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


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


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


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


Local communities, particularly the poorest households, benefit from work and trade opportunities offered by gold mining and agricultural companies but continue to depend on forests for house construction materials, food, and income from the sale of forest resources. However, gold mining and agricultural concessions reduce tree cover, potentially reducing access to forest resources and further marginalizing these households. Our analyses do not provide evidence that long-term resident communities contributed to forest cover loss between 2000 and 2010. We argue that landscape management, which recognizes local community rights to customary community use areas, and appropriate zoning for commercial land uses and protected areas could contribute to both local livelihoods and protect biodiversity throughout Myanmar during economic growth.Myanmar offers unique opportunities for both biodiversity conservation and foreign direct investment due to projected economic growth linked to natural resource exploitation. Industrial-scale development introduces new land uses into the landscape, with unknown repercussions for local communities and biodiversity conservation. We use participatory mapping of 31 communities, focus groups in 28 communities, and analyses of forest cover change during 2000–2010 using MODIS vegetation continuous fields images, to understand the social and environmental impacts of gold mining and agricultural concessions in Myanmar’s Hukaung Valley (~21,800 km Following the lifting of trade embargos since 2011, international economic interests can investigate opportunities offered by Myanmar’s relatively abundant natural resources and human capital14678910One example of an industrial-scale commercial land use which may expand in Myanmar is mining. Myanmar has diverse mineral resources, including gold57662326Like gold mining, the environmental and social impacts of biofuel concessions are hotly debated, even as biofuels are heralded as a sustainable alternative to fossil fuels28293438392 and has already experienced growth in industrial-scale natural resource extraction due to increases in gold mining and the granting of land for agricultural concessions. The interaction between the development of these concessions, gold mining and natural resource use by communities represents a unique landscape that grants the opportunity to understand potential environmental and social impacts of industrial-scale land use and draw lessons for future landscape management in Myanmar. Understanding the complex interactions and feedbacks between socio-economic and ecological systems that emerge through the growth of gold mines and creation of agricultural concessions can be used to propose policies that reconcile economic development and biodiversity conservation in Myanmar.This study focuses on the social and environmental impacts of gold mining and agricultural concessions in the Hukaung Valley in northern Myanmar. With Myanmar’s increased market access and a growing economy based on natural resource exploitation—industrial-scale extractive industries such as mining, logging and agricultural concessionsth century, but gold mining in the Hukaung Valley landscape has substantially increased since the mid-1990s after a ceasefire between the government and Kachin ethnic forces, and by 2001 the nearby Hpakant area was flooded by an estimated half a million migrants from other areas of Myanmar2, were granted in Hukaung Valley in 2006. Although the Jadeland concession remains largely undeveloped, the main crops grown in Yuzana have been jatropha (Jatropha spp.) and cassava (Manihot esculenta), intended for use as biofuels.Mining in Myanmar is nominally the responsibility of the Ministry of Minesth century), where the main livelihood activities are agriculture  and huntingGold mining and agricultural concessions have expanded in Hukaung Valley, but the environmental and social impacts of this expansion for local communities have not been quantified. There are 64 communities in the Hukaung Valley  Which areas of the local environment are used by communities, which natural resources do they value, and how is availability of these resources perceived to have changed as a result of expanding gold mining and agricultural concessions?(2) Do competing land uses  and landscape features  correlate with tree cover change between 2000 and 2010?2, but this ranged from 13.4 km2 to 443.2 km2. The agricultural concessions overlapped with eight mapped CUAs, completely encompassing the CUAs of two communities, which housed around 1100 people. This meant that 15% of the total mapped CUA was inside agricultural concessions. Gold mines were found inside the CUAs of three communities, and two of these communities also overlapped with the Yazana concession. 