has spread from one country to the other. Currently, about 10% to 40% of the electorate in many European countries uses a VAA in the weeks prior to Election Day . BecauseTherefore, a wide range of scholars including political scientists, philosophers, and public opinion researchers are studying VAAs as a societal phenomenon and as an individual decision making tool: what does the design of VAAs reflect about our society’s views on democracy and public opinion formation ? At theallowed in the city centre‘ and ‘Cars should be banned from the city centre‘. Building on to this work in surveys , we also zoom into the effects of the use of explicit negations . Furthermore, we consider political sophistication as a moderator of these effects. The current study is the first to investigate the effect of question polarity of a wide range of contrasts on the answers in Voting Advice Applications. This gives us a sense of how subtle wording variation affects political attitudes, both through reading and answering the questions and of how this relates to levels of political sophistication in this context of motivated use.In the area of survey methodological research, extensive work has been done on the way question wording (unintendedly) affects the answers to attitude and opinion questions. As VAAs consist of survey-like opinion statements, this field of research is critical for VAA design choices. One wording characteristic that has considerable impact on the answers obtained in attitude surveys, is the positive versus implicit negative wording of political attitude questions or statements, such as ‘Cars should be s , ), allowepublic speeches against democracy’ should be ‘forbidden’, and the other half was asked whether they thought, ‘public speeches against democracy’ should be ‘allowed’. The results indicated that fewer respondents were willing to endorse the ‘forbidding’ of those speeches (54%) compared to the number of respondents who indicated ‘no’ to the ‘allow’-version of the question (75%). Hence, respondents seemed to hold more favourable attitudes towards the attitude object ‘public speeches against democracy’ when the question was worded with ‘forbid’ compared to when it was worded with the verb ‘allow’.The first study in which an effect of positive versus negative question wording choice was ‘discovered’, dates back to the 1940s when Rugg  conductespeeches against democracy to X-rated movies, from the use salt on slippery roads in the winter to smoking in public places . ). speechs , whereas , or eveforbid’ wording, i.e. to be more prone to answer ‘no’ to ‘forbid’-questions than ‘yes’ to ‘allow’-questions. This overall effect on the answers is often explained by the tone of wording of ‘forbid’ versus ‘allow’: “The verb ‘forbid’ sounds harsher and may therefore be more difficult to endorse, whereas ‘allow’ in some contexts might seem to encourage deviant behaviour and therefore may invite opposition” .forbid’ vs. ‘allow’: to some other contradictory word pairs such as ‘restrict’ vs. ‘leave free’ ). N = 31,112) answered various manipulated questions containing an implicit linguistic contrast (N = 6), or containing an explicit linguistic contrast (N = 10), each observation is nested within respondents and items at the same time. To account for this, the models distinguish between-item variance , and between-respondent variance –as well as a residue of error variance. Hence, cross-classified models are in operation . ). N = 31N = 21,881).As a second step, we examined to what extent wording effects for contrastive questions can be explained from variation in political sophistication within the population of VAA users. Using the continuous 5-point scale variable Political Sophistication, we added parameters to our two initial multi-level models to assess both the main effect of political sophistication and the interaction between political sophistication and question polarity. The annotation of this more complex model can also be found in Appendix 1. Please note that for this latter analysis only those respondents were included who answered both the question on political interest and on education . As can be seen in A comparison of the main effect of question polarity across questions and respondents revealed an overall effect of question polarity for the set of questions with an d is .13. Another way to classify the effect, is in terms of absolute scale points: 1 out of 20 users shows one scalepoint difference between positive and implicit negative questions.In terms of effect sizes, the mean differences are small compared to the between question variance: Cohen’s The effect varies strongly between questions: using the standard deviation between questions, we can see that the wording effect for 80% of the questions lies between -.59 and +.69 scale points, which means that it is also likely for a question with implicit negations not to affect the answers at all, or to show an effect in a direction opposite to expectations—even though the mean overall effect is in line with theoretical expectations.explicit negation. This analysis also showed an overall mean question wording effect for explicit negative questions . As shown in We ran a comparable model for the set of 10 questions with an d across questions is .05 when compared to the between question varianceOverall, the effect for explicit negatives is significant, but very small: Cohen’s N = 21,811). For our set of 6 implicit negative questions we find a significant interaction effect between question wording and sophistication : the higher the political sophistication of our VAA users, the smaller the difference in mean answers between positive and implicit negative questions . Again, the direction of this interaction effect is similar to expectations from the literature: the higher the political sophistication, the smaller the wording effect of explicit negations (see d being .2 for sophistication level 2).For the set of 10 questions with explicit negations, we also find a significant interaction between political sophistication and question wording  as wellabolish’ vs. ‘maintain’, ‘continue’ vs. ‘stop’). For explicit negations, this is the first study to show a general effect for this type of wording variation for two different types of explicit negations (‘not’ and ‘none’) in political attitude questions. Furthermore, we find these effects are moderated by political sophistication in way that was predicted by the literature. However, the variation between questions in the size and directions of the effects is large; and the interaction effect with political sophistication shows that the overall wording effect is mainly caused by VAA users with a lower political sophistication. The implications for theory and practice are addressed below.All in all, we find consistent effects confirming