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.


Introduction
Lung cancer remains one of the most common malignant tumors and one of the leading causes of cancer-related death worldwide (1).Despite greatly improved prognosis attributed to molecular targeted therapy in recent decades, most of advanced non-small cell lung cancer (NSCLC) patients inevitably develop drug resistance (2, 3).As an innovative therapy, immunotherapy has become an emerging and promising treatment strategy for various cancers, especially lung cancer (4,5).The most commonly utilized immunotherapy is immune checkpoint inhibitors (ICIs) that include blockades for programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic Tlymphocyte associated protein 4 (CTLA-4).However, the response rate to ICIs is still low in NSCLC, and exploring biomarkers to select patients with best clinical response and benefit and to predict prognosis is vital (6,7).
Numerous studies have demonstrated better clinical response and superior survivals in ICIs-treated patients with overexpressed PD-L1 on tumor tissues (8).Until now, the membrane-bound form PD-L1 expressed on tumor cells is the only officially approved and well-established biomarker for patient selection (9).However, the utilization of tumor PD-L1 marker is still in controversy, which is challenged by the highly heterogeneous spatial expression pattern in tumor tissues, highly dynamic changes at different disease stages, and inconvenience of longitudinal tissue sampling owing to the invasive biopsy method (10).Therefore, novel biomarkers that are less heterogeneously expressed, can be detected via less invasive methods and has sufficient prognostic significance are in urgent need for ICIs treatment.
Besides of expression on tumor tissues, PD-L1 can be expressed and detected in peripheral blood, such as the soluble form PD-L1 (sPD-L1) in serum/plasma, PD-L1 expressed in circulating tumor cells (CTC PD-L1) and exosomes (exoPD-L1) (11)(12)(13).These PD-L1 blood markers can be less-invasively detected and then monitored for dynamic changes during treatments.They are usually overexpressed in cancer patients in association with clinicopathological features, and may predict response and prognosis to various anti-cancer treatments, such as c h e m o t h e r a p y , r a d i o t h e r a p y , t a r g e t e d t h e r a p y a n d immunotherapy, in many types of cancers (14)(15)(16).In the scenario of NSCLC patients treated with ICIs, the predictive and prognostic values of PD-L1 blood markers have also been explored (16)(17)(18)).Yet, the conclusions remain controversial due to varieties in sample size, detection methods, biomarker cut-off values, and sampling time points (19,20).With the accumulated evidence in recent years, we performed a systematic review and meta-analysis to explore the prognostic significance of PD-L1 blood markers, including sPD-L1, CTC PD-L1 and exoPD-L1, in NSCLC patients treated with ICIs.

Literature search
This systematic review and meta-analysis was conducted in compliance with the Preferred Reporting Items for Systematic review and Meta-analysis (PRISMA) guideline (21).Candidate studies investigating the prognostic value of blood PD-L1 markers in NSCLC patients undergoing ICIs therapy were comprehensively searched in PubMed, EMBAS, Web of Science, and Cochrane Library from inception to November 30, 2023.The following search terms were applied: (exosome OR exosomal OR extracellular vesicles OR soluble OR circulating tumor cells OR neoplastic circulating cells OR CTCs) AND (programmed cell death ligand 1 OR PD-L1 OR CD274 OR sPD-L1 OR exoPD-L1) AND (NSCLC OR non-small cell lung cancer OR lung adenocarcinoma OR lung cancer OR lung carcinoma).Language was not restricted.The reference lists of included studies and relevant reviews were manually reviewed for additional eligible articles.

Inclusion and exclusion criteria
The eligibility of retrieved studies was judged according to the following criteria (1): NSCLC patients were treated with ICIs alone or in combination with other therapies; (2) at least one of blood PD-L1, including sPD-L1, CTC PD-L1 and exoPD-L1, were detected; (3) pre-treatment level, post-treatment level or dynamic change of blood PD-L1 was determined as prognostic marker; (4) the association of PD-L1 marker with progression-free survival (PFS) and/or overall survival (OS) was investigated.Case reports, reviews, meta-analyses, conference abstracts, and functional studies were excluded.If recruiting various cancers including NSCLC, the study was included only when survival outcomes of NSCLC subgroup were reported.Otherwise, it was discarded.Since we aimed for the prognostic value of functional PD-L1, studies detecting mRNA expression on exosomes were excluded.

