Assessment of the circulating levels of immune system checkpoint selected biomarkers in patients with lung cancer.

BACKGROUND: Lung cancer is recognized as one of the leading causes of cancer-related deaths globally, with a significant increase in incidence and intricate pathogenic mechanisms. This study examines the expression profiles of Programmed Cell Death Protein 1 (PD-1), PD-1 ligand (PDL-1), ß-catenin, CD44, interleukin 6 (IL-6), and interleukin 10 (IL-10), as well as their correlations with the clinic-pathological features and diagnostic significance in lung cancer patients. METHODS AND RESULTS: The research involved lung cancer patients exhibiting various pathological characteristics, alongside demographically matched healthy controls. The expression levels of PD-1, PDL-1, ß-catenin, and CD44 were analyzed using Real-Time PCR, while circulating levels of IL-6 and IL-10 were assessed through ELISA assays. This investigation focused on peripheral blood mononuclear cells (PBMC) to evaluate these factors non-invasively. Findings indicated that levels of PD-1, PDL-1, and CD44 were significantly elevated in patients compared to controls, which coincided with a decrease in ß-catenin levels. Additionally, a concurrent rise in IL-6 and IL-10, both pro-inflammatory cytokines, was observed in patients, suggesting a potential regulatory role for these cytokines on the PD-1/PDL-1 axis, which may help tumors evade immune system checkpoints. The predictive value of these factors concerning lung tumors and metastasis was significant (Regression analysis). Furthermore, these markers demonstrated diagnostic potential in differentiating between patients and healthy controls, as well as between individuals with metastatic and non-metastatic tumors (ROC curve analysis). CONCLUSIONS: This study provides insights into the expression profiles of PD-1/PDL-1 immune system checkpoints and their regulatory factors in lung cancer, potentially paving the way for new therapeutic and diagnostic approaches.

Prognostic value of soluble programmed death-1 and soluble programmed death ligand-1 in severe traumatic brain injury patients.

Patients with traumatic brain injury (TBI) frequently exhibit concomitant immunosuppression. In this study, we evaluated the predictive values of soluble programmed death-1 (sPD-1) and soluble programmed death ligand-1 (sPD-L1) in patients with severe TBI. Peripheral blood sPD-1 and sPD-L1 levels were measured within 48 h of patient admission. A total of 20 healthy volunteers and 82 patients were enrolled in this study. The levels of sPD-1 and sPD-L1 were upregulated in patients with severe TBI (P < 0.001). They were significantly increased in the post-TBI severe pneumonia group and among non-survivors (P < 0.001). The area under the curves (AUCs) for sPD-1 and sPD-L1 levels to predict severe pneumonia were 0.714 and 0.696, respectively, and the AUCs to predict mortality were 0.758 and 0.735. The levels of sPD-1 and sPD-L1 are correlated with the GCS scores at admission, APACHE II scores, length of MV, and time elapsed to mortality. The levels of sPD-1 and sPD-L1 emerged as independent predictive factors for severe pneumonia and mortality. This study demonstrates that upregulation of sPD-1 and sPD-L1 in severe TBI patients is significantly associated with severe pneumonia and mortality, suggesting their potential as predictive biomarkers for these outcomes.

Complete Blood Count-Based Biomarkers as Predictors of Clinical Outcomes in Advanced Non-Small Cell Lung Cancer Patients with PD-L1 < 50% Treated with First-Line Chemoimmunotherapy.

Background: The aim of the study was to investigate a series of complete blood cell count-based biomarkers of systemic inflammation as predictors of clinical outcomes in patients who underwent first-line chemoimmunotherapy for advanced NSCLC. Methods: Consecutive patients with pathologically diagnosed stage III/IV NSCLC and PD-L1 < 50% who underwent first-line chemoimmunotherapy were retrospectively enrolled. The clinical outcomes used for biomarker evaluation were Objective Response Rate (ORR) and Overall Survival (OS). Results: Non-responders had significantly higher values of neutrophil to lymphocyte ratio (NLR, median: 5.36; IQR: 2.78-10.82 vs. 3.31; IQR: 2.15-4.12, p = 0.019), neutrophil to monocyte ratio (NMR, median: 14.00; IQR: 8.82-21.20 vs. 9.20; IQR: 7.45-11.20, p = 0.013), and systemic inflammation index (SII, median: 1395; IQR: 929-3334 vs. 945; IQR: 552-1373, p = 0.025), but only NLR and NMR remained independently associated with clinical response in multivariate logistic regression. In the univariate analysis, white blood cells (OR:1.2202; 95% CI: 1.0339-1.4400, p = 0.019), neutrophils (OR:1.2916; 95% CI: 1.0692-1.5604, p = 0.008), NLR (OR:1.3601: 95% CI: 1.0949-1.6896, p = 0.005) and NMR (OR:1.2159; 95% CI: 1.00396-1.4221, p = 0.015) were significantly associated with survival; Cox regression models confirmed that neutrophils, NLR, and MLR were independently associated with survival; NLR, at a cut-off value of 4.0, showed the better AUC (0.749) in predicting OS. Conclusions: Baseline complete blood cell count biomarkers, especially the NLR, can predict clinical outcomes in patients with advanced NSCLC treated with first-line chemoimmunotherapy.