negativity bias: implicit as well as explicit negatives lead to an overall tendency to disagree, hence suggesting more positive evaluations of the attitude object in the question compared to when the positive equivalent question is asked. For implicit negatives, we find similar wording effects in this VAA context compared to findings reported on the forbid/allow asymmetry in attitude survey questions, this time for a wider range of implicit negative verbs  d) d25]) dpolluting cars should be banned’, the evaluative term ‘banned’ was expected to be too extreme to endorse, whereas it would be more easy to answer ‘no’ to the opposite positive question with the word ‘allow’. But if we zoom out to the content of the question, the attitude object ‘polluting cars’ is something that is generally evaluated negatively, and could hence give rise to an extreme response anyway. So, if linguistic negativity (‘banned’) is combined in one sentence with an attitude object that is conceptually negative, this may lead to subdued polarity effects. To complicate things further, some issues might instantly evoke positive evaluations with left wing voters, and negative evaluations with right wing voters , which Not only do we find quite some variation in the wording effects, we also observe effects in our VAA study that are somewhat smaller than the ones usually reported in the survey literature. These smaller effect sizes might be partly attributed to the five-points scales used in this VAA. Previous research in a survey context established smaller forbid/allow asymmetries when more fine-grained answering scales were offered compared to yes/no-scales .).21]).The smaller effects might also be attributed to the VAA context, as VAA users may generally be more motivated and probably show higher levels of political interest compared to respondents in a general political survey. A VAA differs from a regular opinion survey in that users visit the VAA spontaneously; they are motivated to fill it out and to think about their opinions more thoroughly than when filling out an attitude survey . This sPolitical sophistication is related to motivation. In our study, variation in users’ level of political sophistication was systematically related to the size or occurrence of wording effects. The higher the political sophistication, the smaller the overall wording effect—and the group of VAA users with the highest levels of political sophistication were not susceptible to the effects of question wording at all. This is an interesting result, in line with predictions about the role of attitude strength in survey methodology, as well as with more general theories on information processing and  opinion formation.Voting Advice Applications are not only widely used tools in modern democracies, but they are also a very interesting context with high external validity to study the effect of wording variation. A strong advantage of doing this type of research in the context of VAAs rather than in surveys, is that the context of VAAs automatically implies many checks on the validity of the question wording variation, as not only the researcher, but also the VAA builder(s) and the politicians have to agree that a certain wording variation is relevant and valid in the political context of these specific elections. Furthermore, a large percentage of the electorate will use the VAA because they want to obtain a voting advice, causing these users to be motivated to think about the questions and use the instrument seriously. Our study showed that subtle question wording variation still affects the answers—even in this usage context of likely deep processing.While we conducted our experiment in the context of local elections in the city of Utrecht, the process of VAA construction here was comparable to the construction of VAAs in other municipalities or during different local elections in the Netherlands. In addition, as the demographic characteristics of our VAA users match the typology of VAA users generally established in VAA research , our reThe increasing popularity of VAAs in many democracies stresses the relevance of research into the possible impact of seemingly small design choices in these ‘political decision aids’. The effects of question wording on VAA answers corroborate the idea that VAAs not only inform of the debate during elections, but also contribute to constructing the content of the discussion . The effFuture research might want to address the extent to which question wording variation in VAAs not only affects the answers, but also influences the resulting voting advice. A difficulty in modelling this, however, is that it would be unethical in a field study to construe a VAA version with negative (or positive) questions only.VAAs aim to reach all voters, and also reach out to the less sophisticated voters, as they have most to gain in terms of political knowledge and political efficacy by using a Voting Advice Application. This research shows that VAA builders should be careful in wording the questions in their Voting Advice Applications. Which question wording they should prefer, cannot be advised on conclusively based on this research, however. A next useful step could be to investigate how the party positions for each VAA statement, as established by professional coders working for a VAA builder, are affected by the positive or negative wordings of the questions. If party positions are affected differently by variation in the question wording than VAA users’ answers, the validity of the voting advice is threatened more strongly compared to a situation in which party positions and VAA users’ answers shift in similar ways.All in all, the present study shows convincingly how the choice of wording in an online mass medium such as a VAA contributes to public opinion in election time. It is crucial to deepen our understanding of public opinion formation through more research on the way subtle design characteristics of VAAs affect the answers citizens give.Y(jk) indicates the answer of individual j  to question k , which is the dependent variable we aim to predict. In the model, a constant is used to estimate the mean answer to positive questions (CONS β1), which may vary between persons (uj0), questions (vk0), and the interaction between the two (e(jk)). Next the model adds a fixed parameter to estimate how the mean answer to negative questions deviates from the mean for positive questions Deviation_Neg (β2). The model assumes that the residuals are normally distributed, with an expected value of zero, and a variance of respectively S2e1(jk), S2u1j0, S2v0k.In The second model adds the main effect of political sophistication as well as the interaction between question polarity and political sophistication. This is shown in S1 Appendix(PDF)Click here for additional data file.