230 local natural resources were considered important by at least one of the 1061 participants in 56 focus groups. Over 20% of the 1061 participants in the focus groups valued the bamboo wanet , which is used mostly for walls and floors in house construction (Rusa unicolor), was eaten by participants, but also sold to workers at the gold mines and in the concessions. Most of the ten most important resources  were reported by all groups which discussed their population trends (21 and 26 groups respectively). Discussions of population changes in the other eight most important natural resources were more varied, with some groups reporting little change, while others reported increases or decreases. Two communities reported the bamboo waboe  to be locally extinct. During focus groups it was stated that decreases of natural resources were due to increased demand for charcoal, meat, and house construction materials on migrant workers who came to work in the gold mines and concessions.The community use areas (CUAs) around 31 communities adjacent to Hukaung Valley Wildlife Sanctuary (notified in 2004) were mapped, representing 49% of the 63 communities in the Hukaung Valley landscape. The 31 communities had been founded between seven and more than 200 years before the start of the study, with a mean community age of 50 years. In total, these CUAs supported an estimated 24,650 people, with a mean community size of 747 individuals . These 31 communities had an average CUA of 141.7 ± 102.9 (SD) kmtruction . Anotheresources  are usedQuantitative information on income was not available at the household level, but there was evidence that communities adopted new economic opportunities; ten communities identified gold mining, panning and selling as important income sources, and residents of 11 communities were employed in agricultural concessions. However, income generating activities connected to natural resource extraction and the agricultural concessions were mostly conducted by households perceived by participants as lower income. For example, gold panning and selling of minor forest products was only carried out by lower income households. These households were also more often associated with hunting, rattan and bamboo collection and wage labour for agricultural concessions. In contrast, growing opium and gold dealing were only conducted by households perceived by participants as higher income.st and 3rd quantile of altitude respectively) increased by 2.12% . Although this relationship was statistically significant in 84.5% of models between 2005–2010, it was only statistically significant for 16.5% of models between 2000–2005. Between 2005 and 2010, the two agricultural concessions granted in 2006 were strongly associated with changes in percentage tree cover, with 6.49% greater decreases in tree cover  within agricultural concessions when compared to outside  in 2000 and an annual estimate of 0.094% forest cover loss between 2000 and 2005 for Hukaung Valley Wildlife Sanctuary based on MODIS VCF and Landsat images56575859Dendrocalamus longispathus may reduce the availability of grazing fodder for wild species. Similarly, declines in Sambar deer (Rusa unicolor) population may impact threatened predator species such as the Indochinese tiger (Panthera tigris corbetti), which are found within the Hukaung Valley Wildlife Sanctuary65One drawback of using satellite data of percentage tree cover as an indicator of environmental impacts of land uses is that it does not consider other impacts such as mercury poisoning through the gold extraction process, overhunting and fishing or overuse of specific species. For example, multiple pathways link mining activities and the degradation of ecosystems that support livelihoods in Western Ghana6163Managed populations are consistent with existing governance structures in Myanmar. Governmental recognition of community managed forests in Myanmar is formalized through certified Forest User Groups that manage their own community area and activities such as reforestation. Collection of natural resources is permitted, but private agricultural use is not. The efficiency and inclusiveness of Forest User Groups variesRapid changes in social and environmental conditions will likely occur across Myanmar in the near future as annual rates for economic growth are expected to be as high as 8% and tightly linked to natural resource exploitation242, these protected areas are smaller than the proposed area due to removal of a large central area  includes tropical and temperate broadleaf forests, conifer forests and some areas cleared for human activities including shifting cultivationral area , but remCUAs of 31 communities within the Hukaung Valley landscape were mapped between 2005 and 2010 through collaborations between community inhabitants, the Nature and Wildlife Conservation Division (NWCD) of the Forestry Department of the Ministry of Environmental Conservation and Forests and the Wildlife Conservation Society (WCS) Myanmar field team. Communities adjacent to the Hukaung Valley Wildlife Sanctuary (notified in 2004) were selected for participation. The CUAs were mapped as part of participatory rapid rural appraisals conducted by NCWD and WCS Myanmar to initiate long-term relationships with communities and develop sustainable natural resource management plans. The NCWD and WCS field team met with the head of each community and explained their intention to map the areas used for crops, timber and non-timber forest products. Community elders and those considered knowledgeable were consulted about the traditional CUA boundaries and constructed a sketch map. Knowledgeable individuals in each community accompanied the NCWD and WCS team to map the sketched boundaries with a GPS. Boundaries between neighboring communities were established through joint meetings with both communities. Once the CUA was mapped and approved by community members, a shapefile of all CUAs was produced by WCS Myanmar shown in .ca. 20,000) participated and provided information on the uses and management of key resources, and details of conflicts and issues associated with natural resource use. As part of the participatory rapid rural appraisals, the NCWD and WCS also collected information on the date communities were founded and number of residents.In 28 communities , male and female focus groups were formed  and participants asked to list anything eaten, sold or extracted from forested areas surrounding the community. Focus groups are routinely used in the