Association of MCP-4, NRTN, and PD-L1 with the risk of hepatic fibrosis: A Mendelian randomization study.

Previous studies have confirmed the affiliation between specific inflammatory cytokines and Hepatic fibrosis (HF); however, contradictions remain in the causality. The study implemented a bidirectional two-sample Mendelian randomization (MR) analysis with published statistics derived from Genome-wide Association Studies (GWAS) to investigate casualties between inflammatory cytokines and HF. Additionally, MR analysis was also introduced to consider if 1400 blood metabolites act as the key mediators in this process. Single nucleotide polymorphisms (SNPs) with strong correlations to inflammatory factors were selected for multiple MR analyses in this study. The inverse variance weighted method (IVW) was chosen as the principal analysis, and the others as the supportive. Besides, sensitivity tests were involved to identify potential heterogeneity and pleiotropic level. IVW methods revealed that a relatively high level of prediction-based monocyte chemoattractant protein-4 (MCP-4) (95% CI: 1.014-3.336, P = .045), along with neurturin (NRTN) (95% CI: 1.204-4.004, P = .010), may increase the risk of HF; while programmed cell death 1 ligand 1 (PD-L1) (95% CI: 0.223-0.928, P = .030), showed a protective effect on HF. No significant statistical differences were detected on any other inflammatory cytokines, nor did the impact of HF genetic predisposition on the 91 circulating inflammatory cytokines-related characteristics.

A nomogram for predicting survival based on hemoglobin A1c and circulating tumor cells in advanced gastric cancer patients receiving immunotherapy.

BACKGROUND: Our study aimed to investigate the correlation between hemoglobin A1c (HbA1c), circulating tumor cells (CTCs) and prognosis in advanced gastric cancer (GC) patients who received immunotherapy and explore the potential prognostic predictors to develop a nomogram. METHODS: We retrospectively enrolled 259 patients with advanced GC treated at Beijing Friendship Hospital between September 2014 and March 2024. Patients were divided into the immunochemotherapy cohort (ICT) and the chemotherapy (CT) cohort. Survival rate was calculated by Kaplan-Meier survival curve, and the differences were evaluated by log-rank test. The univariate and multivariate Cox proportional hazards regression model was used to identify factors independently associated with survival. A nomogram was developed to estimate 6-, 12-, and 18-month progression-free survival (PFS) probability based on the ICT cohort. RESULTS: Patients achieved higher PFS in the ICT cohort than the CT cohort. We focused on the ICT cohort and constructed a nomogram based on the multivariate analysis, including five variables: age, PD-L1 expression, HbA1c, CTCs and CEA*. The concordance index value was 0.82 in the training cohort and 0.75 in the validation cohort. Furthermore, we proved the nomogram was clinically useful and performed better than PD-L1 expression staging system. Notably, we found high HbA1c level but not diabetes mellitus significantly affected the efficacy of ICT. CONCLUSION: ICT showed better PFS than CT. In addition, HbA1c and CTCs were novel biomarkers to predict PFS in patients treated with ICT. The nomogram could predict PFS of advanced GC patients receiving ICT with increased accuracy and favorable clinical utility.

Ritlecitinib, a JAK3/TEC family kinase inhibitor, stabilizes active lesions and repigments stable lesions in vitiligo.

