Construction and self-activation detection of yeast two-hybrid bait plasmid of human programmed cell death ligand 1 immunoglobulin variable region domain gene / 中国生物制品学杂志

@#Objective To construct a yeast two-hybrid recombinant bait plasmid of human programmed cell death ligand 1(PD-L1)immunoglobulin variable region(IgV)domain gene,detect its expression in yeast and detect the cytotoxicity and self-activation of PD-L1 IgV protein as well as the interaction between PD-L1 IgV and human thioredoxin(hTrx).Methods Human PD-L1 was analyzed by bioinformatics method,and primers were designed to amplify PD-L1 IgV domain based on the coding region of PD-L1 gene registered in NCBI GenBank database. PCR amplification was carried out with pENTERPD-L1 plasmid as template,and then cloned into yeast two-hybrid bait vector pGBKT7. The recombinant bait plasmid and pGBKT7 empty vector were transformed into Y2HGold yeast cells respectively,and the PD-L1 IgV gene and its expression were detected by PCR and Western blot;Meanwhile,the protein toxicity and self-activation of PD-L1 IgV were detected,and the interaction between PD-L1 IgV and hTrx was detected by drip plate method.Results The bioinformatics analysis results of PD-L1 were consistent with related reports. The recombinant bait plasmid pGBKT7-PD-L1 IgV was correctly constructed,and Y2HGold positive clone was obtained,in which PD-L1 IgV was stably expressed. The empty vector pGBKT7 and recombinant bait plasmid pGBKT7-PD-L1 IgV grew well on SD/-Trp and SD/-Trp/X-α-Gal plates with the same colony size and number and white colony,but they did not grow on SD/-Trp/X-α-Gal/AbA plates,which indicated that PD-L1 IgV protein had no toxicity and no self-activation effect on yeast. The results of drip plates test showed that all experimental groups grew well on SD/-Trp/-Leu plate,while only positive control group grew on SD/-Trp/-Leu/X-α-Gal/AbA plate and showed blue color,which indicated that bait protein PD-L1 IgV and hTrx did not self-activate,and there was no interaction between them.Conclusion Recombinant human PD-L1 IgV bait plasmid was successfully constructed. PD-L1 IgV protein showed no toxicity and self-activation effect on yeast cells,and there was no interaction between PD-L1 IgV and hTrx. Subsequently,hTrx can be used to construct a peptide aptamer library,from which peptide aptamers that specifically bind to PD-L1 IgV can be screened.

Pathological features and immune microenvironment in HER-2 intratumoral heterogeneous breast cancers / 中华肿瘤杂志

Objective: To observe the clinical pathology features, and immune microenvironment of HER-2 intratumoral heterogeneity breast cancer. Methods: Thirty cases of HER-2 intratumoral heterogeneous breast cancer were retrospectively analyzed in Tianjin Medical University Cancer Institute and Hospital from November 2017 to June 2020. HER-2 expression was detected by immunohistochemistry and verified by dual color silver-enhanced in-situ hybridization (D-SISH). HER-2 intratumoral positive and negative regions were divided. The pathological characteristics, subtype, and the level of tumor infiltrating lymphocytes (TILs) and the expression of programmed cell death-ligand 1 (PD-L1) were evaluated respectively. Results: The proportion of HER-2 positive cells of the breast cancer ranged from 10% to 90%. The pathological type was mainly invasive non-special typecarcinoma. Six cases presented different pathological types between HER-2 positive and negative regions. The HER-2-positive areas included 2 cases of carcinoma with apocrine differentiation, and the negative areas included 2 cases of invasive micropapillary carcinoma, 1 case of invasive papillary carcinoma, and 1 case of carcinoma with apocrine differentiation. In HER-2 positive regions, 17 cases were Luminal B and 13 cases were HER-2 overexpressed types. There were 22 cases of Luminal B and 8 cases of triple negative tumors in the HER-2 negative areas. The levels of TILs in HER-2 positive and negative areas accounted for 53.3% (16/30) and 26.7% (8/30), respectively, with a statistically significant difference (P=0.035). The positive expression of PD-L1 in HER-2 positive area and HER-2 negative area were 6 cases and 9 cases, respectively. Among 8 cases with HER-2 negative regions containing triple negative components, 4 cases were positive for PD-L1 expression. Conclusions: In the case of HER-2 intratumoral heterogeneity, it is necessary to pay attention to both HER-2 positive and negative regions, and evaluate subtype separately as far as possible. For HER-2 intratumoral heterogeneous breast cancer containing triple negative components, the treatment mode can be optimized by refining the intratumoral expression of PD-L1.

Salvianolic acid B exerts its anti-tumor immunity by targeting USP2 and reducing the PD-L1 level / 药学学报