Blockade of the PD-1/PD-L1 immune checkpoint pathway is emerging as a promising immunotherapeutic approach for the management and treatment of head and neck cancer patients who do not respond to 1st/2nd line therapy. However, as checkpoint inhibitors are cost intensive, identifying patients who would most likely benefit from anti PD-L1 therapy is required. Developing a non-invasive technique would be of major benefit to the patient and to the health care system.We report the case of a 56 year old man affected by a supraglottic squamous cell carcinoma (SCC). A CT scan showed a 20 mm right jugulodigastric node and suspicious lung lesions. The lung lesion was biopsied and confirmed to be consistent with SCC. The patient was offered palliative chemotherapy. At the time of presentation, a blood sample was taken for circulating tumour cell (CTC) analysis. The dissemination of cancer was confirmed by the detection of CTCs in the peripheral blood of the patient, measured by the CellSearch System (Janssen Diagnostics). Using marker-independent, low-shear spiral microfluidic technology combined with immunocytochemistry, CTC clusters were found in this patient at the same time point, expressing PD-L1.This report highlights the potential use of CTCs to identify patients which might respond to anti PD-L1 therapy.The online version of this article (doi:10.1186/s12885-017-3316-3) contains supplementary material, which is available to authorized users. Head and neck cancer (HNC) patients often present with advanced metastatic disease. Whilst there have been improvements in the management of locoregional disease, distant metastatic spread remains a challenge in the field –3. PalliThe programmed cell death-1/programmed cell death-1 ligand (PD-1/PD-L1) pathway has shown to play a crucial role in tumour immune invasion. Recent literature suggests that PD-L1 over expression in solid tumour types has shown direct tumour protection. Recent studies have shown that antibodies targeting PD-1/PD-L1 have significant anti-tumour activity with a much lower toxicity profile and are currently being investigated in a number of tumour types , 6. PembIn the 2014, American Society of Clinical Oncology (ASCO) meeting, it was reported that in a majority 77.9%) of pre-treated HNSCC patients, PD-L1 is expressed in the tumour, defined by ≥1% stained cells in the tumour microenvironment 7.9% of p, 8. The Metastatic sites have shown unique genomic alterations, which can be quite different from the primary site , 10. InvWe report the case study of a 56 year old man who had been diagnosed with a supraglottic SCC and treated with chemoradiotherapy. The patient was assessed for CTCs by the FDA-approved CellSearch (Janssen Diagnostics) and spiral microfluidics platform. Single CTCs were detected by both platforms. Using the spiral approach, CTC clusters were identified expressing PD-L1. We propose the notion that CTC PD-L1 assessment may be an avenue to identify patients who would be suitable candidates for anti-PDL1 therapy.A 56 year old Caucasian male with a background history of Crohn’s disease, treated with azathioprine, presented at the end of 2013 with a supraglottic T3N2b squamous cell carcinoma which was treated with upfront chemoradiotherapy, utilising Cisplatin. In October 2014, a CT scan showed a 20 mm right jugulodigastric node. Lung lesions were observed which were queried to be fungal/distant metastasis by the MDT clinic. Biopsied specimen of the lung lesions confirmed moderately differentiated SCC. Progressive disease to the lungs and pelvic bone were observed whilst on Taxol and Carboplatin, given weekly. Having limited options, a trial of infusional 5-FU (fluorouracil) was given at the end of 2015. The patient died in February 2016.Nodules were projected over the left infrahilar region and within the right upper lobe suspicious for SCC metastasis Fig. .Fig. 1ChThe Patient presented with CellSearch-positive CTCs (2CTCs/7.5 ml) in circulation at time of presentation to clinic  Fig.  and 2 CTThe antibody against PD-L1 showed a relatively high mean intensity for  was mixed with 6.5 ml of CellTracks™ buffer and centrifuged at 800 x g for 10 mins. The sample was placed on the AutoPrep™ system and the protocol followed as per manufacturer’s instructions. The system uses a positive selection enrichment based on EpCAM antibodies and characterizes the selected cells for pan-cytokeratin (pan-CK), CD45 and DAPI staining. A CTC was determined based on the following parameters: positive for EpCAM, pan-CK, DAPI, at least a diameter of 4x4μm and negative for CD45 (common leukocyte marker). The results were reported as the number of CTCs/7.5 ml whole blood.8 mL of whole blood was collected in EDTA tubes . To reduce the cellular components passing through the spiral chip, a red blood cell (RBC) lysis was performed. Briefly, post RBC lysis , the sample was centrifuged and the pellet resuspended in 10 mL of sheath buffer . Tygon® tubing was inserted into the inlet/outlets of the spiral chip, and the inlet tubing connected to a syringe pump. The spiral chip was fixed into position on a phase contrast microscope . The outlet tubing was connected to sterile 15 