Quality assessment of included studies
Newcastle-Ottawa Scale (NOS) for cohort study was used to assess the quality of included studies (22).The risk of bias with regard to selection, comparability, and outcome was judged.Selection and outcome domains contained 4 and 3 items, respectively, and 0 or 1 score was awarded to each item.In comparability domain, 0, 1 or 2 scores were awarded.Thus, the total score ranged from 0 to 9. A study with ≥7, 4-6, and ≤5 scores were deemed to have a high, moderate, and low quality, respectively.Quality assessment was performed by two independent authors (JC, PL), and conflicts were resolved by a third author (XT).

Data extraction
Two independent authors (JC, PL) extracted the following information of each study: first author, publication year, study design, region, cancer stage, sample size, age, percentage of male, percentage of smoker, type and treatment line of ICIs, CTC enrichment method, PD-L1 detection method, enzyme-linked immunosorbent assay (ELISA) kit, cut-off value, cut-off determination method, time point of marker detection, hazard ratio (HR) with corresponding 95% confidence interval (CI) of survival outcomes (PFS, OS), and follow-up duration.We extracted HR and 95%CI from multivariate analysis if available.Otherwise, HR results from univariate analysis were extracted.If HR was not directly reported, we estimated it after extracting the survival data from Kaplan-Meier curve if available using Engauge Digitizer v12.1.Disagreement was resolved by a third author (XT).

Statistical analysis
The statistical analysis was conducted by using STATA 16.0 (Stata Corporation, TX, USA).The model for quantitative synthesis was determined by between-study heterogeneity.A fixed-effect model was applied when there was low heterogeneity (I 2 <50% and Q test P>0.10).Otherwise, a random-effect model was used.The association strength between PD-L1 blood markers was calculated by pooled HR with 95%CI.The prognostic values of pre-treatment, post-treatment, and dynamic change of PD-L1 blood markers in predicting PFS and OS was assessed separately.Subgroup analysis and meta-regression were performed to explore the potential source of heterogeneity.Subgroup analyses were stratified by study design, region, PD-L1 detection method, ELISA kit, CTC enrichment method, cut-off value, cut-off determination method, HR analysis, HR extraction, and sample size.Meta-regression analysis was done to explore the modulation effect of baseline variables, including median age, percentage of males, percentage of smokers, sample size, percentage of first-line immunotherapy, and cut-off value, on prognostic value of PD-L1 blood markers.Funnel plot was viewed for symmetry and Egger's test was performed to assess potential publication bias.In case of potential publication bias, we implemented a trim-and-fill analysis to explore the impact of publication bias on pooled results.P<0.05 indicated statistical significance.

