Immune checkpoint ligands expressed on mature high endothelial venules predict poor prognosis of NSCLC: have a relationship with CD8+ T lymphocytes infiltration.

Background: An insufficient number of intratumoral CD8+ T lymphocytes is a major barrier to antitumor immunity and immunotherapy. High endothelial venules (HEVs) are the major sites through which lymphocytes enter tumors; however, the molecular mechanism through which HEVs mediate CD8+ T lymphocyte infiltration remains poorly understood. Methods: Forty-two patients with stage IIIA lung adenocarcinoma, who underwent surgery, were recruited. Multiplex immunohistochemical staining was conducted on tumor tissues to detect the immune checkpoint ligands (ICLs) expressed in the HEVs, blood vessels, and lymphatics. A new ICL score model was constructed to evaluate ligand expression. The relationship between ICL score, tumor-infiltrating CD8+ T cell frequency, and survival of patients was investigated. Results: Mature HEVs, but not blood vessels or lymphatics, mediated CD8+ T cell infiltration. However, the ICLs expressed on mature HEVs could negatively regulate CD8+ T cell entry into tertiary lymphoid structures (TLSs). In addition, according to the results obtained using our ICLtotal score model, the expression of ICLs on HEVs was observed to be a predictor of both CD8+ T cell infiltration and survival, in which a high ICLtotal score > 1 represent a weak CD8+ T cell infiltration and a high ICLtotal score > 2 predicts poor survival. Conclusion: Using the ICL score model, we discovered that ICLs expressed on HEVs are indicative of CD8+ T cell subset infiltration in TLSs, as well as of patient survival with lung cancer.

Effect of Chemotherapeutics on In Vitro Immune Checkpoint Expression in Non-Small Cell Lung Cancer.

Objectives: Immune checkpoint (ICP) expression in tumor cells could directly or indirectly affect the results of immunotherapy. ICP ligands on tumor cells usually bind their immune cell receptors to inhibit the activity, resulting in tumor immune escape. Thus, the purpose of this study was to ascertain the impact of various chemotherapeutic drugs on ICP expression in non-small cell lung cancer (NSCLC) cell lines with different pathological subtypes to provide a basis for the development of a superior regimen of chemotherapy combined with ICP blockade. Methods: Several first-line chemotherapy agents (cisplatin, carboplatin, paclitaxel, gemcitabine, vinorelbine, and pemetrexed) were selected to treat different NSCLC cell lines (squamous carcinoma H1703, adenocarcinoma A549, and large cell cancer H460) for 72 hours, and then the changes in ICP expression in the tumor cells were observed through flow cytometry. Results: Cisplatin, carboplatin, and paclitaxel upregulated the expressions of programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2) in A549 and H460 cell lines. Meanwhile, vinorelbine and pemetrexed upregulated PD-L1 and PD-L2 in H1703, A549, and H460 cell lines. Paclitaxel, gemcitabine, vinorelbine, and pemetrexed significantly upregulated the expressions of both galectin-9 and high-mobility group box protein 1 (HMGB1) in the A549 cell line. Cisplatin and paclitaxel significantly upregulated the expressions of major histocompatibility complex-II (MHC-II), galectin-3, α-synuclein, and fibrinogen-like protein 1 (FGL1) in A549 and H460 cell lines. In addition, cisplatin and vinorelbine significantly upregulated the expressions of both CD155 and CD112 in the H460 cell line. Vinorelbine upregulated MHC-I in all three cell lines. Conclusion: Chemotherapy agents have different effects on the expression of ICP ligands in tumor cells with different pathological types, and this may affect the efficacy of combined immunotherapy. These results provide a theoretical basis for further selection and optimization of the combination of chemotherapy and immunotherapy.

TP53 Exon 5 Mutation Indicates Poor Progression-Free Survival for Patients with Stage IV NSCLC.

BACKGROUND: Genetic mutations are quite common in non-small cell lung cancer (NSCLC), however, their prognostic value remains controversial. METHODS: This study explored the mutational landscape of tumor samples from patients with advanced NSCLC by next-generation sequencing (NGS). A total of 101 NSCLC patients in stage III or IV receiving first-line treatment were included. RESULTS: TP53 mutation was the most frequent genetic alteration in NSCLC tumors (68%), followed by EGFR (49%), CDKN2A (12%), LRP1B (9%), and FAT3 (9%) mutations. Among 85 patients with stage IV NSCLC, first-line targeted therapy remarkably prolonged progression-free survival (PFS) of patients compared with first-line chemotherapy (p = 0.0028). Among 65 patients with stage IV NSCLC whose tumors harbored EGFR, ALK, ROS, or BRAF mutations, first-line targeted therapy substantially prolonged the PFS of patients (p = 0.0027). In patients with TP53 mutations who received first-line targeted therapy or chemotherapy, missense mutation was the most common mutation type (36/78), and exon 5 represented the most common mutated site (16/78). CONCLUSIONS: TP53 mutation in exon 5 could independently predict poor PFS of patients with stage IV NSCLC after the first- line treatment. Moreover, mutations in TP53 exon 5 and LRP1B were associated with shorter PFS of such patients whether after first-line chemotherapy or targeted therapy, respectively. Thus, these patients should be given immunotherapy or immunochemotherapy.