mL BD falcon collection tubes. To run the patient sample, the sample was carefully loaded into a 10 ml syringe and pumped through the spiral chip at a flow rate of 1.7 ml/min . The outFive head and neck cell lines were used to develop a dynamic range of PD-L1 expression. FaDu (ATCC®HTB43™), CAL27 (ATCC®CRL2095™), SCC25 (ATCC®CRL-1628™) were from the American Type Culture Collection (ATCC™). SCC15 (ATCC®CRL-1623™) a generous gift from Dr. Glen Boyle  and 93-VU-147 T (CVCL_L895) (HPV-positive) cell line from Dr. Johan de Winter . The human chronic myelogenous leukemia K562 (ATCC®CCL-243) cells were used as a negative control . Briefly, cytospins  were prepared using aliquots of 1000 cells/slide by centrifugation at 300 x g for 5 mins. The slides were stained with PD-L1 as per the immunocytochemistry protocol below.Briefly, the initial sample was stained using the CellSearch antibody cocktail (Janssen Diagnostics) and anti-EGFR antibody  as previously described –20. A fuThe predictive value of PD-L1 expression in primary tissues is limited . FurtherThe patient presented with CTCs by enrichment using both CellSearch and spiral technologies. Importantly, the spiral technology was able to enrich for CTC clusters which are rarely detectable by CellSearch . These mIn the 2016 AACR meeting, in locally advanced HNC patients, PD-L1+ CTCs were associated with shorter progression free survival (PFS) and overall survival (OS), and were proposed to “select and monitor patients for PD-1 checkpoint inhibitors” , 29. TheThis case report highlights the potential of using of CTCs to (i) identify patients ‘at-risk’ of developing metastasis (ii) identifying HNC patients that are likely to benefit from anti PD-L1 therapy and (iii) the development of a CTC PD-L1 immune-score for HNC. Further studies are warranted comparing patient tumour and CTC PD-L1 expression to develop predictive biomarkers.


Correlation analysis with clinicopathological parameters was undertaken; the cause-specific outcome was modeled with competing risk analysis; multivariate Cox regression was used to determine independent predictors of survival. Membranous PD-L1 was expressed in a minority of tumors, defined by HPV-negativity. Its presence geographically correlated with immunocyte-rich regions of cancer islets and was an independent prognostic factor for poor outcome. Our data support the notion that vulvar cancer is an immunomodulatory tumor that harnesses the PD-1/PD-L1 pathway to induce tolerance. Accordingly, immunotherapeutic approaches might have the potential to improve outcome in patients with vulvar cancer and could complement conventional cancer treatment.Vulvar cancer is rare but incidence rates are increasing due to an aging population and higher frequencies of young women being affected. In locally advanced, metastatic or recurrent disease prognosis is poor and new treatment modalities are needed. Immune checkpoint blockade of the PD-1/PD-L1 pathway is one of the most important advancements in cancer therapy in the last years. The clinical relevance of PD-L1 expression in vulvar cancer, however, has not been studied so far. We determined PD-L1 expression, numbers of CD3 Howevernced age . Howevernced age , 8. Surgnced age . Patientnced age .The recent success of checkpoint blockade caused a paradigm shift in cancer treatment. It focuses on disinhibiting tumor-specific immune responses. One of the most important immune-inhibitory checkpoint pathways consists of (i) the programmed death 1 (PD-1) receptor, expressed on activated T cells; and (ii) the programmed death ligand 1 (PD-L1), expressed on tumor cells . Their i+ T cells, CD20+ B cells and CD68+ monocytes/macrophages to be present in both compartments (Table + regulatory T cells (Treg cells) and CD163+ tumor-associated macrophages (TAMs), shown to play a crucial role in various cancer types [We first evaluated whether VSCC is a malignancy able to elicit an inflammatory response using samples that cover the wide range of the disease Table . Severaler types , 22. TheIn the next step, we aimed to determine whether additional immune escape mechanisms of the tumor are present. Membranous expression of PD-L1 was identified in 23.3% (24/103) VSCC samples. The intensity of continuously stained cell membranes ranged from low (3 cases) to moderate (4 cases) to high (13 cases); four cases lacked confluence of staining Figure . The amo+, CD20+ and CD68+ intra-tumor immunocytes  to identify VSCCs driven by viral infection [INK4a. With the exception of PD-L1, no difference was found for immune cells between viral and non-viral etiology  hinfection . We founy Figure .Given our observation that PD-L1 correlates with non-HPV pathogenesis, we analyzed whether the presence of PD-L1 would be of use to prognosticate poor outcome in VSCC. Since our cohort had an average age of 64 years, in which deaths from non-cancer illnesses are expected to occur in a higher proportion, the effect of mortality from causes other than VSCC was modeled as competing risk. PD-L1 expression was able to discriminate between recurrence-free survival (RFS) rates relating a positive staining to a significantly more unfavorable outcome Figure . AdditioWhile immune checkpoint blockade may offer a promising novel therapeutic approach with regard to locally advanced, metastatic or recurrent disease, little is published about the immune landscape in VSCC and no data are available on the clinical relevance of PD-L1. We found PD-L1 to be expressed in VSCC within an immune-rich environment. The correlation of PD-L1 with HPV-negativity and poor outcome points towards immune escape mechanisms in a distinct subset of VSCC.