social sciences as a cost-effective method of gathering data on specific themes. They can be used to generate a broad range of data, including highlighting consistent and contrasting attitudes, perceptions and beliefs2 grid cells consistent with the MODIS VCF products. The VCF 2000 product was cross-validated using a ground-truthed supervised land cover classification of Landsat 7ETM+ imagery from February 2000–20012 = 0.9708) with percent tree cover calculated from a Landsat 5 image811155We conducted separate analyses for the periods 2000–2005 and 2005–2010 to examine tree cover changes after two agricultural concessions were granted in 2006. We used the Moderate Resolution Imaging Spectroradiometer (MODIS) vegetation continuous fields (VCF) product for the years 2000, 2005, and 20102 raster grid squares which were in forested areas of the Hukaung Valley landscape in 2000 (including inside CUAs). For each grid square, dependent and independent variables were extracted from the rasters using the package ‘raster’ in R 3.2.1To investigate the impact of different human activities on forest across the whole landscape, analyses were conducted on the 230 mTo access the impact of different drivers of forest change within community use areas, we generated 40 random points inside each mapped CUA within the Hukaung Valley landscape. Additional independent variables (population density in the CUA and number of years before 2010 that the community was founded) were extracted from the participatory rapid rural appraisals at each community. For consistency in the analysis, two communities were excluded as the majority of their CUA lay outside the Hukaung Valley landscape, leaving a total of 29 communities. For each community, the mean distance to gold mines, accessibility and water bodies were calculated from the 40 random locations, and each community was classified as either inside (8 communities) or outside (21 communities) an agricultural concession. For the eight communities classified as inside an agricultural concession, between 2% and 100% of the CUA lay inside the concession (median = 54%). As there were only 29 communities, altitude and slope could not be considered in this analysis, due to insufficient degrees of freedom for robust analyses. Altitude and slope were excluded as an exploratory analysis did not suggest any relationship between mean altitude or slope and changes in tree cover within CUAs.How to cite this article: Papworth, S. et al. The impact of gold mining and agricultural concessions on the tree cover and local communities in northern Myanmar. Sci. Rep.7, 46594; doi: 10.1038/srep46594 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Numerical calculations show good qualitative agreement with the experimentally obtained results for the photoresponse of G-FET.Graphene/semiconductor Schottky junctions are an emerging field for high-performance optoelectronic devices. This study investigates not only the steady state but also the transient photoresponse of graphene field-effect transistor (G-FET) of which gate bias is applied through the Schottky barrier formed at an n-type Si/graphene interface with a thin oxide layer, where the oxide thickness is sufficiently thin for tunneling of the charge carrier. To analyze the photoresponse, we formulate the charge accumulation process at the n-Si/graphene interface, where the tunneling process through the SiO A graphene/semiconductor heterojunction is an emerging field of recent optoelectronic devices because of their low light absorption of graphene of 2.3%2, which is appropriate for high-performance transparent electrodes of optoelectronic devices such as photosensors and photovoltaic applications13. In such applications, the charge carrier transport through the graphene/semiconductor heterojunction is crucially important to assess the device performance. Generally, a graphene contact to the doped semiconductor substrates is described as a Schottky contact, which is useful in high-performance optoelectronics and photovoltaic devices21. In addition, the presence of the thin tunnel barrier at graphene/semiconductor interface modifies the current-voltage characteristics of the junction. In the case of a metal/Si Schottky junction with a 2–10-nm-thick oxide layer at the interface, which is sufficiently thin for tunneling of charge carriers, the open circuit voltage and short circuit current under light irradiation are strongly suppressed because the photogenerated carriers recombine at the interface states in the thin oxide followed by tunneling23. Photoresponse similar to the ideal Schottky diode is observed only in a reverse bias regime. Similar characteristics were observed for a graphene-Si Schottky contact20. In this case, both the trap state at the interface and the unique density of states of graphene should be considered for detailed analyses, where the energy position of Fermi level of graphene can be modulated by the field effect through the oxide layer.Graphene is anticipated for use in high-performance electronic devices because of its extraordinary carrier transport property with single-atomic-layer thickness11. Although detailed analysis of current–voltage characteristics of the graphene/Si Schottky junction with thin oxide layer has been conducted14, detailed analysis of the G-FET with the Si Schottky junction with the thin oxide gate remains an open subject.The transfer characteristics of graphene field-effect transistor (G-FET) are governed by the induced charge carrier density by application of the gate voltage. For a graphene/doped Si junction with thin oxide layer at reverse bias, the induced charge carrier in graphene should be balanced to that of Si depletion layer induced by the reverse bias corresponding to the