The efficacy of ritlecitinib, an oral JAK3/TEC family kinase inhibitor, on active and stable lesions was evaluated in patients with active non-segmental vitiligo in a phase 2b trial (NCT03715829). Patients were randomized to placebo or daily ritlecitinib 50 mg (with or without 4-week 100-mg or 200-mg loading dose), 30 mg, or 10 mg for 24 weeks. Active lesions showed greater baseline expression of inflammatory/immune markers IFNG and CCL5, levels of CD103, and T-cell infiltrates than stable lesions. Patients with more active than stable vitiligo lesions showed higher baseline serum levels of CXCL9 and PD-L1, while patients with more stable than active lesions showed higher baseline serum levels of HO-1. At Week 24, ritlecitinib 50 mg significantly stabilized mean percent change from baseline in depigmentation extent in both active lesions and stable lesions vs. placebo-response, with stable lesions showing greater repigmentation. After 24 weeks of treatment, ritlecitinib 50 mg increased expression of melanocyte markers in stable lesions, while Th1/Th2-related and co-stimulatory molecules decreased significantly in both stable and active lesions. Serum from patients with more active than stable lesions showed decreased levels of ICOS and NK cell activation markers. These data, confirmed at transcription/protein levels, indicate that stable lesion repigmentation occurs early with ritlecitinib, while active lesions require stabilization of inflammation first. ClinicalTrials.gov: NCT03715829.

Glyco-signatures in patients with advanced lung cancer during anti-PD-1/PD-L1 immunotherapy.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1/programmed cell death ligand-1 (PD-1/PD-L1) have significantly prolonged the survival of advanced/metastatic patients with lung cancer. However, only a small proportion of patients can benefit from ICIs, and clinical management of the treatment process remains challenging. Glycosylation has added a new dimension to advance our understanding of tumor immunity and immunotherapy. To systematically characterize anti-PD-1/PD-L1 immunotherapy-related changes in serum glycoproteins, a series of serum samples from 12 patients with metastatic lung squamous cell carcinoma (SCC) and lung adenocarcinoma (ADC), collected before and during ICIs treatment, are firstly analyzed with mass-spectrometry-based label-free quantification method. Second, a stratification analysis is performed among anti-PD-1/PD-L1 responders and non-responders, with serum levels of glycopeptides correlated with treatment response. In addition, in an independent validation cohort, a large-scale site-specific profiling strategy based on chemical labeling is employed to confirm the unusual characteristics of IgG N-glycosylation associated with anti-PD-1/PD-L1 treatment. Unbiased label-free quantitative glycoproteomics reveals serum levels' alterations related to anti-PD-1/PD-L1 treatment in 27 out of 337 quantified glycopeptides. The intact glycopeptide EEQFN 177STYR (H3N4) corresponding to IgG4 is significantly increased during anti-PD-1/PD-L1 treatment (FC=2.65, P=0.0083) and has the highest increase in anti-PD-1/PD-L1 responders (FC=5.84, P=0.0190). Quantitative glycoproteomics based on protein purification and chemical labeling confirms this observation. Furthermore, obvious associations between the two intact glycopeptides (EEQFN 177STYR (H3N4) of IgG4, EEQYN 227STFR (H3N4F1) of IgG3) and response to treatment are observed, which may play a guiding role in cancer immunotherapy. Our findings could benefit future clinical disease management.

Clinical significance of circulating biomarkers of immune-checkpoint molecules with atezolizumab plus bevacizumab therapy in unresectable hepatocellular carcinoma.