With the development of small-molecule immunotherapy drugs, its combination with the programmed cell death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) antibodies would provide a new opportunity for cancer treatment. Therefore, targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity and considered as the next generation of tumor immunotherapy. In the present study, we investigated the anti-tumor role of salvianolic acid B (SAB) by regulating the PD-L1 level in tumors. Changes of total PD-L1 and membrane PD-L1 levels were determined by Western blot, flow cytometry and PD-1/PD-L1 interaction assays. The expression of mRNA level of PD-L1 was detected by real-time PCR. The cytotoxicity of activated peripheral blood mononuclear cell (PBMC) cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment. Surface plasma resonance technique was used to analyze the direct interaction between SAB and ubiquitin carboxyl-terminal hydrolase 2 (USP2). The antitumor effect of SAB in vivo was examined by C57BL/6 mice bearing MC38 xenograft tumor (all animal experiments were conducted in accordance with the Animal Ethics Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences). Western blot and flow cytometry assay showed that SAB can significantly downregulate the abundance of PD-L1 in RKO and PC3 cells in dose- and time-dependent manner. PD-1/PD-L1 binding assay revealed that SAB reduces the binding of tumor cells to recombinant PD-1 protein. Mechanism studies revealed that SAB can bind directly to USP2 protein and inhibit its activity, thus promote the ubiquitin-proteasome pathway degradation of PD-L1 proteins. In addition, Cell impedance and crystal violet staining indicated that SAB enhances the killing activity of co-cultured PBMC cells toward tumor cells. MC38 tumor transplanted mouse experiments revealed that SAB treatment displayed significant suppression in the growth of MC38 tumor xenografts in C57BL/6 mice with an inhibition rate of 63.2% at 20 mg·kg-1. Our results demonstrate that SAB exerts its anti-tumor activity by direct binding and inhibiting the activity of USP2 and reducing the PD-L1 level. Our study provides an important material basis and scientific basis for the potential application of SAB in tumor immunotherapy drug targeting USP2-PD-L1 axis.

Preparation of CD73/PD-L1 bispecific antibody and evaluation of its antitumor function in vitro / 中国生物制品学杂志

@#Objective To prepare bispecific antibody targeting cluster of differentiation 73(CD73) and programmed cell death-ligand 1(PD-L1),and evaluate its binding ability and killing ability in vitro.Methods Using genetic engineering method,PD-L1 single-chain fragment variable(scFv) was inserted into the hinge region of CD73 monoclonal antibody to construct anti-CD73/PD-L1 bispecific antibody(BS-21),which was screened by CHO GS expression system to obtain highly expressed cell line.After purified by Protein A and molecular sieve,the purity of antibody was detected by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),the binding ability of antibody in vitro was detected by flow cytometry,and the killing ability in vitro was detected by using peripheral blood mononuclear cell(PBMC) to kill Calu 1 lung cancer cells in vitro.Results High-yield cell lines were obtained by pressure screening.A bispecific antibody BS-21 with a purity of 99.6% was obtained by purification,which bound to CD73 and PD-L1 molecules simultaneously.Compared with anti CD73 and anti PD-L1 groups,BS-21 group significantly increased the killing rate of immune cells to Calu 1 tumor cells(F=30.36,each P<0.001).Conclusion Bispecific antibody BS-21 reduced the immunosuppressive effect of CD73 and PD-Ll on immune cells simultaneously,and showed good anti-tumor function.

PD-L1 expression in invasive solid papillary breast carcinomas

Background: Invasive solid papillary carcinomas (ISPC) are rare malignant neoplasms in the classification of WHO 2019 breast tumors. Aims: We aimed to investigate the correlations between programmed cell death ligand-1 (PD-L1) expression status of tumor and immune cells and clinicopathological parameters by molecular classification of this rare morphological subtype. This study will contribute to the literature about the PD-L1 expression state of ISPCs for the first time. Material and Methods: The study included 19 invasive solid papillary carcinoma cases diagnosed between 2009 and 2019 in Pathology Department. Molecular subtyping was performed in 19 cases by immunohistochemical studies (ER/PR, Her-2/neu, Ki-67), and PD-L1 expression was evaluated in neoplastic and immune cells. Results: PD-L1 expression was detected in 4 (21%) cases, 3 (75%) of them were in luminal B and 1 (25%) were in the luminal A group. The correlation between molecular subtypes and PD-L1 expression was statistically significant (P = 0.016). Patients with PD-L1 expression had a higher Ki-67 index than patients without PD-L1 expression (P = 0.037). In addition, there was a statistically significant correlation between PD-L1 expressions of intratumoral lymphocytes and PD-L1 expressions of neoplastic cells (P = 0.004). Conclusions: While predicting the group that will benefit more from immunotherapy in solid papillary carcinoma cases, not only PD-L1 expression of tumor cells but also PD-L1 expression in tumor infiltrating lymphocyte (TIL) can help. In addition, PD-L1 staining rates of tumor cells as well as clinicopathological parameters (molecular subtype, high Ki-67 index, presence of TIL) can be predictive about immunotherapy.

Role of immunotherapy in metastatic EGFRm NSCLC: Is it relevant?

EGFR?TKIs have changed the landscape of metastatic NSCLC treatment with a significant improvement in survival of EGFRm patients compared to wild?type EGFR. Even with the newer third generation EGFR TKIs like, Osimertinib, which has proven efficacy against the resistance mutation of EGFRm T790M, progression eventually occurs. There are limited treatment options for patients with metastatic EGFRm NSCLC with other acquired resistance. Therefore, novel therapeutic combination strategies are being researched to overcome potential resistance to EGFR?TKI?targeted therapy. The ICIs targeting the programmed cell death?1 pathway in patients with EGFRm NSCLC were greatly anticipated based on preclinical studies showing increased PD?L1 expression. In clinical settings, this increased expression did not translate into a survival benefit. Treatment with ICIs failed to positively affect EGFRm patients because of multiple reasons: nonsynonymous tumor mutational burden, lower PD?L1 expression in tumors, and cancer cells utilizing alternate immune escape mechanisms. The NCCN guidelines currently do not recommend immunotherapy in patients with metastatic EGFRm NSCLC. Recently, a subgroup analysis in the IMpower150 study provided a signal for overall survival of atezolizumab with bevacizumab plus chemotherapy in EGFRm?TKI progressed patients. Based on these encouraging findings, several combinations of ICIs and EGFR?TKIs are being evaluated in TKI?failed EGFRm patients. These regimens might provide a favorable therapeutic effect by combining higher response rates of TKIs and durable disease control of ICIs. However, further research is warranted to understand the exact underlying molecular and cellular mechanisms responsible for the clinical benefits. In this article, we explored the TKI failed metastatic EGFRm NSCLC, reviewed the available clinical data of ICI use in metastatic EGFRm NSCLC, and discussed its emerging role as a combination regimen in this patient population