gate bias. Under light irradiation, the excess charge carrier generated in the depletion layer by light irradiation is accumulated at the Si/oxide interface because of the presence of the thin oxide layer, which increases the charge carrier density in the graphene and which consequently increases the drain current of the G-FET. This effect can be used to amplify the photoresponse of the graphene/Si Schottky junction, where the photoresponse appears on the drain current of G-FETx layer is considered in addition to the recombination of the accumulated holes and the electrons in the graphene at the surface states on SiOx layer.This study investigates not only the steady state but also the transient photoresponse of G-FET of which gate bias is applied through the Schottky barrier formed at the n-type Si/graphene interface with a thin oxide layer, where the oxide thickness is sufficiently thin to tunnel the charge carrier. To analyze the photoresponse, we formulate the charge accumulation process at n-Si/graphene Schottky interface, where the tunneling process through the SiOx layer acts as the insulating layer of the gate. All processes were performed in air, so that the channel part of the graphene is contacted to the Si surface through a 2–5 nm thick native oxide layer, which is sufficiently thin to tunnel the charge carrier23. As depicted schematically in Fig. VGS) corresponding to the reverse bias of the Si/graphene Schottky junction.Figure VGS > 0, the photoinduced hole–electron pair at the depletion layer is separated by the build-in potential, as presented schematically in Fig. −1, so that the penetration depth of the 623 nm light is ~3 μm, which is much greater than the depletion layer thickness. Consequently, the hole–electron pair is generated throughout the depletion layer. The photo-excited electrons at the depletion region drift to the back gate electrode that is positively biased. The holes at the depletion region also drift toward the graphene channel. Thus, the either direct or trap controlled recombination of photogenerated hole and electron in the depletion region is suppressed. For an ideal Schottky junction, the holes are swept out to the graphene without restriction. However, under the presence of the thin SiOx, the holes are accumulated at the interface, followed by tunneling to the graphene, which closely resembles the inversion layer at MOS interface for n-type semiconductor. In this case, the electrons are induced in the graphene as the counter charge of the accumulated holes at the interface, which engenders the Fermi level shift of graphene. Additionally, one must consider recombination of the photogenerated holes and electrons in the graphene through the recombination center at SiOx. Consequently, the output current (IDS) of the G-FET is increased by light irradiation depending on the accumulated holes at the Si/SiO2 interface. Similar concept of the trap controlled band modification for the enhancement of photosensitivity has been reported in organic photodiodes28.At IDS, which is determined by the carrier density of graphene nG. For simplicity, we assume that the recombination life-times of both of electrons and holes are the same to match τR of the Shockley–Read–Hall (SRH) recombination model29. Furthermore, we assume that the intrinsic carrier density is negligibly small. Therefore, the resultant rate equation of accumulated holes, pSi, at the Si/SiO2 interface can be expressed asx and the SRH recombination of the accumulated holes and the electrons in graphene at SiOx. Also, τT−1 stands for the tunneling probability of the accumulated holes, τR−1 expresses the recombination rate of the accumulated holes and the electrons in graphene at SiOx, and g denotes the photo-generation rate of holes in the depletion layer. The density of electrons in graphene at Fermi level EFG is known to be32.EDP is the energy position of the charge neutral point, the Dirac point, vF represents the Fermi velocity of charge carriers in graphene (~108 cm/s). The tunneling probability τT−1 of holes into graphene is proportional to the graphene density DG(ETG) as final states for tunneling, where ETG is the energy position for hole tunneling. The graphene density at energy E isEFG − EDP . At VGS higher than that at the gate voltage corresponding to the Dirac point (VDirac) under the dark condition, the excess VGS (=VGS − VDirac) is applied for development of the depletion layer, so that one can assume EFG ~ EDP . At this condition, the energy state for tunneling of accumulated holes into graphene, which corresponds to the energy position of valence band top of Si at the SiO2/Si interface, is defined as Ev0. To clarify the tunneling process under light irradiation, we investigate the relation between ETG and Ev0, where we consider the Fermi level shift of graphene ΔEFG = EFG − EDP and the additional potential drop at the thin SiOx, ΔVOX, because of nG as the counter charge of accumulated holes induced by photoexcitation, pSi. The relation between ETG and Ev0 under the light irradiation is given asBased on the model described above, we will formulate the light-intensity dependence of nG at dark is negligible. In this case, one can expect nG ~ pSi. As described above, tunneling probability τΤ−1 is proportional to the final density of states asα is the constant for proportional factor including the transmittance of tunneling electrons through the thin SiO2. Inserting this equation to Eq.  dependence of pSi should be expressed as gθ with θ = 1 at the steady state, i.e., dpSi/dt = 0. For the case in which β2 originating from the voltage across the thin oxide layer is dominant at stronger light intensity, θ is approx. 