BACKGROUND: The aims of this study were to identify clinically significant biomarkers of a response to atezolizumab plus bevacizumab (ATZ + BV) therapy and to develop target strategies against unresectable hepatocellular carcinoma (u-HCC). METHOD: We first investigated the potential of circulating tumor DNA (ctDNA) to serve as a biomarker for predicting the therapeutic outcome in 24 u-HCC patients treated with ATZ + BV therapy. Next, we analyzed levels of immune-related cytokines in blood samples from 134 u-HCC patients who received ATZ + BV. For this, serum immune-related molecules or cancer-immune cycle-related molecules that have been reported in HCC patient sera, namely CD274, LAG-3, CCL2, 4, 5, CXCL1, 9, 10, 12, 13, CX3CL1, CCR5, IFNγ and IL-6, 8 were measured using enzyme-linked immunosorbent assay. RESULTS: More than 1% of variant read frequency (VRF) mutations were found in TP53, APC, PIK3CA and VHL, although with no correlation with treatment response. Among the 15 cytokines evaluated, CXCL9 and LAG-3 levels were significantly different between patients with objective response (OR), stable disease (SD), and progressive disease (PD) following ATZ + BV treatment. Receiver-operating characteristic curve analyses of CXCL9 (cut-off value: 419.1 pg/ml) and LAG-3 (cut-off value: 3736.3 pg/ml) indicated areas of 0.779 and 0.697, respectively, for differentiating PD from non-PD and OR from non-OR. In multivariate analysis of progression-free survival (PFS) and overall survival (OS), high serum CXCL9 (hazard ratio (HR) and 95% confidence interval (CI): 0.412 (0.251-0.677) (p = 0.0005) for PFS and 0.252 (0.125-0.508) (p = 0.0001) for OS), and low serum LAG-3 (HR and 95% CI 0.419 (0.249-0.705) (p = 0.0011) for PFS and 0.294 (0.140-0.617) (p = 0.0012) for OS) were independent positive predictive factors. CONCLUSION: Although, as far as we examined, no ctDNA mutations in blood were found to be related to ATZ + BV treatment efficacy, serum CXCL9 and LAG-3 levels, which are related to the cancer-immune cycle, were associated with treatment efficacy and could be predictive markers of the efficacy of ATZ + BV treatment in HCC patients.

Prognostic significance of soluble PD-L1 in prostate cancer.

Purpose: The aim of this study was to assess the role of sPD-L1 and sPD-1 as potential biomarkers in prostate cancer (PCa). The association of the values of these soluble proteins were correlated to the clinical data: stage of disease, Gleason score, biochemical recurrence etc. For a comprehensive study, the relationship between sPD-L1 and sPD-1 and circulating immune cells was further investigated. Methods: A total of 88 patients with pT2 and pT3 PCa diagnosis and 41 heathy men were enrolled. Soluble sPD-L1 and sPD-1 levels were measured in plasma by ELISA method. Immunophenotyping was performed by flow cytometry analysis. Results: Our study's findings demonstrate that PCa patients had higher levels of circulating sPD-L1 and sPD-1 comparing to healthy controls (p < 0.001). We found a statistically significant (p < 0.05) relationship between improved progression free survival and lower initial sPD-L1 values. Furthermore, patients with a lower sPD-1/sPD-L1 ratio were associated with a higher probability of disease progression (p < 0.05). Additionally, a significant (p < 0.05) association was discovered between higher Gleason scores and elevated preoperative sPD-L1 levels and between sPD-1 and advanced stage of disease (p < 0.05). A strong correlation (p < 0.05), between immunosuppressive CD4+CD25+FoxP3+ regulatory T cells and baseline sPD-L1 was observed in patients with unfavorable postoperative course of the disease, supporting the idea that these elements influence each other in cancer progression. In addition to the postoperative drop in circulating PD-L1, the inverse relationship (p < 0.05), between the percentage of M-MDSC and sPD-L1 in patients with BCR suggests that M-MDSC is not a source of sPD-L1 in PCa patients. Conclusion: Our findings suggest the potential of sPD-L1 as a promising prognostic marker in prostate cancer.

Dual-signal output biosensor for the detection of program death-ligand 1 and therapy progress monitoring of cancer.

A disposable dual-output biosensor to detect program death-ligand 1 (PD-L1) was developed for immunotherapy progress monitoring and early cancer detection in a single experimental setup. The aptamer probe was assembled on rGO composited with carboxylated terthiophene polymer (rGO-pTBA) to specifically capture PD-L1 protein labeled with a new redox mediator, ortho-amino phenol para sulphonic acid, for amperometric detection. Each sensing layer was characterized through electrochemical and surface analysis experiments, then confirmed the sensing performance. The calibration plots for the standard PD-L1 protein detection revealed two dynamic ranges of 0.5-100.0 pM and 100.0-500.0 pM, where the detection limit was 0.20 ± 0.001 pM (RSD ≤5.2%) by amperometry. The sensor reliability was evaluated by detecting A549 lung cancer cell-secreted PD-L1 and clinically relevant serum levels of soluble PD-L1 (sPD-L1) using both detection methods. In addition, therapeutic trials were studied through the quantification of sPD-L1 levels for a small cohort of lung cancer patients. A significantly higher level of sPD-L1 was observed for patients (221.6-240.4 pM) compared to healthy individuals (16.2-19.6 pM). After immunotherapy, the patients' PD-L1 level decreased to the range of 126.7-141.2 pM. The results indicated that therapy monitoring was successfully done using both the proposed methods. Additionally, based on a comparative study on immune checkpoint-related proteins, PD-L1 is a more effective biomarker than granzyme B and interferon-gamma.