Effect of JQ1 combined with siPD-L1 on proliferation and apoptosis of oral squamous cell carcinoma cells / 中国医师杂志

Objective:To investigate the effects of bromodomain and extraterminal domain (BET) inhibitor JQ1 combined with siPD-L1 on the proliferation and apoptosis of oral squamous cell carcinoma (OSCC) Scc-25 cells and its mechanism.Methods:Scc-25 cells were cultured in vitro and treated with different concentrations of JQ1 (0, 0.2, 1, 5 μmol/L). Cell proliferation was detected by cell count kit-8 (CCK-8) assay; the expression levels of programmed cell death ligand 1(PD-L1) and forkhead box M1(FoxM1) protein were detected by Western blot. Appropriate concentration of JQ1 was selected for subsequent experiments. Scc-25 cells were divided into four groups: control group (without any treatment), siPD-L1 group (transfected with siPD-L1), JQ1 group (added JQ1 after transfected with non-specific siRNA), and combined treatment group (added JQ1 after transfected with siPD-L1). CCK-8 assay was used to detect the proliferation ability of Scc-25 cells in each group. Western blot was used to detect the expression levels of cleaved caspase-3, PD-L1 and FoxM1, and flow cytometry was used to detect the apoptosis rate of cells in each group. Results:With the increase of JQ1 concentration, the proliferation ability of SCC-25 cells and the expression levels of PD-L1 and FoxM1 decreased gradually (all P<0.01). JQ1 concentration of 1 μmol/L had obvious inhibitory effect on cell proliferation and the expression levels of PD-L1 and FoxM1, so JQ1 concentration of 1 μmol/L was selected for subsequent experiments. The proliferation ability of Scc-25 cells, the expression of PD-L1 and FoxM1 proteins in JQ1 group, siPD-L1 group and combination treatment group were significantly lower than those in the control group (all P<0.01), and the expression of cleaved caspase-3 protein and the rate of apoptosis were significantly higher than those in the control group (all P<0.01); Moreover, the effect of the combination treatment group was more significant than that of siPD-L1 group, JQ1 group (all P<0.01). Conclusions:The combination of JQ1 and siPD-L1 could effectively inhibit the proliferation and promotes the apoptosis of OSCC Scc-25 cells, and its mechanism may be related to the suppression of PD-L1 and FoxM1 signaling pathways.

Tumor immune checkpoint therapy and the drug delivery strategies / 药学学报

Tumor immune checkpoint therapy is a clinical treatment strategy developed based on the new principle of the inhibition of negative immune regulation. In this article, the tumor immune checkpoint therapy and the drug delivery strategies were reviewed, mainly including immunity and tumor therapy, tumor immune checkpoint therapy and its mechanism of action, clinical application of tumor immune checkpoint therapy and therapeutic drugs, immune resistance of programmed cell death protein 1 (PD1)/programmed cell death ligand 1 (PDL1) treatment and countermeasures, drug delivery strategies for tumor immune checkpoint therapeutic agents, etc. As a revolutionary new immunotherapy strategy, tumor immune checkpoint therapy has shown obvious superior therapeutic efficacy in a variety types of tumor. However, tumor immune checkpoint therapy is also faced with a big challenge, namely, immunotherapy resistance. With the discovery of new mechanism, the continuous development of new therapeutic drugs and delivery strategies, tumor immune checkpoint therapy is expected to further improve the clinical efficacy of tumor.

The IL-6/STAT3 signaling pathway mediated by radiotherapy regulates the expression of PD-L1 in esophageal cancer cells / 中华肿瘤杂志

Objective: To explore the possible mechanism of radiotherapy regulating the expression of PD-L1 in esophageal carcinoma. Methods: Three esophageal cancer cell lines (Eca109, Kyse150, TE1) were irradiated with different doses of X-rays, and 6 Gy+ AG490 group was set. The mRNA expression of PD-L1 was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein expressions of PD-L1, STAT3, p-STAT3 were detected by western blotting and the protein level of IL-6 was detected by ELISA. Results: The mRNA expressions of PD-L1 in Eca109, Kyse150 and TE1 were 2.86±0.30, 960.01±21.27 and 106.78±6.67, higher than 1.07±0.15 in normal esophageal cell line HET-1A (P<0.01). The protein expressions of PD-L1 in Eca109, Kyse150 and TE1 were 0.091±0.036, 1.533±0.079 and 0.914±0.035, higher than 0.063±0.01 in normal esophageal cell line HET-1A (P<0.01). After 48 hours of 6 Gy irradiation, the protein expression levels of PD-L1 in Eca109, Kyse150 and TE1 were 0.135±0.007, 1.66±0.06 and 1.32±0.06, higher than 0.09±0.01, 1.21±0.05 and 0.93±0.03 of the 0 Gy group (P<0.01), while the protein expression levels of p-STAT3 in Eca109, Kyse150 and TE1 were 1.44±0.26, 0.75±0.04 and 1.92±0.17, higher than 0.18±0.05, 0.48±0.02 and 0.36±0.06 of the 0 Gy group (P<0.01). IL-6 protein expression increased significantly after different doses of irradiation (P<0.01). After the IL-6/STAT3 signaling pathway was blocked by the specific inhibitor AG490, the expressions of PD-L1 of Eca109, Kyse150 and TE1 in the 6 Gy+ AG490 groups were 0.11±0.03, 1.07±0.08 and 0.96±0.11, without significant differences of 0.09±0.01, 0.96±0.05 and 0.85±0.09 of the 0 Gy group (P>0.05), while the protein expressions of p-STAT3 were 0.76±0.11, 0.59±0.06 and 0.96±0.12, without significant differences of 0.67±0.08, 0.54±0.06 and 0.84±0.11 of the 0 Gy group (P>0.05). Conclusion: Radiotherapy may regulate the expression of PD-L1 in esophageal cancer cells through IL-6 / STAT3 signaling pathway.