0.5. As presented in Fig. Supplementary Information, the numerically calculated light-intensity dependence of pSi shows power law dependence with two slopes. It is noteworthy that nG ~ pSi. Thereby, one can expect that the light-intensity dependence of IDS shows power law dependence of gθ with θ < 1.Using the combination of Eqs  and 4),,4), one mentclass2pt{minimCG–VGS) characteristics of the Schottky junction were investigated with or without light irradiation, where the measured capacitance CG is a series capacitance consisting of a quantum capacitance of graphene CQ, the thin native oxide layer COX, and the depletion layer at the Si/SiOx interface, CD. As presented in Fig. VGS < −0.5 V corresponding to the accumulation condition for MOS junction, the gate capacitance CG decreases with increasing VGS because of the decrease of CQ34. The maximum capacitance of CG−1 = Cox−1 + CQ−1 at VGS < −1 V is greater than 0.65 μF/cm2. The SiOx layer thickness is estimated as 5 nm or less. The corresponding band diagram is portrayed in Fig. Supplementary Information. To clarify the light irradiation effect on CG, the CG difference between the dark and the light conditions, ΔCG, is shown against VGS in Fig. VGS < −0.5 V, ΔCG shows no marked light-intensity dependence.To investigate the depletion layer at the junction, the capacitance–voltage  by the Schottky barrier. Under the light irradiation, IG increases concomitantly with increasing light intensity to 63 nA at 641 μW/cm2. In this region, the photo-excited electrons are swept out through the depletion layer of n-Si, whereas the photo-excited holes are tunneled to the graphene through SiOx layer or recombined at the graphene-Si interface at the thin SiOx layer, as presented in Fig. IG at −0.5 V < VGS < 0.2 V under light illumination is suppressed, unlike the common Schottky junction behavior. This behavior is commonly observed at the Schottky junction of lightly doped n-Si with thin SiOx layer23. It originated mainly from the presence of thin SiOx layer at the interface, resulting in the increase of the direct carrier recombination of the photogenerated carriers at the depletion layer of n-Si14.Figure 1 at VG < VDirac. The drain current IDS at VG > VDirac in a dark condition remains almost constant, which implies that EFG ~ EDP, as assumed in the numerical calculations. Under light irradiation, IDS increases concomitantly with increasing light intensity of 8–11 μA with saturation behavior at higher VGS, where VDirac (VGS ~ 0.15 V) is the bias voltage for the charge neutrality point of G-FET determined at minimum IDS. The IDS difference between the dark-current and photo-current, ΔIDS, is 3 μA at 641 μW/cm2, which is 2–3 orders of magnitude larger than that of ΔIG depicted in Fig. IDS is negligible.The transfer characteristics of the G-FET under an illumination condition are presented in Fig. IDS is governed by the accumulated holes at SiOx/Si interface, even under light irradiation, as described earlier. The ionized donor (positively charged) in the depletion layer gives limited impact to IDS at VGS > VDirac under light irradiation because of the slight increase of IDS in dark conditions. The light-intensity dependence of the saturated IDS under light irradiation at VG > VDirac indicates that the charge carrier density in graphene is determined by the accumulated photo-excited excess hole at the interface, where the density of the accumulated photo-excited excess hole is regulated by the recombination rate of excess holes through the thin SiOx barrier, as described above. The field effect mobility of hole in graphene was estimated as 800 cm2V−1s−1 from CG of around VGS = −0.5 V.Based on the band model depicted in Fig. IG and ΔIDS, both showing power law dependence of the light intensity. The light-intensity dependence of ΔIG can be fitted by one slope of 0.73. By contrast, the light-intensity dependence of ΔIDS comprises two slopes as predicted theoretically earlier. The experimental data are well fitted to the numerically calculated result shown in Fig. vF = 0.8 × 106 m/s, τR = 18 ms, Evo = 0.05 eV, α = 2 × 10−34, SiOx thickness = 5 nm are used. The actual psi, which is proportional to ΔIDS, is unknown. For that reason, the vertical axis was adjusted with an arbitrary ratio of ΔIDS/psi. From a practical perspective for the photosensor, the ΔIDS is 2–3 orders of magnitude larger than ΔIG. This photocurrent amplification of G-FET with the Si Schottky gate gives rise to easier measurement of the light intensity than the simple graphene/Si Schottky diode, where the noise equivalent power (NEP) of the G-FET obtained at 8.9 μW/cm2 is 7 × 10−14 W/Hz1/2 (corresponding specific detectivity: 1 × 1011 Jones).Figure CD, internal series resistance RT, and load resistance RL, as portrayed schematically in Fig. IL is a current source determined by the light intensity. In our device structure, RL and RT consist of contact resistance, tunnel resistance, and resistance of the Si substrate. Based on the simplified model presented here, the time constant for rise time, τ0, is defined simply by CDRT, expressed as 2 interface, which induces the time dependence of IL determined by Eq. pSi corresponding to IL under various light intensities, where the used parameters are the same for those of light-intensity dependence in a steady state. The calculated data are well fitted by τrise, which depends on the light intensity. Consequently, one can expect that the transient photoresponse at light-on is given as the product of Transient response of G-FET is important for practical applications. The photoresponse time of the Schottky diode is usually explained by the simplified model with junction capacitance IDS on the G-FET, where the light intensity was varied from 0.91 to 257 μW/cm2. As might be readily apparent, the response time at light-on τrise