The Role of Programmed Cell Death 1/Programmed Death Ligand 1 (PD-1/PD-L1) Axis in Sepsis-Induced Apoptosis.

Background and Objectives: Sepsis involves a dysregulated host response, characterized by simultaneous immunosuppression and hyperinflammation. Initially, there is the release of pro-inflammatory factors and immune system dysfunction, followed by persistent immune paralysis leading to apoptosis. This study investigates sepsis-induced apoptosis and its pathways, by assessing changes in PD-1 and PD-L1 serum levels, CD4+ and CD8+ T cells, and Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE II) severity scores. Materials and Methods: This prospective, observational, single-centre study enrolled 87 sepsis patients admitted to the intensive care unit at the County Emergency Clinical Hospital in Târgu Mureș, Romania. We monitored the parameters on day 1 (the day sepsis or septic shock was diagnosed as per the Sepsis-3 Consensus) and day 5. Results: Our study found a statistically significant variation in the SOFA score for the entirety of the patients between the studied days (p = 0.001), as well as for the studied patient groups: sepsis, septic shock, survivors, and non-survivors (p = 0.001, p = 0.003, p = 0.01, p = 0.03). On day 1, we found statistically significant correlations between CD8+ cells and PD-1 (p = 0.02) and PD-L1 (p = 0.04), CD4+ and CD8+ cells (p < 0.0001), SOFA and APACHE II scores (p < 0.0001), and SOFA and APACHE II scores and PD-L1 (p = 0.001 and p = 0.01). On day 5, we found statistically significant correlations between CD4+ and CD8+ cells and PD-L1 (p = 0.03 and p = 0.0099), CD4+ and CD8+ cells (p < 0.0001), and SOFA and APACHE II scores (p < 0.0001). Conclusions: The reduction in Th CD4+ and Tc CD8+ lymphocyte subpopulations were evident from day 1, indicating that apoptosis is a crucial factor in the progression of sepsis and septic shock. The increased expression of the PD-1/PD-L1 axis impairs costimulatory signalling, leading to diminished T cell responses and lymphopenia, thereby increasing the susceptibility to nosocomial infections.

Plasma metabolomics of immune-related adverse events for patients with lung cancer treated with PD-1/PD-L1 inhibitors.

BACKGROUND: Metabolomics has the characteristics of terminal effects and reflects the physiological state of biological diseases more directly. Several current biomarkers of multiple omics were revealed to be associated with immune-related adverse events (irAEs) occurrence. However, there is a lack of reliable metabolic biomarkers to predict irAEs. This study aims to explore the potential metabolic biomarkers to predict risk of irAEs and to investigate the association of plasma metabolites level with survival in patients with lung cancer receiving PD-1/PD-L1 inhibitor treatment. METHODS: The study collected 170 plasmas of 85 patients with lung cancer who received immune checkpoint inhibitors (ICIs) treatment. 58 plasma samples of 29 patients with irAEs were collected before ICIs treatment and at the onset of irAEs. 112 plasma samples of 56 patients who did not develop irAEs were collected before ICIs treatment and plasma matched by treatment cycles to onset of irAEs patients. Untargeted metabolomics analysis was used to identify the differential metabolites before initiating ICIs treatment and during the process that development of irAEs. Kaplan-Meier curves analysis was used to detect the associations of plasma metabolites level with survival of patients with lung cancer. RESULTS: A total of 24 differential metabolites were identified to predict the occurrence of irAEs. Baseline acylcarnitines and steroids levels are significantly higher in patients with irAEs, and the model of eight acylcarnitine and six steroid metabolites baseline level predicts irAEs occurrence with area under the curve of 0.91. Patients with lower concentration of baseline decenoylcarnitine(AcCa(10:1) 2, decenoylcarnitine(AcCa(10:1) 3 and hexanoylcarnitine(AcCa(6:0) in plasma would have better overall survival (OS). Moreover, 52 differential metabolites were identified related to irAEs during ICIs treatment, dehydroepiandrosterone sulfate, corticoserone, cortisol, thyroxine and sphinganine 1-phaosphate were significantly decreased in irAEs group while oxoglutaric acid and taurocholic acid were significantly increased in irAEs group. CONCLUSIONS: High levels of acylcarnitines and steroid hormone metabolites might be risk factor to development of irAEs, and levels of decenoylcarnitine (AcCa(10:1) 2, decenoylcarnitine (AcCa(10:1) 3 and hexanoylcarnitine (AcCa(6:0) could be used to predict OS for patients with lung cancer received ICIs treatment.