Mechanism of mixed probiotics relieves food allergy in infant mice through the programmed cell death 1/programmed cell death ligand 1 pathway / 中华实用儿科临床杂志

Objective:To investigate the effects of mixed probiotics on food allergy and the underlying mechanism.Methods:BALB/c mice on the 15 th day of pregnancy were randomly (random number table method) classified into the control group, food allergy model group and mixed probiotics group.Mice in the food allergy model and mixed pro-biotics group were subjected to ovalbumin (OVA) sensitization after birth, and those in the mixed probiotics group were then given probiotic solution by gavage from day 21 to day 35.Mice in control group were similarly given 9 g/L saline.Twenty-four hours after the last OVA sensitization, intestinal histopathological sections were prepared to observe intestinal pathological changes.Blood smears were prepared to detect eosinophil count.In addition, serum samples were collected to measure cytokine levels and OVA specific antibodies.The number of dendritic cells (DCs) and regulatory T cells (Tregs) in mouse mesenteric lymph nodes was calculated.Differences among 3 groups were compared by the One- Way ANOVA or Kruskal- Wallis H test. Results:Compared with those of food allergy model group, diarrhea score, the ratio of eosinophils and serum levels of interleukin(IL)-4, IL-5, IL-13, mast cell protease 1 (MCPT-1), and OVA specific antibodies IgE and IgG were significantly lower in mixed probiotics group[(2.00±0.71) points vs.(3.22±0.97) points, (2.28±1.61)% vs.(10.99±2.26)%, (413.68±22.81) ng/L vs.(708.78±27.66) ng/L, (36.64±3.74) ng/L vs.(46.05±4.95) ng/L, (201.37±65.61) ng/L vs.(495.22±96.66) ng/L, (31 924.15±1 177.77) ng/L vs.(36 175.77±618.29) ng/L, (9.10±8.08) ng/L vs.(19.69±0.84) ng/L, (30.50±8.81) ng/L vs.(190.32±6.40) ng/L], while IL-10 level was significantly higher[(164.12±3.88) ng/L vs.(123.90±7.31) ng/L] ( t=3.37, 8.72, 16.07, 3.90, 7.40, 7.95, 3.91, 44.00 and 7.76, respectively, all P<0.01). Compared with those of food allergy model group, programmed cell death ligand 1 (PD-L1) level on the surface of CD 103+ DCs and CD 103+ CD 80-CD 40-DCs, the proportion of Tregs in CD4 + T cells, and the level of programmed cell death 1 (PD-1) on the surface of Tregs were significantly higher in mixed probiotics group[(75.59±0.45)% vs.(45.60±4.73)%, (67.56±1.87)% vs.(37.12±6.07)%, (8.24±0.69)% vs.(6.20±0.66)%, (11.25±3.12)% vs.(4.08±2.33)%]( t=7.88, 4.48, 3.63 and 3.71, all P<0.01). Conclusions:Mixed probiotics can alleviate the symptoms of food allergy and inflammatory response of young rats through mediating Tregs via the PD-1/PD-L1 pathway.

Excavation and evaluation of adverse reaction signals of 4 kinds of imported PD- 1/PD-L1 inhibitors / 中国药房

OBJECTIVE To exc avate and evaluate the adverse reaction signals of 4 kinds of imported programmed cell death 1 （PD-1）and its ligand （PD-L1）inhibitors，and to guide rational drug use in clinic. METHODS OpenVigil 2.1 software was used to obtain the adverse event reports of four drugs as nivolumab ，pembrolizumab，atezolizumab and durvalumab from the first quarter of 2013 to the fourth quarter of 2020 in the US FDA adverse event reporting system. The reporting odds ratio and proportional reporting ratio were used for signal mining to evaluate new or potential adverse reaction signals. RESULTS A total of 46 840 reports of adverse events with PD- 1/PD-L1 inhibitors as the primary suspected drug were collected ，including 28 896 reports of nivolumab，13 298 reports of pembrolizumab ，3 398 reports of atezolizumab ，and 1 248 reports of durvalumab. From the general characteristics of these reports ，the gender distribution was more men than women ，and the age distribution was mainly in the range of 51-85 years old. The reporting year was mainly in the nearly 4-5 years，and the main reporting countries were the US and Japan，with“death”and“hospitalization or prolonged hospitalization ”as the main serious adverse events which were over 50％ of the whole of 4 kinds of adverse events. A total of 1 597 adverse reaction signals were obtained ，involving 26 systems，focusing on “benign，malignant and unspecified neoplasms （cystic and polypoid tumor ）”，“infections and infestations ”and“investigations”， etc. The analysis of the top 50 adverse reaction signals showed that the largest number of report was endocrine system disease ，the most frequency signal was “malignant neoplasm progression ”and the strongest adverse reaction signal was “radiation pneumonitis ”. And it was also found that 13 adverse reaction signals ，such as “radiation pneumonitis ”“cholangitis”“fulminant type 1 diabetes mellitus” “blood creatine phosphokinase increased ” “disseminated intravascular coagulation ”“cardiac failure ”and “cerebral infarction ”，etc.，were not reported in the drug instructions. CONCLUSIONS PD-1/PD-L1 inhibitors mediate a large number of adverse reaction signals，resulting in high safety risks in “benign，malignant and unspecified neoplasms （cystic and polypoid tumor ）”，“infections and infestations ”and “investigations”，etc. The newly discovered 13 adverse reaction signals ，such as “radiation pneumonitis ”“cholangitis”“blood creatine phosphokinase increased ”“cardiac failure ”and“cerebral infarction ”are of great significance for guiding rational drug use in clinic.