+ T cells, CD20+ B cells and CD68+ monocytes/macrophages as well as Foxp3+ Treg cells and CD163+ TAMs to be present in peri- and intra-tumor areas. Data on the role that the immune system plays in VSCC are still scarce and conflicting. In accordance with our results, earlier studies have discovered evidence of CD4+ and CD8+ T cells in cancer nests and the peri-tumoral stroma [+ T cells did not correlate with better outcome suggesting counteracting inhibitory mechanisms. In accordance with this hypothesis, higher levels of indoleamine 2,3-dioxygenase (IDO) expression were shown to be associated with poor outcome [We observed CD3l stroma –19. Howe outcome . These f outcome .+ T cells at the tumor site itself was shown to be crucial for treatment response to PD-L1 blockade [reg cells [+ TAMs in melanoma [Our findings point towards a vital role of PD-L1 in the immune escape of VSCC. The observed correlation between tumor-associated immune cells and PD-L1 expression suggests that the VSCC-directed immune response provokes tolerance. The more stringent association with immunocytes in cancer cell islets is in accordance with previous data showing that intra-tumor leukocytes reflect immune reactions against cancer with higher accuracy . Of noteblockade , 30. Theeg cells . Similarmelanoma . PotentiOverall, we found PD-L1 to be present in VSCC, albeit at a low frequency. A previous study evaluated PD-L1 in a small number of 23 VSCCs . HoweverWe observed an unfavorable outcome in women with PD-L1-expressing tumors, suggesting impaired T cell-mediated immune responses and a microenvironment more permissive to tumor recurrence. Further support for this hypothesis comes from the observation that PD-L1 expression did not correlate with any commonly recognized feature of greater malignant potential. Our data suggest that PD-L1-based immune evasion mechanisms occur primarily in HPV-negative VSCCs. In penile cancer, PD-L1 expression was also found to be mainly expressed in HPV-negative tumors . In EpstImmune checkpoint blockade has been a game-changer in the field of tumor treatment and the identification of a biomarker that predicts the response to PD-L1 inhibition is essential. While PD-L1-negative tumors might also benefit from checkpoint inhibitors and some PD-L1-positive tumors do not respond to treatment, an increased probability of therapeutic response has been observed in tumors defined by the presence of PD-L1 , 42. AccTo the best of our knowledge, this is the first study analyzing the clinical impact of PD-L1 expression in VSCC. The strength of our analysis lies in the compilation of immunological markers and diverse clinical as well as pathological data. A potential limitation of our study is the utilization of TMA. Although reliable for the determination of antigen expression, a heterogeneous protein distribution could potentially influence results . In cervIn summary, our study provides evidence that inhibitory pathways are operational in VSCC, arresting the tumor-specific immune response and influencing outcome. Our findings will hopefully boost the development of immunotherapy in VSCC. While further efforts must be made to validate our findings of PD-L1 as a prognostic factor and determine its value as a predictive biomarker, checkpoint blockade could be the leading path to increased treatment success in VSCC.th TNM classification of the Union for International Cancer Control (UICC) was used to determine the tumor stage. For tumors diagnosed before 2009, stages were re-classified according to the updated version. The study was approved by the Institutional Review Board of the Medical Faculty of the University of Bonn, Germany (228/15).The retrospective study population included 103 patients with primary VSCC diagnosed at the University of Bonn between 2002 and 2013. Women who underwent neoadjuvant treatment were excluded. Patients were treated according to the S2k guideline of the German Cancer Society and the German Society for Gynecology and Obstetrics. Our cohort had a high number of women not treated with lymph node dissection. Due to cardiovascular comorbidities, 20 patients with early-stage tumors (pTIb/II) did not undergo lymphonodectomy; clinically and sonographically lymph nodes appeared to be unaffected. Additionally, the presence of lymphovascular space invasion was recorded, known to be a risk factor for lymph node metastasis . In our 2) was taken from the selected cancer nests and arranged in TMA blocks.The TMA was created from archival formalin-fixed paraffin-embedded tissue (FFPE) specimens. Sections stained with hematoxylin and eosin (HE) were used to identify representative tumor areas. For each case, one 1 mm core biopsy , mouse anti-human CD3 IgG2a monoclonal antibody , mouse anti-human CD20 IgG2a monoclonal antibody , mouse anti-human CD68 IgG3 monoclonal antibody , mouse anti-human CD163 IgG1 monoclonal antibody , mouse anti-human Foxp3 IgG1 monoclonal antibody .Immunostaining of PD-L1, CD3, CD20 and CD68 was performed on TMAs using an automated staining system  and the Ventana amplifier detection kit for visualization. Additionally, in a subset of our cohort (42 samples) staining of CD163 and Foxp3 was performed on serial 4μm sections using an automated staining system  and the streptavidin-biotin-peroxidase/DAB technique (DAKO) for visualization. To determine PD-L1 expression we employed an antibody that is FDA-approved for selecting non-small cell lung cancer patients for treatment with the anti-PD-1 antibody pembrolizumab . PrimaryINK4a, a continuous strong nuclear plus cytoplasmic labeling of the basal cells with extension upward involving ≥30% of the epithelial thickness was regarded as positive [reg cells by nuclear staining of Foxp3. CD3+ T cells, CD20+ B cells and CD68+ monocytes/macrophages were enumerated using the whole core area of the TMA . Treg cells and TAMs were determined in three high-power fields (HPFs) with maximum infiltration and set in relation to the relative amount of tumor and stroma of these HPFs . Treg cells were enumerated; TAMs were recorded digitally using a semi-automated computerized method as described before [Immunostained cells were analyzed with a Leica DM LB2 microscope  with the Pannoramic Viewer  or the Axio Observer D1 microscope  with the AxioVision 4.7 software (Zeiss). As a negative control for PD-L1 staining, we used sections of kidney, as a positive control sections of tonsil . The mempositive , 51, 52.d before .After deparaffinization, tumor tissue was macrodissected from unstained slides and tissue was lysed with proteinase K overnight. Subsequently, extraction of DNA from FFPE-embedded tissue DNA was carried out with the BioRobot M48 Robotic Workstation and the corresponding MagAttract DNA Mini M48 Kit  following the manufacturer's protocol. Quality of genomic DNA was assessed by agarose gel electrophoresis. DNA concentration of each sample was determined using Nanodrop 2000 .The HPV Type 3.5 LCD-Array Kit was used for the determination of HPV subtypes by hybridization to HPV-specific DNA probes . Amplification of HPV-specific DNA segments (L1 region) was achieved using the primer sets HPV ‘125′ and HPV MY09/MY11. Ten microliters of the amplification products were hybridized to HPV type-specific capture probes fixed to an LCD array chip. All steps were performed according to manufacturer's recommendations.Statistical analysis was carried out using ‘R’ version 3.2.3 . Comparisons between PD-L1 expression and continuous data were performed using the Kruskal-Wallis test; comparisons between PD-L1 expression  and categorical variables using the Fisher's exact test. Ki-67 staining in ≥ 30% of the epithelium was used as a cut-off point to assign tumors into high and low reactivity groups . Poor-di


Benefiting from the induced image charge on metal film, the light energy is confined on a film surface under metal nanoparticle dimer, which is called electromagnetic field redistribution. In this work, electromagnetic field distribution of metal nanoparticle monomer or dimer on graphene is investigated through finite-difference time-domain method. The results point out that the electromagnetic field (EM) redistribution occurs in this nanoparticle/graphene hybrid system at infrared region where light energy could also be confined on a monolayer graphene surface. Surface charge distribution was analyzed using finite element analysis, and surface-enhanced Raman spectrum (SERS) was utilized to verify this phenomenon. Furthermore, the data about dielectric nanoparticle on monolayer graphene demonstrate this EM redistribution is attributed to strong coupling between light-excited surface charge on monolayer graphene and graphene plasmon-induced image charge on dielectric nanoparticle surface. Our work extends the knowledge of monolayer graphene plasmon, which has a wide range of applications in monolayer graphene-related film.The online version of this article (10.1186/s11671-018-2535-0) contains supplementary material, which is available to authorized users. As a pioneer of two-dimensional 2D) material in the twenty-first century, graphene possesses plenty of excellent properties, such as electronic performance, superior thermal conductivity, robust mechanical strength, and high surface area. Based on the aforementioned characteristics, graphene has attracted great interests and has been applied in various research fields, such as optoelectronics , 2, optiD materia3, polyvinylpyrrolidone (PVP), and sodium borohydride were purchased from Aldrich Chemical Co. The way to synthesize silver nanoparticles was through sodium borohydride reduction of AgNO3. The monolayer graphene was grown on clean copper foils in a tube-style chemical vapor deposition (CVD) system. Under atmospheric pressure, mixture gas of 25% methane in hydrogen  was introduced into the chamber, after substrate temperature increased