DC101, an anti-VEGFR2 agent, promotes high-endothelial venule formation and immune infiltration versus SAR131675 and fruquintinib.

There is an increasing interest in combining immune checkpoint inhibitors (ICIs) with anti-angiogenic drugs to enhance their anti-tumor effects. In this study, three anti-angiogenic agents, DC101 (acting on VEGFR2), SAR131675 (acting on VEGFR3), and fruquintinib (a small-molecule inhibitor acting on multiple targets) were administered to B16F1-OVA-loaded C57BL/6 mice. Immune cells infiltration in the tumor tissues, vascular normalization, and high-endothelial venule (HEV) formation were assessed to provide evidence for drug combination. Both DC101 and fruquintinib significantly slowed the melanoma growth and increased the proportion of CD3+ and CD8+ T cells infiltration compared with SAR131675, of note, the effect of DC101 was more pronounced. Moreover, DC101 and fruquintinib increased the interferon-γ and perforin levels, meanwhile, DC101 increased the granzyme B levels, whereas fruquintinib and SAR131675 did not. Only the fruquintinib-treated group showed decreased regulatory T cells infiltration. We found upregulation of PD-L1 expression in tumor cells and CD45+ immune cells in DC101-treated group as well as upregulation of PD-1 expression on CD3+ T cells. However, fruquintinib only increased PD-L1 expression in tumors. Both DC101 and fruquintinib reduced the proportion of CD31+ vessels, while DC101 increased the ratio of α-SMA +/CD31+ cells and reduced the expression of HIF-1α more than fruquintinib. Moreover, DC101 enhanced the infiltration of dendritic cells and B cells, and local HEV formation. In conclusion, our data indicate that DC101 may be a better choice for the combined clinical application of ICIs and anti-angiogenic agents.

Prediction of immune-related adverse events in non-small cell lung cancer patients treated with immune checkpoint inhibitors based on clinical and hematological markers: Real-world evidence.

Clinical and hematological parameters can predict immune-related adverse events (irAEs) caused by immune checkpoint inhibitors (ICIs). However, the exact correlation between these parameters and irAEs is unclear. This study aimed to establish a prediction model for irAEs in patients with non-small cell lung cancer (NSCLC) treated with ICIs. This retrospective study included patients with NSCLC treated with a minimum of one dose of ICIs at the Tianjin Medical University Cancer Hospital and Shanxi Bethune Hospital from 2016 to 2020. Baseline characteristics, treatment details, and adverse events were evaluated. The Student's t-test, Chi-square test, and logistic regression were used to identify risk factors for irAEs to establish a prediction model. A total of 667 patients were included; the median age was 62.47 (range, 27-85) years. Most patients were men (74.5%) with stage IV cancer (93.1%). The incidence of any grade and grade 3 or higher irAEs was 21.74% (145/667) and 5.25% (35/667), respectively. A total of 145 patients experienced 220 irAEs; the incidence of endocrinopathies (35.91%, 79/220) was highest in all grade irAEs, while that of pneumonitis (7.73%, 17/220) was the highest in grade 3 or higher irAEs. A prediction model based on treatment lines, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), absolute lymphocyte count (ALC), and systemic immune inflammation index was established. The area under the receiver operator characteristic curve was 0.722 (95% confidence interval: 0.650-0.793), with a cut-off value of 0.247 and a sensitivity and specificity of 62.9% and 74.6%, respectively. The multivariate logistic regression analysis showed that the risk of irAEs was higher in patients undergoing second-line therapy than in those undergoing treatment with adjuvant therapy (odds ratio [OR] = 8.239, p = 0.011). AST (OR = 1.053, p = 0.007) and ALC (OR = 2.556, p = 0.001) showed a positive correlation with the risk of irAEs, while LDH showed a negative correlation with irAEs (OR = 0.994, p = 0.007). The model showed good prediction efficiency, whereas the treatment lines, AST, ALC, and LDH were independent risk factors for the onset of irAEs.

[Effects of tillage mode on black soil's penetration resistance and bulk density].