Comparative Study of PD-L1 Expression in Different Sites of Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#The expression of programmed cell death ligand 1 (PD-L1) as a biomarker for immunotherapy in non-small cell lung cancer (NSCLC) is routinely detected in clinical pathology department. However, the spatial heterogeneity of PD-L1 expression in intrapulmonary tumors and extrapulmonary metastases is still a challenge for the clinical testing. This study aims to explore the differences of PD-L1 expression in test samples obtaining from different sites of NSCLC. This study may contribute to the detection strategy of PD-L1 in patients with advanced lung cancer.@*METHODS@#One hundred and thirty-one cases of consecutively detected PD-L1 (22c3 assay, Dako) staining in metastatic NSCLC and 972 cases of non-paired intrapulmonary NSCLC were collected. The discrepancies of tumor proportion score (TPS) of PD-L1 expression in intrapulmonary samples and extrapulmonary metastatic samples of different sites were compared.@*RESULTS@#The positive expression rate of PD-L1 in extrapulmonary metastatic NSCLC (TPS ≥ 1%) was 61.83%, and the TPS was significantly higher than that in intrapulmonary tumors (P=0.03). The PD-L1 scores of the specimens obtained from different sites were significantly different (P=0.007). The positive rates of PD-L1 in liver and adrenal metastases were 85.71% and 77.78% respectively, and their TPS were significantly higher than that of the intrapulmonary samples (P<0.05). The positive rates of PD-L1 in lymph node, bone, brain, soft tissue, and pleural metastases was 40.00%-66.67%, with no significant differences compared to intrapulmonary tumors. The analysis of histological subtype and sample type showed that the PD-L1 score of extrapulmonary samples of adenocarcinoma subtype or surgical specimen was significantly higher than that of intrapulmonary tumors. The analysis of clinicopathological parameters showed that the PD-L1 positive expression or high expression were significantly correlated with male patients, smoking history, and epidermal growth factor receptor (EGFR) wild type.@*CONCLUSIONS@#The expression of PD-L1 in metastatic NSCLC is generally higher than that in intrapulmonary tumor, and the positive rate of PD-L1 expression was discrepant in different sites of specimen. The differences of PD-L1 score between extrapulmonary metastatic samples and intrapulmonary samples may be associated with different metastatic sites, histological subtype, and specimen type.

Desbalances genómicos del locus 9p24.1 en pacientes argentinos con linfoma de Hodgkin clásico / Genomic imbalances at 9p24.1 locus in Argentine patients with classical Hodgkin lymphoma

Resumen Introducción: El linfoma de Hodgkin clásico presenta escasas células de Reed Sternberg/Hodgkin inmersas en un abundante microambiente tumoral. Los desbalances genómicos del locus 9p24.1 han sido asociados con alteraciones en la expresión de los genes del ligando de muerte celular 1 y 2, ambos reguladores de la respuesta inmune. Objetivo: Evaluar desbalances genómicos del locus 9p24.1 en células de Reed Sternberg/Hodgkin y del microambiente tumoral en biopsias de pacientes con linfoma de Hodgkin clásico y correlacionarlo con la expresión del ligando de muerte celular 1 y la presentación de la enfermedad. Materiales y Métodos: Se efectuó hibridación in situ en biopsias de 22 pacientes con linfoma de Hodgkin clásico dirigida a los genes del ligando de muerte celular 1 y 2. Las alteraciones se clasificaron en: amplificación, ganancia y polisomía. La expresión se evaluó mediante inmunohistoquímica. Resultados: Todos los pacientes mostraron alteraciones del número de copias. Se diferenciaron dos grupos: con amplificación (32%) y sin amplificación (68%); este último subdividido en: rico en ganancia (53%) y rico en polisomías (47%). El grupo rico en polisomías mostró mayor edad (p=0,027). El 40% de los pacientes con amplificación y rico en ganancias no presentó masa bulky. La expresión proteica mostró score +3 sólo en estos últimos. El title% de los casos ricos en polisomías presentaron monosomía del cromosoma 9 en los linfocitos circundantes respecto al 36,4% de los otros dos grupos. Conclusiones: Nuestros datos constituyen un aporte a la caracterización biológica del LHC, de interés en el marco de las nuevas modalidades terapéuticas. MÉD.UIS.2020;34(1):35-44.

Abstract Background: Classical Hodgkin lymphoma shows scarce tumor Reed-Sternberg/Hodgkin cells surrounded by a dense immune microenvironment. Genetic alterations at the 9p24.1 locus result in genomic imbalances in the copy number of PD-L1/PD-L2 genes, both of them being immune response regulators. Aim: To characterize genomic imbalances at the 9p24.1 locus in Reed-Sternberg/Hodgkin cells and immune microenvironment in biopsies of patients with Classical Hodgkin lymphoma and correlate it with PD-L1 protein expression and disease presentation. Material and Methods: Paraffin embedded biopsies of 22 patients with CHL were retrospectively evaluated by fluorescence in situ hybridization using SPEC CD274/PDCD1LG2/CEN9 DNA probe. The frequency of 9p24.1 alterations, amplification, copy gain and polysomy, were determined taking into account the number of gene copies with respect to the centromere. PD-L1 protein expression was evaluated by immunohistochemistry. Results: All cases presented alterations in the number of copies of PD-L1 / PD-L2, which are differentiated in two groups: with amplification (32%) and without amplification (68%). The latter was subdivided into rich in gains (RG) (53%) and rich in polysomies (RP) (47%). Groups with amplification and RG were younger than the RP group (p = 0.027). The latter was not associated with bulky disease, a fact observed in 40% of patients with amplification and RG. Protein expression showed score +3 only in the latter. All RP cases presented chromosome 9 monosomy in the surrounding lymphocytes, compared to 36.4% of the other two groups. Conclusions: Our data contributes to the biologic characterization of CHL, of interest in the context of new therapeutic modalities. MÉD.UIS.2020;34(1):35-44