decreases concomitantly with increasing light intensity, which is consistent with the calculated results based on the proposed model shown in Fig. τ0 determined from CDRT was fixed. Note that the lowest photoresponse obtained at 0.91 μW/cm2 is quite noisy and close to the dark signal, so that we could not obtain the reliable fitting curve. The experimentally obtained results were not fitted well by the simple exponential function. The time constant τrise obtained from the fitting clearly depends on the light intensity: it decreases from 12.5 to 1.6 ms with increasing light intensity, as portrayed in Fig. τrise obtained from the numerically calculated results presented in Fig. 2 could not be well reproduced based on our simplified model. This is most likely that the contribution of recombination process becomes more dominant at low carrier density. This is a subject for further study.Figure t > 3 ms, the decay can be expressed by τf = 17.8 ms for all light intensities, mainly because of the recombination time constant τR  of the G-FET obtained at 8.9 μW/cm2 is 7 × 10−14 W/Hz1/2. This amplification function is useful for practical applications. To analyze the photoresponse, we formulated the charge accumulation process at the n-Si/graphene interface, where the tunneling process through the SiOx layer was considered in addition to the recombination of the accumulated holes and the electrons in the graphene at the surface states on SiOx layer. The accumulated holes induce the Fermi level shift of graphene, which produces variation of the tunneling probability. The analytical framework proposed here is well described by the experimentally obtained results for both steady states and transient states. Moreover, it provides an effective means of analyzing charge carrier transport through the grapheme-based heterostructure through a thin tunneling barrier, which represents one path to achieving ultrathin opto-electronic devices in the future.We demonstrated a photosensor consisting of G-FET with gate bias applied through the Schottky barrier formed at an n-Si/graphene interface, where a thin SiO2 layer using conventional photolithography processing. The resistivity of the n-type Si substrate used in this experiment is 8 Ωcm. Unlike common back-gate G-FETs, we removed the SiO2 layer (300 nm thick) between the electrodes using buffered HF. All processes were performed in air, so that the channel part of the graphene is contacted to the Si surface through a 2–5 nm thick native oxide layer. Subsequently, after a monolayer graphene was transferred onto the substrate using polymethyl methacrylate, it was trimmed using oxygen plasma etching to form the G-FET channel, where the graphene was synthesized using low-pressure chemical vapor deposition at 1000 °C using Cu foil as catalyst36. The channel length and width are, respectively, 70 and 100 μm.The device fabrication process was the following. First, metal electrodes consisting of Cr/Au (5 nm/30 nm) as a source and drain electrodes were fabricated on a Si substrate with a 300 nm-thick SiOSupplementary Information


PD-L1/PD-1 interaction leads to a down-regulated immune response and it is one of the most promising targets for gastric cancer immunotherapy. PD-L1/PD-1 and CD8 expression were immunohistochemically investigated in a series of 169 FFPE GCs, including 33 EBV+, 59 MSI and 77 MSS/EBV− cases. PD-L1 membrane immunoreactivity in more than 5% of tumor cells was present in 31/169 GCs and was associated with high levels of CD8 intraepithelial lymphocytes . PD-L1+ cases were mainly poorly differentiated (71%), intestinal type (85%) and high lymphoid response  tumors. PD-L1 expression was only present in EBV⁺ (46%), MSI (24%) and rare MSS/EBV− (3%) GCs with high CD8+ TILs (p < 0.001). Despite being associated with a better prognosis both in the whole series (p < 0.05) and in the MSI subset, PD-L1 is not an independent prognostic factor. PD-L1 gene amplification was detected in 3/17 cases, including 2/7 EBV+ and 1/8 MSI GC. PD-1⁺ TILs were significantly higher in EBV⁺ than MSI and MSS/EBV− cases. PD-L1/PD-1 pathway is selectively activated in HLR GCs and could be considered an emerging therapeutic target, particularly for EBV and MSI GCs.Both EBV Gastric cancer (GC) is a heterogeneous disease characterized by different molecular and histological profiles ,2, whichAs in other malignancies such as melanoma and non-small cell lung cancer, GC tumor cells can also activate immune checkpoints to inhibit and escape an immune system response. One of these checkpoints is programmed cell death-ligand 1/programmed cell death protein-1 (PD-L1/PD-1) interaction. PD-L1 (B7-H1/CD274) is a type I transmembrane glycoprotein constitutively expressed on macrophages, T, B and dendritic cells, and its expression is enhanced by pro-inflammatory cytokines. In normal tissue, PD-L1 protein is less synthesized compared to its mRNA which is present in various human tissues . PD-1 is+ T cells [In cancers, up-regulation of PD-L1 expression could depend on intrinsic cellular control directed by genomic alterations, or it could be the consequence of an adaptive extrinsic control induced by CD8 T cells . In addi T cells ,6,7,8,9.The role of PD-L1 expression as a prognostic factor remains unclear because of the absence of uniform evaluating criteria for its analysis and the different tumor characteristics analyzed and correlated to survival in the various studies ,11,12,13+ tumor infiltrating lymphocytes (TILs) in whole formalin-fixed, paraffin-embedded tissue sections of a large series of 169 GCs which were well characterized histologically and enriched for EBV+ and MSI cases. The role of PD-L1 in predicting survival was also investigated. In this work we evaluated PD-L1/PD-1 