up to 1000 °C. The graphene growth maintained for 10–30 min, and then, the samples were cooled to room temperature rapidly. Finally, the graphene film was transferred onto the silicon substrate using poly(methyl-methacrylate) (PMMA) polymer [AgNO polymer . Then, tE0) of plane source was set as 1 V/m. Then, the conditions of FDTD boundary were set to perfectly matched layer (PML), which absorbed total incident light. Moreover, the frequency-domain field profile monitors were added to superintend the electromagnetic field distributions for succedent analysis. The excited light entered from the Ag nanoparticle side normal to the substrate plane. The polarization of incident light is along the dimer axis, which can effectively excite the plasmon coupling modes in the dimers.In this work, all theoretical analyses were simulated adopting the FDTD method to calculate the distributions of electric field and the surface charge. The Ag nanoparticle dimer was located 1 nm above the graphene, and the gap of Ag nanoparticle dimer was also set as 1 nm. The PVP on the particles is 0.5 nm, and the spacing of the graphene is 0.5 nm. Thus, we set the gap as 1 nm. The Si is underneath the monolayer. Then, the 633, 2000, and 3000 nm plane light source perpendicularly irradiated to the substrate, respectively. The electric component


Programmed cell death ligand-1 (PD-L1) expression may predict the response to both programmed cell death-1 and PD-L1 inhibitors in lung cancer. However, the extent of intratumoral heterogeneity of PD-L1 expression, which may cause false negative results, is largely unexplored. We aimed to assess the intratumoral heterogeneity of PD-L1 expression in surgically resected lung cancer specimens by applying a novel method of tissue microarray, namely Spiral Arrays, which enables us to observe the heterogeneity in spiral-shaped tissue cores. Adenocarcinoma and squamous cell carcinoma specimens were obtained from consecutive patients with lung cancer who had undergone surgical resection at Nagasaki University Hospital  since 2009. Small cell lung cancer and large cell carcinoma specimens were selected from patients in the same archive who had undergone resection since 1998. Spiral Arrays were constructed of spiral-shaped cores, prepared from representative blocks of each case, which were subjected to immunohistochemistry using an anti-PD-L1 antibody. Each core was divided into 8 segments and each segment was classified as either PD-L1-positive or PD-L1-negative using thresholds of 1.0%, 5.0%, 10.0%, and 50.0%, respectively. In total, 138 specimens were selected, including 60 adenocarcinomas, 59 squamous cell carcinomas, 12 small cell lung cancers, and 7 large cell carcinomas. The majority of specimens with PD-L1-positive segments exhibited heterogeneous expression  irrespective of the threshold  of cases with heterogeneous PD-L1 expression. In conclusion, intratumoral heterogeneity of PD-L1 expression was frequently observed in cases of lung cancer. Thus, multiple tumor biopsy specimens may be needed to accurately determine the PD-L1 expression status. Lung cancer is the leading cause of cancer-related mortality. The 5-year relative survival rate is 10.0–15.0% worldwide . AlthougIn the present study, we aimed to assess the intratumoral heterogeneity of PD-L1 expression in surgically resected lung cancer specimens by employing a unique tissue microarray technique, Spiral Arrays, which enables us to observe the heterogeneity in spiral-shaped tissue cores –18.The study protocol was approved by the Ethical Review Board Committee  of Nagasaki University  on July 26, 2016. Informed consent was obtained from each patient at the time of surgery.Adenocarcinoma and squamous cell carcinoma specimens were prospectively obtained from consecutive patients with lung cancer who had undergone surgical resection at Nagasaki University Hospital  since 2009. Small cell lung cancer and large cell carcinoma specimens were also selected from patients in our institutional archive, but who had undergone surgical resection since 1998, because of the low number of cases due to the infrequency of these histological types. Pathological reports were reviewed and patients with only one of these histological components were included. Hematoxylin and eosin (H&E)-stained slides were also reviewed, and patients with insufficient numbers of malignant cells to construct the Spiral Arrays were excluded. Patients lacking sufficient formalin-fixed, paraffin-embedded tissue were also excluded.