Effect of dendritic cells on immune function regulated by programmed cell death-1/programmed cell death-ligand 1 in sepsis / 中华危重病急救医学

Objective:To study the effect of programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) regulating dendritic cells (DC) on the immune status of sepsis, and analyze the effect of PD-1/PD-L1 on prognosis.Methods:Twenty-five patients with sepsis in the intensive care unit (ICU) of the Affiliated Hospital of Zunyi Medical University from October 2018 to September 2019 were collected and followed up for 28 days. According to the 28-day survival of patients, patients were divided into survival group and death group. Among them, 10 cases were in the survival group and 15 cases were in the death group. Simultaneously, 20 healthy subjects in our hospital during the same period served as the healthy control group. Peripheral blood of patients with sepsis was taken within 24 hours after diagnosis, and the healthy control group was taken at the time of enrollment. Flow cytometry was used to detect the proportion of CD4 +T and CD8 +T cells, the ratio of T cell subsets (CD4/CD8), the expression of PD-1 on CD4 +T and CD8 +T cells, and the expression of PD-L1 and CD86 in DC. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of interleukin-10 (IL-10) and tumor necrosis factor-α(TNF-α) in serum. Spearman correlation analysis was used to analyze the correlation between CD11c +PD-L1 and CD4 +PD-1, CD8 +PD-1, TNF-α, DC, CD11c +CD86, T cell subpopulation ratio, CD4 +T cells, CD8 +T cells, and IL-10. Binary Logistic regression was used to analyze the risk factors affecting the death of patients with sepsis, and receiver operator characteristic curve (ROC curve) was drawn to evaluate the predictive value of independent risk factors on the prognosis of patients. Results:The scores of acute physiology and chronic health evaluationⅡ (APACHEⅡ) and sequential organ failure assessment (SOFA) in the death group were higher than that in the survival group (APACHEⅡ score: 27.0±7.3 vs. 17.0±3.9, SOFA score: 15.1±4.1 vs. 10.7±2.7, both P < 0.05). The ratio of T cell subsets in the survival group and the death group was less than 1, the death group was lower than that in the survival group (CD4/CD8: 0.54±0.15 vs. 0.79±0.09, P < 0.05), and the ratio of T cell subsets in the healthy control group was greater than 1. Compared with healthy control group, the levels of CD4 +T cells, CD8 +T cells, CD11c +DC, CD11c +CD86, IL-10 and TNF-α in survival group and death group were significantly decreased, the level of CD4 +PD-1, CD8 +PD-1, CD11c +PD-L1 were significantly increased, and the changes in the above indicators in the death group were significant compared with the survival group [CD4 +T cells: 0.14±0.07 vs. 0.22±0.08, CD8 +T cells: 0.24±0.07 vs. 0.28±0.10, CD11c +DC: 0.84±0.14 vs. 0.93±0.03, CD11c +CD86: (58.83±20.77)% vs. (78.24±9.39)%, IL-10 (ng/L): 34.22±13.98 vs. 18.49±5.55, TNF-α(ng/L): 95.30±29.33 vs. 67.00±20.16, CD4 +PD-1: (39.58±10.08)% vs. (27.03±6.35)%, CD8 +PD-1: (38.77±11.91)% vs. (29.15±8.37)%, CD11c +PD-L1: (21.13±11.54)% vs. (12.11± 8.34)%, all P < 0.05]. Spearman correlation analysis showed that CD11c +PD-L1 was positively correlated with CD4 +PD-1, CD8 +PD-1, and IL-10 ( r values were 0.748, 0.713, 0.898, all P < 0.05), while was negatively correlated with DC, CD11c +CD86, T cell subpopulation ratio, CD4 +T cells, CD8 +T cells, and TNF-α( r values were -0.587, -0.906, -0.840, -0.706, -0.513, -0.820, all P < 0.05). Multivariate binary Logistic regression analysis showed that CD4 +T PD-1 was an independent risk factor for the prognosis of sepsis patients [odds ratio ( OR) = 1.463, 95% confidence interval (95% CI) = 1.032-2.074, P = 0.033]. ROC curve analysis showed that CD4 +TPD-1 had certain predictive value for the prognosis of patients with sepsis [area under ROC curve (AUC) = 0.857, 95% CI was 0.709-1.000, P < 0.01). When the best predictive value was 34.48%, the sensitivity, specificity, and accuracy were 66.7%, 90.0%, and 85.7% respectively. Conclusions:Up-regulation of PD-1/PD-L1 in peripheral blood could inhibit the activation and proliferation of DC, affect the activation of T cells, and induce immunosuppressive state. PD-1/PD-L1 can reflect the immune status of patients with sepsis. The expression of PD-1 on CD4 +T cells may have important significance for the evaluation of prognosis.