expression and CD8PD-L1 immunoreactivity was observed in tumor cells and in immune cells, lymphocytes and macrophages infiltrating and surrounding tumor nests. + tumor cells, varying from 1% to 90%, showed weak to strong immunoreactivity prevalently along the cellular membrane or both along the membrane and in the cytoplasm. Five cases displayed only cytoplasm immunoreactivity and were considered negative for PD-L1.PD-L1+, showing 5% or more PD-L1+ tumor cells. They were mainly poorly differentiated GCs, intestinal in type according to the Lauren Classification, with the extent of the primary lesion (pT) being low and a prevalence of TNM stage I and II (Thirty-one out of 169 (18.3%) GCs were PD-L1I and II . They oc+ cases were high lymphoid response (HLR) GCs, 2 were ordinary cohesive  and one was an anaplastic MSI GC with a moderate number of TILs. Noteworthy, no ordinary or invasive mucinous carcinomas, including MSI cases, showed PD-L1 expression.According to the histotype-based prognostic classification , 28/31  and MSI  than in MSS/EBV−  GCs (p < 0.001). When we considered the cut-off of 1%, instead of 5% of immunoreactive tumor cells, the percentage of PD-L1+ cases increased to 61% for EBV+ and 37% for MSI GCs, whereas it remained unvaried in MSS/EBV− GCs. PD-L1+ cases, PD-L1+ tumor cells were spread out along the tumor and varied from 1% to 90%  of EBV+ and 37% (22/59 cases) of MSI GCs. Regarding the pattern of infiltration, in addition to the invasive margin the PD-L1+ immune cells infiltrated tumor in 58% and 54% of EBV+ and MSI GCs, respectively  of the cases. They were few and prevalently localized along the invasive margin (IM). TI pattern, in fact, was present in only 1/19 .In addition to PD-L1vely TI, . On the + lymphocytes were present both within tumor-cell nests and in intratumoral and peritumoral stroma. The mean number of CD8+ TILs varied from 0 to 167 and was significantly (p < 0.001) higher in EBV+  and MSI  GCs than in MSS/EBV− tumors  GCs with low CD8+ TILs, p < 0.001; Considering the previously defined cut-off value of 9.5 CD8 for HPF , PD-L1+ + TILs was observed in 137/169 GCs (81%), more frequently in EBV+  and MSI  than MSS/EBV−  GCs  compared to MSI (7 cells/HPF) and MSS/EBV⁻  GCs (The presence of PD-1.05) GCs . Overall.01) GCs .+, 8 MSI and 2 MSS/EBV⁻ GCs), including 12 high PD-L1 expression and 5 PD-L1 negative GCs with only cytoplasm immunoreactivity. The probe used for this study identified CD274,PDCD1LG2 gene cluster amplifications; high levels of amplification of this region were observed in three out of 12 PD-L1+ cases  and HLR (p < 0.0001) histotypes, low TNM stage (p < 0.0001), low pT (p < 0.0001), absence of lymph node metastases (p = 0.002) and high CD8 TILs (p = 0.01) were significantly correlated with a better outcome  gastric cancers, correlations with survival were analyzed only for advanced  GCs (AGCs). Follow up data were available for 146 AGCs. According to the literature, at univariate analysis intestinal  and high number of CD8+ TILs (p < 0.05) or HLR histotype (p < 0.05) were identified as independent prognostic factors . EBVnstrated . The hosnstrated ,17. To onstrated  and in tne cells ,12,19,20+ cases belong to the HLR group [+ T-cells infiltrating tumoral epithelium and stroma and that includes EBV+, MSI and a small number of GCs with high lymphoid infiltration but absence of virus infection and a defective MMR system. In our series about 50% of EBV+ and 25% of MSI GCs expressed PD-L1 in more than 5% of tumor cells. Outside these two distinct subgroups PD-L1 was observed in only two (3%) other cases, both MSS/EBV− GCs characterized by a high lymphoid infiltration that affects the whole tumor area or only a part of it. In addition, EBV+ cases showed a higher number of PD-L1+ cells, CD8+ TILs and PD-1+ cells than MSI and both compared to MSS/EBV− GCs. By confirming previous observations on smaller series of GCs [+ IC were also significantly more frequent in EBV+ and MSI than MSS/EBV− cases. In the last subset of carcinomas, positive IC, if present, were confined at the invasive front, whereas they infiltrate tumor in EBV+ and MSI GCs. The majority of PD-L1LR group , which is of GCs , PD-L1+ + GCs were prevalently EBV+ or MSI, their clinic-pathological profile is a mixture of both EBV and MSI characteristics. Furthermore, our GCs do not belong to a consecutive series of cases, but rather are enriched in both EBV and MSI carcinomas to compare them to MSS/EBV− subgroup. Therefore, the frequency of some features may be altered by such enrichment. The high frequency of low TNM stages in PD-L1+ cases, for instance, could be more likely related to the presence of early GCs (pT1) in EBV, but not in MSS/EBV− group, rather than to PD-1/PD-L1 pathway activation. Anyway, PD-L1+ carcinomas were prevalently poorly differentiated intestinal GCs, with larger diameter, lower pT and stage, with a preference for male and older patients. As previously observed by Ma and colleagues [+ GCs remains the presence of high CD8+ lymphocytes. Since PD-L1lleagues  all muci+ T-cells infiltration [+ lymphocytes via γ interferon secretion or by an intrinsic resistance due to oncogenic signaling. Derks et al. [− GCs. In a recent paper, Mimura and colleagues [+ T cells and tumor IFN-γ. All these data seem to suggest that an adaptive immune resistance contributes to PD-L1 expression rather than an innate immune resistance.Previous papers reported the association between PD-L1 expression and CD8ltration ,9,20, bolleagues  demonstr+ cases. Also, Clavè and colleagues [+ GCs showed a very strong immunoreaction involving a high number of tumor cells. FISH analysis demonstrated PD-L1 gene amplification in 3 out of these cases, which were two EBV+ and one MSI GCs. The induction of