® Aperio CS2 slide scanner . The scanned whole-slide image of each H&E-stained slide was reviewed to select and mark two continuous straight regions of interest (X and Y axes) prior to constructing the Spiral Arrays. Two 120.0-μm-thick sections were subsequently cut from each block and arranged together on the Spiral Array Constructor  as the X or Y axis on each section was aligned in the same direction. The aligned sections were rolled together into a cylindrical form and cut along the line reflecting the X and Y axes. One of the separated reels was embedded vertically into a recipient block to construct the Spiral Arrays. Finally, 4.0-μm-thick sections were prepared from the Spiral Array blocks for further histopathological evaluation using H&E staining and IHC.Spiral Arrays were constructed as described previously  17]. Br. Br17]. ® Aperio CS2 slide scanner (Leica Microsystems).Well-characterized anti-PD-L1  and anti-cluster of differentiation 68 (CD68)  antibodies were selected. IHC was performed using an automated staining system . The antibody dilutions were optimized to 1:100 for anti-PD-L1 and 1:400 for anti-CD68. The slides were dewaxed and rehydrated using distilled water, and were subsequently processed for PD-L1  or CD68 (proteolytic treatment). After incubation with the primary antibodies , the tissue sections were rinsed, and the sections for PD-L1 staining were further incubated with EnVision FLEX+ Rabbit LINKER (DAKO) and EnVision+ HRP Labelled Polymer (DAKO). The sections for CD68 staining were incubated with the Bond Polymer Refine Detection Kit (Leica Microsystems). Staining was visualized using diaminobenzidine, and counterstaining was performed using hematoxylin. Formalin-fixed, paraffin-embedded tissue blocks of human placenta and tonsil were prepared as positive controls. The stained slides were scanned as whole-slide images using a ScanScopePD-L1 expression was only evaluated in tumor cells. We attempted to exclude intratumoral immune cells, such as macrophages and lymphocytes. PD-L1 positivity was defined as any tumor cell that expressed PD-L1 on the cell membrane at any intensity. The tumor cells’ PD-L1 positivity was scored using four different positivity thresholds  in any tumor that included a minimum of 100 tumor cells.To evaluate tissue heterogeneity, each Spiral Array core was divided into 8 segments as shown in Six cases were randomly selected from those with positive PD-L1 staining in the Spiral Array. The staining heterogeneity was compared between the Spiral Array core and the whole tissue section in each case.The patients’ characteristics at diagnosis were presented as the frequency and percentage for categorical data, and as the median and interquartile range for continuous data. Interobserver agreement for PD-L1 IHC scoring was assessed using the kappa statistic for all possible pairs among the three observers. An average kappa statistic was also calculated for each value , 20.Descriptive and comparative analyses were performed using Microsoft Excel 2010  and JMP software for the patients’ characteristics . The kappa statistics were calculated using Microsoft Excel 2010 (Microsoft Corporation) and Easy R software  , a graphvs. 26.1%), and the median age was 70  years. The majority of patients were current or former smokers . The median smoking index was 900 . Almost all of the patients had Stage I, II, or IIIA disease .The patients’ clinicopathological characteristics are shown in Representative images of PD-L1 expression for each criterion are shown in vs. negative) for each threshold  were 814, 824, 850, and 869, respectively. The average kappa statistics were 0.76, 0.75, 0.78, and 0.79 for the 1.0%, 5.0%, 10.0%, and 50.0% thresholds, respectively.The interobserver agreement for PD-L1 IHC scoring among the three observers is shown in et al. [In the present study, using a newly developed tissue microarray method, namely Spiral Arrays, we revealed that intratumoral heterogeneity of PD-L1 expression was common in lung cancer. In past studies, the intratumoral heterogeneity of PD-L1 expression has been reported in solid tumors, including lung cancer, breast cancer, melanoma, and renal cell carcinoma , 22–29. et al. , for inset al. .et al. [et al. [vs. 7.6%) was observed between the two reports. Our findings were comparable to those of Ilie et al. [To our knowledge, this is the first study to evaluate the intratumoral heterogeneity of lung cancer using a segmental approach with several thresholds. We used a Spiral Array technique with 8 segmented cores to evaluate intratumoral heterogeneity. Specimens with both PD-L1-positive and PD-L1-negative segments within the cores were defined as having heterogeneous PD-L1 expression. Our findings suggest that PD-L1 frequently exhibits heterogeneity in its expression . Intereset al.  and Kita [et al.  comparede et al. , suggeste et al. , 31. ThuInterobserver agreement regarding PD-L1 expression scores slightly varies according to the thresholds. However, all the thresholds produced good . DisagreThis study has several limitations. First, we retrospectively evaluated surgically resected specimens, and PD-L1 expression is usually assessed in the clinic using small biopsy specimens. Second, we used only one antibody clone 28–8) against PD-L1 to limit the confusion caused by differences in antigen recognition between different clones. Clone 28–8 has been characterized by several investigators and is used in clinical trials of lung cancer –8 agains–37. FinaIntratumoral heterogeneity of PD-L1 expression was frequently observed in cases of lung cancer. Thus, our findings suggest that a relatively large number of tumor biopsy specimens may be needed to accurately determine PD-L1 expression status.S1 FigIdentical staining patterns were observed in all 6 cases.(PDF)Click here for additional data file.