Cardiotoxicity of Anti-PD-L1 Antibody and the Effect of Levothyroxine in Attenuating the Related Mortality in Mice / 中国肺癌杂志

BACKGROUND@#Immune checkpoint inhibitors (ICIs) such as antibodies against programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1), have shown remarkable efficacies in many subtypes of cancers. However, ICIs may also cause severe immune-related adverse events in the recipient patients. Recently, ICI-associated myocarditis have been reported in hundreds of patients worldwide, with a mortality rate of approximately 50% in these cases. This study aims to recapitulate the cardiotoxicity and explore the detoxicifying approaches to attenuate mortality caused by PD-1/PD-L1 inhibitors in healthy mice.@*METHODS@#Six to eight-week-old C57BL/6 mice were inoculated with anti-PD-1 antibody (12.5 μg/g every 5 days for 6 injections), anti-PD-L1 antibody (10 μg/g once a week for 6 weeks), anti-PD-L1 antibody (with the same dosage described above) in combination with levothyroxine (0.25 μg/g, intraperitoneally injected half an hour before anti-PD-L1 antibody injection), or isotype control immunoglobulin IgG (10 μg/g once a week for 6 weeks). The ejection function of the hearts was detected by echocardiography, body temperature and blood pressure were detected by Mouse MonitorTM and non-invassive blood pressure minotor, and serum free thyroxine concentration was detected by The enzyme linked immunosorbent assay (ELISA).@*RESULTS@#PD-L1 was expressed at different levels by the cardiomyocytes of the mice. The isotype control immunoglobulin and anti-PD-1 antibody did not cause death of the mice. The 12 mice receiving 3-6 injections of anti-PD-L1 antibody showed a significant increase in the heart-to-tibial ratio and cardiomyoctye degeneration, hyalinization and extravascular inflammatory cell infiltration. In addition, the serum thyroxine was mardedly decreased to 1/3 of that in the control group mice, and the blood pressure and body temperature were abnormally decreased in mice upon treatment with PD-L1 blockade. Eight of the 12 (66.7%) mice died from multiple intravenous injection of anti-PD-L1 antibody.Intraperitoneal injection of levothyroxine 30 min before the injection of anti-PD-L1 antibody significantly attenuated the mortality rate of the anti-PD-L1 antibody-treated mice.@*CONCLUSIONS@#The anti-PD-L1 antibody is cardiotoxic and lethal, and levothyroxine is able to rescue the mice from this immune checkpoint inhibitor-caused mortality.

Clinical utility of PD-L1 expression in circulating tumor cells in non-small cell lung cancer patients treated with immunotherapy / 中国胸心血管外科临床杂志

@# Lung cancer is the most frequent cancer and the leading cause of cancer death all around the world. Anti-programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) therapies have significantly improved the outcomes of non-small cell lung cancer (NSCLC) patients in recent years. However, the objective response rate in non-screened patients is only about 20%. It is very important to screen out the potential patients suitable for immunotherapy. Immunohistochemical staining of tumor tissue biopsies with PD-L1 antibodies can predict the therapeutic response to immunotherapy to some extent, but it still has some limitations. Recently some clinical studies have shown that PD-L1 expression in circulating tumor cells (CTC-PD-L1) is a potential independent biomarker and may provide important information for immunotherapy in NSCLC. This article will review technology for CTC-PD-L1 detection and the predictive value of CTC-PD-L1 for immunotherapy in NSCLC and review the latest clinical research progress.

CD45RO⁺ Memory T Lymphocytes As A Candidate Marker for Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#Lung cancer is the most common malignancy world-wide. There are a variety of immune infiltrating cells in tumor microenvironment, which is an important component of tumor immunity and has clinical significance for the prognosis of patients. CD45RO is a surface marker of memory T cells. The expression of CD45RO⁺ tumor infiltrating lymphocytes (TILs) is associated with the prognosis of many tumors. The purpose of this study was to evaluate the relationship between the density of CD45RO⁺ TILs in tumor and stromal area and the clinical characteristics of patients with non-small cell lung cancer (NSCLC) and its impact on the prognosis of patients. We aimed to explore the clinical value of CD45RO⁺ TILs and programmed cell death ligand 1 (PD-L1) as prognostic markers.@*METHODS@#Multiple fluorescent immunohistochemical staining was used to stain the tissue microarray chips of 167 patients with NSCLC, marking CD45RO, cytokeratin (CK) and PD-L1. Using artificial intelligence image recognition technology and tumor cell-specific CK staining, divide the tumor and stromal area in the tissue, evaluate the density of CD45RO⁺ TILs in the tumor and stromal area, and the expression level of PD-L1 in tumor cells. The non-parametric test was used to analyze the relationship between CD45RO⁺ TILs and the clinical characteristics of patients, and the Kaplan-Meier method and Cox risk ratio model were used to analyze the relationship between CD45RO⁺ TILs independently or in combination with PD-L1 and tumor prognosis.@*RESULTS@#The density of CD45RO⁺ TILs was significantly associated with patient age, smoking, tumor stage, and pathological type. Single-factor survival analysis showed that NSCLC (P=0.007) stromal region and lung adenocarcinoma (LUAD) (P<0.001) with CD45RO⁺ TILs high density had better OS. Multivariate survival analysis showed that the high density of CD45RO⁺ TILs in the stromal region of NSCLC (HR=0.559, 95%CI: 0.377-0.829, P=0.004) and lung adenocarcinoma (HR=0.352, 95%CI: 0.193-0.641, P=0.001) were independent prognostic factors for overall survival time (OS). Combined with PD-L1 score of tumor cells in tumor tissues and infiltration score of CD45RO⁺ TILs in all tumor tissues, the patients were divided into 4 groups: patients with PD-L1⁺/CD45RO⁺ had the longest disease-free survival (DFS) time, and patients with PD-L1⁺/CD45RO- had the shortest DFS time. Multivariate Cox regression analysis showed that PD-L1⁺/CD45RO- was an independent prognostic factor for DFS and had a higher risk of poor prognosis compared to the other three groups (HR=2.221, 95%CI: 1.258-3.919, P=0.006).@*CONCLUSIONS@#In tumor tissues, the density of CD45RO⁺ TILs, as well as the combination of CD45RO⁺ TILs and PD-L1 in tumor areas, significantly correlated with clinicopathological features and prognosis of NSCLC, which can be used as a new prognosis marker.