PD-L1 expression by inflammatory cytokines may be more efficient in tumors with gene amplification and the knowledge of the presence of an oncogenic upregulation of PD-L1, in addition to an adaptive immune response, could be useful for choosing the appropriate immunotherapy.On the other hand, the TCGA research network  reportedlleagues  describe+ GCs and is associated with high levels of CD8+ TILs, all parameters related to a more favorable prognosis. Interestingly, subgroup analyses demonstrated that cancer-specific survival was better for PD-L1+ cases in the MSI but not in the EBV+ subgroup. It could be due to the low number of EBV+ advanced GCs or the significantly higher presence of PD-1+ lymphocytes in EBV+ than MSI GCs. A more favorable prognosis was also observed when PD-L1 expression was combined with high CD8+ TILs. This result might be explained again by the CD8+ TILs positive influence on prognosis. Multivariate analysis, however, showed that PD-L1 expression is not an independent prognostic factor. Tumor stage and HLR histological type or high levels of CD8+ lymphocytes are the only independent prognostic parameters, stronger than MSI and EBV status.Controversial data are reported in the literature about the impact of PD-L1 expression on survival in GCs ,13,19,20+, MSI and rare MSS/EBV− gastric cancers characterized by abundant CD8+ lymphocyte infiltration. In these tumors the use of immune checkpoint inhibition can restore the efficiency of immune system. The presence of PD-L1 gene amplification can contribute to PD-L1 upregulation. In conclusion, PD-L1/PD-1 expression is a distinctive biological feature of EBV+ TILs, but not PD-L1 expression, are independent positive prognostic factors.Low TNM stage and high CD8The study was performed on 169 cases of advanced and early gastric carcinomas (GCs) operated on at the Ospedale di Circolo Fondazione Macchi in Varese and Fondazione Policlinico San Matteo in Pavia  between 1980 and 2012. Patients who had undergone preoperative chemotherapy or radiotherapy were excluded from the study.+) and MSI cases had to be excluded because there was no remaining tumor tissue; thus, to make the groups comparable, new cases were added from the Pavia series. Altogether, 33 cases where EBV+, 59 were MSI and 77 were microsatellite stable-EBV negative (MSS/EBV−) carcinomas.Most of the cases belong to the Varese series of GCs, previously well characterized both histologically and for the presence of EBV infection, microsatellite instability (MSI) and MMR protein expression . Some EBThe tumors were classified according to the Lauren criteria  and histPatients were followed-up either until death or for a mean period of 164 months (range 3–340).The clinico-pathological data of the GCs are summarized in The study was performed in accord with the ethical standards established by the Local Institution Review Board, and in accord with the Helsinki Declaration of 1975.2O2 in water for 15 min. Slides were treated for antigen-retrieval with pH8 EDTA buffer in a domestic microwave oven for 20 min. Immunohistochemical reactions for PD-L1, PD-1 and CD8 were performed on whole tissue sections obtained from formalin-fixed, paraffin-embedded tumor blocks. Three μm thick sections were deparaffinized and rehydrated. Endogenous peroxidase activity was inhibited in 3% HPrimary antibodies were applied overnight at −4 °C. In particular, PD-L1 rabbit monoclonal antibody  was used at a working dilution of 1/100; PD-1 mouse monoclonal antibody,  was undiluted, while CD8 monoclonal antibody  was used at a dilution of 1/20. UltraVision Quanto Detection System HRP  was used as amplification system according to the manufacturer’s instructions and with 3-3′-Diaminobenzidine as the chromogen. + immune cells were recorded as tumor infiltrating (TI) or located only along the invasive margin (IM), as reported previously [+ and CD8 TILs were counted at 400×  in ten consecutive fields and the mean value of immunoreactive TILs per high power fields was reported. The count area was selected according to the maximal number of neoplastic cells with minimal desmoplastic stroma and necrosis and only the immunoreactive lymphocytes in direct contact with tumor cells were included in the count.PD-L1 immunoreactivity was evaluated separately in tumor and immune cells. For tumor cells, only membrane staining was considered, and the percentage of positive cells was recorded, independently of the intensity. A case was considered positive if PD-L1 membrane immunostaining was present in at least 5% of tumor cells. For immune cells, both the pattern of infiltration and the intensity of immunoreaction were evaluated. The intensity was scored as absent/weak, moderate or strong, whereas PD-L1eviously . PD-1+ aAll the cases showing intense and abundant (more than 30% of tumor cells) membrane or cytoplasmic immunoreactivity for PD-L1 were tested for the presence of gene amplification. Interphasic FISH analysis was performed on 3–4 μm-thick sections used for conventional histologic examination as reported in the guidelines of the European Cytogeneticists Association  and the p value of <0.05 was considered significant. The GraphPad v.5.0  and MedCalc 11.2.0.0  software were used for statistical analyses.Statistical analysis was performed by using Fisher’s exact test or Chi-square test and Wilcoxon rank-sum test for unpaired data. Survival curves were calculated using the Kaplan-Meier estimator test. Statistical differences were tested using the log-rank test. Multivariate analysis was performed using Cox regression analysis. A + and MSI gastric carcinomas are characterized by high PD-L1/PD-1 expression and abundant CD8+ tumor infiltrating lymphocytes. PD-L1 expression is not an independent prognostic factor.EBV