Persistence of lung structural and functional alterations at one year post-COVID-19 is associated with increased serum PD-L2 levels and altered CD4/CD8 ratio.

BACKGROUND: Persistent respiratory symptoms and lung abnormalities post-COVID-19 are public health problems. This study evaluated biomarkers to stratify high-risk patients to the development or persistence of post-COVID-19 interstitial lung disease. METHODS: One hundred eighteen patients discharged with residual lung abnormalities compatible with interstitial lung disease (COVID-ILD patients) after a severe COVID-19 were followed for 1 year (post-COVID-ILD patients). Physical examination, pulmonary function tests, and chest high-resolution computed tomography (HRCT) were performed. Soluble forms (s) of PD-L1, PD-L2, TIM-3, and GAL-9 were evaluated in serum and cell culture supernatant, as well as T-cells subsets and the transmembrane expression of PD-L1 and PD-L2 on the cell surface. RESULTS: Eighty percent of the post-COVID-ILD patients normalized their lung function at 1-year follow-up, 8% presented COVID-independent ILD, and 12% still showed functional and HRCT alterations. PD-L2 levels were heterogeneous during acute COVID-19 (aCOVID); patients who increased (at least 30%) their sPD-L2 levels at 1 year post-COVID-19 and exhibited altered CD4/CD8 ratio showed persistence of chest tomographic and functional alterations. By contrast, patients who decreased sPD-L2 displayed a complete lung recovery. sPD-L1, sTIM-3, and sGAL-9 increased significantly during aCOVID and decreased in all patients after 1-year follow-up. CONCLUSION: Increased sPD-L2 and an altered CD4/CD8 ratio after 12 months of aCOVID are associated with the persistence of lung lesions, suggesting that they may contribute to lung damage post-COVID-19.

Elevated serum soluble programmed death ligand 1 (sPD-L1) level correlate with clinical characteristics in breast cancer patients: A study at Hospital Universiti Sains Malaysia.

BACKGROUND: Elevated serum levels of soluble PD-L1 (sPD-L1) have been reported in many cancers; however, there is limited data of sPD-L1 in breast cancer, especially those representing Asian (Malay) women. The purpose of this study was to evaluate sPD-L1 serum levels and analyze its correlation with clinical characteristics in breast cancer patients at Hospital Universiti Sains Malaysia (HUSM). METHODS: Blood specimens were obtained from 92 malignant, 16 benign breast cancer patients and 23 healthy controls. The serum concentrations of sPD-L1 were assessed by enzyme-linked immunosorbent assay (ELISA). RESULTS: The median serum sPD-L1 concentration of malignant and benign breast cancer patients was significantly elevated compared to the healthy cohorts (12.50 ng/mL vs 13.97 ng/mL vs 8.75 ng/mL, p < 0.05). Optimal cut-off value of serum sPD-L1 for predicting disease progression was 8.84  ng/mL. Elevated serum sPD-L1 levels were significantly associated with menarche age, ethnicity, birth control usage, comorbidity and HER2 status (p < 0.05). Multivariate analysis showed the menarche age and birth control were the independent factors affecting sPD-L1 expression. CONCLUSION: Elevated serum levels of sPD-L1 were significantly associated with several clinical characteristics and warrant further investigation in evaluating patients pre-diagnosed with breast cancer.

Ultrasensitive quantification of PD-L1+ extracellular vesicles in melanoma patient plasma using a parallelized high throughput droplet digital assay.