Evaluation of Response to Immune Checkpoint Inhibitor Monotherapy or Combination with Chemotherapy for Patients with Advanced Non-small Cell Lung Cancer and High PD-L1 Expression / 中国肺癌杂志

BACKGROUND@#Immunotherapy represented by immune checkpoint inhibitors (ICIs) has been widely used in the treatment of lung cancer. There are controversies in clinical practice for patients with advanced non-small cell lung cancer (NSCLC) and high programmed cell death-ligand 1 (PD-L1) expression receiving ICIs monotherapy or combination chemotherapy.@*METHODS@#This study retrospectively analyzed the clinical data of 49 patients with advanced NSCLC and high PD-L1 expression. Immunohistochemistry was performed with 22C3 antibody, and the expression level of PD-L1 was evaluated according to tumor proportion score (TPS). Objective response rate (ORR) and progression free survival (PFS) were compared by groups of different clinical characteristics.@*RESULTS@#ORR of monotherapy and combination therapy group was 47.1% (8/17) and 43.8% (14/32), respectively, without statistical difference (P=0.825). The median PFS of monotherapy and combination therapy group was 8.0 months and 6.8 months, respectively, without statistical difference (P=0.502). Statistical analysis of predictors of immunotherapy for the patients showed first-line immunotherapy had better ORR than subsequent immunotherapy (12/19, 63.2% vs 10/30, 33.3%, P=0.041), however no difference in PFS. And there were no differences in ORR or PFS among groups of age, gender, smoking status, performance status (PS), pathological type, tumor size and tumor-node-metastasis (TNM) stage.@*CONCLUSIONS@#The therapeutic effect is similar between ICIs monotherapy and combination chemotherapy for patients with advanced NSCLC and high PD-L1 expression. ORR of first-line immunotherapy was better in patients with advanced NSCLC and high PD-L1 expression. The optimal treatment for this population remains further prospective clinical studies.

Expression of PD-1/PD-L1 in esophageal squamous cell carcinoma and its relationship with survival prognosis: A systematic review and meta-analysis / 中国胸心血管外科临床杂志

@# Objective    To systematically evaluate the expression of programmed cell death receptor 1 (PD-1) and programmed cell death ligand 1 (PD-L1) in esophageal squamous cell carcinoma and its relationship with prognosis. Methods    The literature from PubMed, EMbase, The Cochrane Library, Web of Science, CNKI and Wanfang data from inception to February 22, 2020 was searched by computer. Data were extracted and the quality of literature was evaluated using RevMan 5.3 software for meta-analysis. Egger's and Begg's tests were used to evaluate publication bias, and Stata 15.1 software was used for sensitivity analysis. Results     A total of 16 articles were included, and there were 3 378 patients with esophageal squamous cell carcinoma. The methodological index for nonrandomized studies (MINORS) scores were all 12 points and above. The meta-analysis results showed that the positive expression rates of PD-1 and PD-L1 in tumor cells were 37.8% (190/504) and 41.7% (1 407/3 378), respectively. The positive expression of PD-L1 in tumor immune infiltrating cells was 41.7% (412/987). The overall survival (OS) of the tumor cell with high PD-L1 expression was lower than that with low PD-LI expression (HR=1.30, 95%CI 1.01-1.69, P=0.04). The OS of the tumor immune infiltrating cell with high PD-L1 expression was significantly higher than that with low PD-LI expression (HR=0.65, 95%CI 0.53-0.80, P<0.000 1). Conclusion    PD-L1 has a high expression rate in esophageal squamous cell carcinoma and is an important factor for the prognosis of esophageal squamous cell carcinoma.

Chlorogenic acid down-regulates the expression of PD-L1 in esophageal squamous cell carcinoma via IFN-γ signaling pathway / 药学学报

In this study, the regulatory effects of chlorogenic acid (CGA) on the expression of programmed cell death ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC), as well as the role of interferon γ (IFN-γ), has been discussed using both in vitro and in vivo animal models. ESCC murine model was established according to the standard operating procedures (SOP) of Animal Experiment Center of Institute of Materia Medica, Chinese Academy of Medical Sciences. The expression of PD-L1 in esophageal tissues of murine models was analyzed using the microarray assay. Then, the results were verified by qRT-PCR, Western blot and immunohistochemistry (IHC) staining, the molecular mechanism was explored in KYSE180 and KYSE510 ESCC cells in vitro. The results showed that CGA could suppress the expression of PD-L1 in tumor tissues in murine models significantly, rather than the expression in KYSE180 and KYSE510 ESCC cells in vitro. However, after the pretreatment of IFN-γ, the expression of PD-L1 was significantly increased, then it was down-regulated by CGA in both dose- and time-dependent manner. Meanwhile, the expression of interferon regulatory factor 1 (IRF1), an upstream regulatory factor of PD-L1, was suppressed by CGA in both KYSE180 and KYSE510 pretreated with IFN-γ, which was consistent with the expression of PD-L1. These results indicate that CGA down-regulates the expression of PD-L1 in ESCC via IFN-γ-IRF1 signaling pathway, providing the molecular theoretical basis for exploration of new treatment of ESCC.