Baseline characteristics of studies included in meta-analysis
As illustrated in Figure 1, we obtained a total of 487 articles by comprehensive literature search and discarded 440 after reviewing titles and abstracts as they did not fulfill the inclusion criteria.We further reviewed the full texts of the remaining 47 studies for eligibility and excluded 21 with the following reasons: survival outcomes not investigated (n=5), non-ICI treatments (n=6), various treatments including ICIs (n=2), ICIs treatment unknown (n=2), no NSCLC patients (n=1), survival results not provided (n=3), survival analysis at specimen level (n=1), PD-L1 mRNA expression in exosomes (n=1).These articles with detailed exclusion reasons were listed in Supplementary Table 1.Finally, 26 studies comprising 1606 NSCLC patients receiving ICIs treatment were included in the meta-analysis (16,17,19,20,.The prognostic significance of sPD-L1, CTC PD-L1 and exoPD-L1 were investigated in 16, 9 and 3 studies, respectively.The correlations of survival outcomes with pre-treatment blood PD-L1 markers, post-treatment markers and dynamic changes from baseline were reported by 21, 5 and 7 studies, respectively.There were 4 retrospective studies and 22 prospective studies.These studies were conducted in East Asian countries (n=12), European countries (n=12) or America (n=2).The sample size ranged greatly from 14 to 233.PFS outcome was analyzed in 26 studies while OS outcome was analyzed in 19 studies.According to NOS, all included studies had high methodological quality.The baseline characteristics of all studies included in meta-analysis were summarized in Table 1.
For pre-or post-treatment sPD-L1 and exoPD-L1, cut-off value of PD-L1 concentrations was determined to categorize high and low groups.For dynamic change, cut-off value of change fold was set to segregate patients with up-regulation and without up-regulation after immunotherapy.For pre-or post-treatment CTC PD-L1, cutoff value was used to define PD-L1 + CTCs.Seven studies determined the cut-off value by using the median value of PD-L1 concentrations.Nine studies established the cut-off value as the optimal point showing the best performance by various analyses, such as receiver operating characteristic (ROC) curve, Classification and regression tree (CART) analysis, and or log-rank test.Details regarding CTC enrichment, PD-L1 detection, cut-off determination, HR analysis and extraction, and follow-up duration were shown in Supplementary Table 2.

Prognostic significance of sPD-L1
The association between pre-treatment sPD-L1 and PFS was investigated in 11 studies involving 1011 NSCLC patients treated with ICIs.There was substantial level of between-study heterogeneity (I 2 = 60.3%,P=0.005), and a random-effect model was used.Pooled analysis demonstrated patients with high pretreatment sPD-L1 concentrations had significantly worse PFS than those with low concentrations (HR=1.49,95%CI 1.13-1.95,P=0.004, Figure 2).The correlation of pre-treatment sPD-L1 with OS was analyzed in 10 studies comprising 995 patients.Using a random-effect model, meta-analysis revealed significantly shorter OS in patients with high sPD-L1 levels compared with those with low sPD-L1 levels (HR=1.83,95%CI 1.25-2.67,P=0.002, Figure 3).Further analysis stratified by baseline characteristics were performed (Table 2).There were no significant between-subgroup differences (all P values >0.05), indicating that baseline features were not the main sources of heterogeneity.
The prognostic value of post-treatment PD-L1 + CTCs and dynamic change of PD-L1 + CTCs was both evaluated in only 1 study.Ikeda M et al. found patients with ≥7.7% PD-L1 positivity rate in CTCs had a longer PFS than those with <7.7% positivity rate but did not have OS benefit (30).Spiliotaki M et al. observed longer PFS in patients with decreased PD-L1low CTCs after pembrolizumab treatment (33).

Meta-regression analysis of pre-treatment sPD-L1
We conducted meta-regression to analyze whether baseline features modulated the association between pre-treatment sPD-L1 and survival outcomes in NSCLC patients treated with ICIs (Supplementary Table 4).Meta-regression showed that median age, percentage of males, percentage of smokers, sample size, percentage of first-line ICIs, and cut-off value did not modulate the association of sPD-L1 with PFS.Moreover, these factors did not modulate the correlation between sPD-L1 and OS except for cut-off value.After excluding an outlier (3357 ng/ ml) that was extremely larger than the other cut-offs (27), metaregression demonstrated a positive linear relationship between cut-off value and effect size (P=0.019,Supplementary Figure 6).Cut-off value may partially explain the source of heterogeneity in analysis of pretreatment sPD-L1 associated with OS.Forest plot of pre-treatment sPD-L1 levels in association with progression-free survival.

FIGURE 3
Forest plot of pre-treatment sPD-L1 levels in association with overall survival.