The expression of programmed death-ligand 1 (PD-L1) on extracellular vesicles (EVs) is an emerging biomarker for cancer, and has gained particular interest for its role mediating immunotherapy. However, precise quantification of PD-L1+ EVs in clinical samples remains challenging due to their sparse concentration and the enormity of the number of background EVs in human plasma, limiting applicability of conventional approaches. In this study, we develop a high-throughput droplet-based extracellular vesicle analysis (DEVA) assay for ultrasensitive quantification of EVs in plasma that are dual positive for both PD-L1 and tetraspanin (CD81) known to be expressed on EVs. We achieve a performance that significantly surpasses conventional approaches, demonstrating 360× enhancement in the limit of detection (LOD) and a 750× improvement in the limit of quantitation (LOQ) compared to conventional plate enzyme-linked immunoassay (ELISA). Underlying this performance is DEVA's high throughput analysis of individual EVs one at a time and the high specificity to targeted EVs versus background. We achieve a 0.006% false positive rate per droplet by leveraging avidity effects that arise from EVs having multiple copies of their target ligands on their surface. We use parallelized optofluidics to rapidly process 10 million droplets per minute, ∼100× greater than conventional approaches. A validation study on a cohort of 14 patients with melanoma confirms DEVA's ability to match conventional ELISA measurements with reduced plasma sample volume and without the need for prior EV purification. This proof-of-concept study demonstrates DEVA's potential for clinical utility to enhance prognosis as well as guide treatment for cancer.

Serum sPD-1 and sPD-L1 as predictive biomarkers for HBsAg clearance in HBeAg-negative CHB patients undergoing IFN-based therapy.

BACKGROUND AND AIMS: For chronic hepatitis B (CHB) patients, there is still a need to improve hepatitis B surface antigen (HBsAg) clearance rates. This study aimed to assess the predictive effectiveness of soluble programmed cell death-1 (sPD-1) and soluble programmed cell death ligand-1 (sPD-L1) for HBsAg clearance in HBeAg-negative CHB patients undergoing peginterferon (Peg-IFN)-based antiviral treatment. METHODS: This study encompassed 280 patients undergoing treatment with Peg-IFNα. Serum levels of sPD-1 and sPD-L1 were measured using ELISA kits at baseline, as well as at 12, 24 and 48 weeks. The primary endpoint of the study was the determination of HBsAg clearance at 48 weeks. Logistic regression analysis was employed to identify predictors of HBsAg clearance. RESULTS: The clearance group demonstrated significantly lower serum sPD-L1 levels compared to the non-clearance group. While both groups exhibited an increase in sPD-1 levels, only the clearance group showed a rise in sPD-L1 levels. Multivariate analysis identified sPD-L1 increase at 24 weeks, and HBsAg decline at 24 weeks as predictors for HBsAg clearance at 48 weeks. The combined use of these indicators showed a predictive performance for HBsAg clearance with an AUROC of 0.907 (95% CI: 0.861-0.953, p < 0.001). CONCLUSIONS: The study revealed an inverse relationship between the trends of sPD-1/sPD-L1 and HBsAg clearance during combined IFN and NAs treatment. Moreover, the magnitude of HBsAg reduction and sPD-L1 increase emerged as significant predictors for HBsAg clearance.

A Mesoporous Gold Sensor Unveils Phospho PD-L1 in Extracellular Vesicles as a Proxy for PD-L1 Expression in Lung Cancer Tissue.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death ligand 1 (PD-L1), or its receptor, PD-1 have improved survival in patients with non-small-cell lung cancer (NSCLC). Assessment of PD-L1 expression requires tissue biopsy or fine needle aspiration that are currently used to identify patients most likely to respond to single agent anti-PD-1/PD-L1 therapy. However, obtaining sufficient tissue to generate a PD-L1 tissue proportion score (TPS) ≥ 50% using immunohistochemistry remains a challenge that potentially may be overcome by liquid biopsies. This study utilized a mesoporous gold sensor (MGS) assay to examine the phosphorylation status of PD-L1 in plasma extracellular vesicles (EV pPD-L1) and PD-L1 levels in plasma from NSCLC patient samples and their association with tumor PD-L1 TPS. The 3-dimensional mesoporous network of the electrodes provides a large surface area, high signal-to-noise ratio, and a superior electro-conductive framework, thereby significantly improving the detection sensitivity of PD-L1 nanosensing. Test (n = 20) (Pearson's r = 0.99) and validation (n = 45) (Pearson's r = 0.99) cohorts show that EV pPD-L1 status correlates linearly with the tumor PD-L1 TPS assessed by immunohistochemistry irrespective of the tumor stage, with 64% of patients overall showing detectable EV pPD-L1 levels in plasma. In contrast to the EV pPD-L1 results, plasma PD-L1 levels did not correlate with the tumor PD-L1 TPS score or EV pPD-L1 levels. These data demonstrate that EV pPD-L1 levels may be used to select patients for appropriate PD-1 and PD-L1 ICI therapy regimens in early, locally advanced, and advanced NSCLC and should be tested further in randomized controlled trials. Most importantly, the assay used has a less than 24h turnaround time, facilitating adoption of the test into the routine diagnostic evaluation of patients prior to therapy.