Taking an eight-year field experiment site in Dehui County of Jilin Province, Northeast China as test object, this paper studied the effects of different tillage modes (no tillage and ploughing in autumn) on the penetration resistance and bulk density of black soil. No tillage increased the soil penetration resistance, especially at the soil depth of 2.5-17.5 cm. In the continuous cropping of maize and the rotation of maize-soybean, the maximum soil penetration resistance at planting zone under no tillage and ploughing in autumn was 2816 and 1931 kPa, and 2660 and 2051 kPa, respectively, which had no restriction on the crop growth. The curve of soil penetration resistance under ploughing in autumn changed with ridge shape, while that under no tillage changed less. Comparing with ploughing in autumn, no tillage increased the bulk density of 5-20 cm soil layer significantly. Under no tillage, the bulk density of 5-30 cm soil layer changed little, but under ploughing in autumn, soil bulk density increased gradually with increasing soil depth. There was no significant correlation between soil bulk density and soil penetration resistance.

[Prediction of soil organic carbon in different soil fractions of black soils in Northeast China using near-infrared reflectance spectroscopy].

The soil organic carbon (SOC) associated with different soil fractions varies in the composition and dynamics. The present work is aimed to evaluate the potential of near infrared spectroscopy (NIRS) to predict SOC content in different soil fractions of black soils. SOC contents of 136 black soil samples in China were analyzed and the NIR spectra were collected using a VECTOR/22 (Fourier transform infrared spectroscopy). Partial least squares (PLS) regression with cross validation was used to develop calibrations between reference data and NIRS spectra (n = 100) which were validated using an independent set of samples (n = 36). Predictions for water-sieved aggregate associated organic carbon were generally good with R2 (coefficient of determination) ranging from 0.69 to 0.82 and the RPD (residual prediction deviation) from 1.2 to 1.8. NIRS well predicted the SOC in < 53 microm mineral fraction (R2 = 0.97, RPD = 5.4), but the prediction for SOC in 250-2 000 microm or in 53-250 microm particulate matter fractions was poor. However, the prediction for the SOC in 53-2 000 microm fraction was good (R2 = 0.79, RPD = 2.2). In addition, NIRS very well predicted the SOC in fine particle fraction (< 20 microm) (R2 = 0.93, RPD = 3.8). Accordingly, NIRS showed a good potential to predict SOC in some soil fractions and could reduce tedious laboratory analysis.

[Field sampling methods for earthworm: a review].

As a critical component of soil ecosystem, earthworm can improve soil structure and relates closely to soil nutrient cycling, playing an important role in promoting soil quality and productivity. However, there is lack of systematic study on the field sampling methods for earthworm, especially in China. This paper reviewed the operational processes of commonly used field sampling methods for earthworm, and discussed their corresponding merits, efficacy, and potential influence on research results. To achieve a complete and accurate characterization of earthworm community size and structure, the method of chemical repellent combined with hand-sorting could work well at the sites where physical disturbance was acceptable, while the AITC (allyl isothiocyanate) method would be a favorable option at the sites where soil destruction was not feasible.

[Near infrared spectroscopy in determining organic carbon and total nitrogen in black soil of Northeast China].

In this study, near infrared reflectance spectroscopy (NIRS) was used to determine the organic carbon (OC), total nitrogen (TN), and C/N ratio in black soil of Northeast China. Based on the 3699-12000 cm(-1) NIRS of 136 black soil samples collected in 2004-2005, and by using partial least square (PLS), the related quantitative models were established. Leave-one-out cross validation showed that the OC and TN were well predicted, with the values of coefficient of determination (R2) being 0.92 and 0.91, RPD (the ratio of standard deviation of validation set to root mean square error of cross validation) being 3.45 and 3.36, and correlation coefficient (r) being 0.94 and 0.93 respectively, suggesting that NIRS had the potential to predict the OC and TN in black soil of Northeastern China. However, the C/N ratio was poorly predicted, with R2 = 0.61, RPD = 1.61, and r = 0.74, indicating that NIRS could not give reasonable prediction for the C/N ratio in black soil.

[Characters of infiltration and preferential flow of black soil in Northeast China under different tillage patterns].

By using dye tracer and double-ring infiltrometer techniques, the characters of infiltration and preferential flow of black soil under no-tillage (NT) and fall moldboard plow (MP) were compared after six years continuous management. The results showed that the infiltration rate was higher under NT than under MP. When the infiltration reached steady, the infiltration rate and accumulative infiltration capacity under NT were 1.35 and 1.44 times as high as those under MP, respectively. The penetration depth of methylene blue reached a depth of 43 cm in NT soil, being 16 cm deeper than that in MP soil. Comparing with MP soil, NT soil had better development of pore structure and more biological pores, and presented better preferential flow character, which were of importance for water infiltration and soil and water conservation.