Prognostic significance of programmed cell death ligand 1 blood markers in non-small cell lung cancer treated with immune checkpoint inhibitors: a systematic review and meta-analysis.

Background: Immune checkpoint inhibitors (ICIs) are effective for non-small cell lung cancer (NSCLC) treatment, but the response rate remains low. Programmed cell death ligand 1 (PD-L1) in peripheral blood, including soluble form (sPD-L1), expression on circulating tumor cells (CTCs PD-L1) and exosomes (exoPD-L1), are minimally invasive and promising markers for patient selection and management, but their prognostic significance remains inconclusive. Here, we performed a meta-analysis for the prognostic value of PD-L1 blood markers in NSCLC patients treated with ICIs. Methods: Eligible studies were obtained by searching PubMed, EMBAS, Web of Science, and Cochrane Library prior to November 30, 2023. The associations between pre-treatment, post-treatment and dynamic changes of blood PD-L1 levels and progression-free survival (PFS)/over survival (OS) were analyzed by estimating hazard ratio (HR) and 95% confidence interval (CI). Results: A total of 26 studies comprising 1606 patients were included. High pre- or post-treatment sPD-L1 levels were significantly associated with worse PFS (pre-treatment: HR=1.49, 95%CI 1.13-1.95; post-treatment: HR=2.09, 95%CI 1.40-3.12) and OS (pre-treatment: HR=1.83, 95%CI 1.25-2.67; post-treatment: HR=2.60, 95%CI 1.09-6.20, P=0.032). High pre-treatment exoPD-L1 levels predicted a worse PFS (HR=4.24, 95%CI 2.82-6.38, P<0.001). Pre-treatment PD-L1+ CTCs tended to be correlated with prolonged PFS (HR=0.63, 95%CI 0.39-1.02) and OS (HR=0.58, 95%CI 0.36-0.93). Patients with up-regulated exoPD-L1 levels, but not sPD-L1, after ICIs treatment had significantly favorable PFS (HR=0.36, 95%CI 0.23-0.55) and OS (HR=0.24, 95%CI 0.08-0.68). Conclusion: PD-L1 blood markers, including sPD-L1, CTCs PD-L1 and exoPD-L1, can effectively predict prognosis, and may be potentially utilized for patient selection and treatment management for NSCLC patients receiving ICIs.

Construction of a sensitive SWCNTs integrated SPR biosensor for detecting PD-L1+ exosomes based on Fe3O4@TiO2 specific enrichment and signal amplification.

Programmed cell death-ligand 1 positive (PD-L1+) exosomes play a crucial role in the realm of cancer diagnosis and treatment. Nevertheless, due to the intricate nature of biological specimens, coupled with the heterogeneity, low refractive index (RI), and scant surface coverage density of exosomes, traditional surface plasmon resonance (SPR) sensors still do not meet clinical detection requirements. This study utilizes the exceptional electrical and optical attributes of single-walled carbon nanotubes (SWCNTs) as the substrate for SPR sensing, thereby markedly enhancing sensitivity. Furthermore, sp2 hybridized SWCNTs have the ability to load specific recognition elements. Additionally, through the coordination interaction of Ti with phosphate groups and the ferromagnetism of Fe3O4, efficient exosomes isolation and enrichment in complex samples are achievable with the aid of an external magnetic field. Owing to the high-quality and high-RI of Fe3O4@TiO2, the response signal experiences amplification, thus further improving the performance of the SPR biosensor. The linear range of the SPR biosensor constructed by this method is 1.0 × 103 to 1.0 × 107 particles/mL, with a limit of detection (LOD) of 31.9 particles/mL. In the analysis of clinical serum samples, cancer patients can be differentiated from healthy individuals with an Area Under Curve (AUC) of 0.9835. This study not only establishes a novel platform for exosomes direct detection but also offers new perspectives for the sensitive detection of other biomarkers.