Publication bias
Publication bias was assessed by viewing the symmetry of funnel plots and Egger's test (Supplementary Table 5).There was potential publication bias in meta-analysis of dynamic change of sPD-L1 concentrations associated with PFS and OS (P<0.05).Trim-and-fill analysis after imputing 1 and 2 studies showed that the association between dynamic change of sPD-L1 and survivals remained insignificant (PFS: HR=0.86, 95%CI 0.54-1.37;OS: HR=1.00, 95%CI 0.61-1.63;Supplementary Figure 7).Thus, publication bias had no obvious impact on results of pooled analysis.We did not observe obvious publication bias in other meta-analyses (Supplementary Figure 8).between up-regulation of sPD-L1 and survivals were inconsistent (19,23,29,31,35).Oh S et al. found NSCLC patients with >100% increase in sPD-L1 after ICIs treatment had longer PFS and OS, but the results were opposite in melanoma (31).Yet, they could not draw a definite conclusion as most of these studies have very small sample size.Our meta-analysis demonstrated no significant correlation between sPD-L1 dynamic change and survivals in ICItreated NSCLC patients.A recent patient-level meta-analysis yielded similar conclusion that sPD-L1 changes during ICIs treatment did not influence the prognosis of NSCLC patients regardless of sex, age and ICI type (55).PD-L1 can be detected on CTCs in many malignant tumors and is correlated with treatment response and prognosis (15,(56)(57)(58).Yet, the clinical relevance of CTCs PD-L1 seems to be treatment dependent.PD-L1 expression on CTCs predicted significantly poor survivals in patients treated with non-ICIs treatment but not in ICItreated patients (59,60).The present meta-analysis, incorporating more eligible studies, revealed patients with PD-L1 + CTCs prior to ICIs treatment had a significantly prolonged OS (HR=0.58,95%CI 0.36-0.93,P=0.024) and tended to have a longer PFS (HR=0.63,95%CI 0.39-1.02,P=0.062).Post-treatment PD-L1 + CTCs showed clinical significance in patients receiving chemotherapy or radiotherapy, which predicted significantly longer PFS and OS (58,61).Only one study was performed in NSCLC patients receiving ICIs, showing improved PFS patients with posttreatment PD-L1 + CTCs (30).The prognostic value of posttreatment PD-L1+ CTCs and the dynamic change needs investigation in more studies.
Exosomes belong to a subtype of extracellular vesicles with a diameter of 30~150 nm, which can be purified from blood of cancer patients (62).Tumor cells can secret PD-L1 via exosomes and exoPD-L1 exerts immunosuppressive function through inhibiting activation and promoting apoptosis of T cells, suppressing immune memory, and promoting tumor growth (11,63).Up-regulated exoPD-L1 induces immune escape to promote tumor progression and mediates immunotherapy resistance by competitively binding to anti PD-(L)1 antibody (64).In NSCLC patients treated with immunotherapy, significantly improved PFS was achieved in patients with low pre-treatment exoPD-L1 levels than those with high levels (16,34).Moreover, the increase of exoPD-L1 levels after ICIs treatment was found to be associated with favorable PFS (34,35).This increase after several cycles of treatment may reflect successful anti-tumor immunity of immunotherapy as more tissue PD-1/PD-L1 interaction are blocked and more PD-L1 are secreted to exome.Our meta-analysis demonstrated high pretreatment exoPD-L1 levels predicted poor survival while the increase after treatment predicted better PFS, suggesting exoPD-L1 as biomarker for ICIs treatment.
Our meta-analysis has several strengths than previous ones (53,65,66).We focus on NSCLC patients treated with ICIs and include more eligible studies with the largest sample size.Hence, the populations are more homogeneous and the statistical power is larger.Secondly, we evaluate several PD-L1 blood markers at different assessment time-points, i.e. pre-treatment, posttreatment and the dynamic changes.Our study firstly shows significant associations of high post-treatment sPD-L1 levels and pre-treatment PD-L1 + CTCs with survivals.Despite promising prognostic value, there are several practical challenges and considerations associated with the clinical implementation of PD-L1 blood markers in ICI-treated NSCLC patient management, and the limitations of our study should be noted.Given the diversity in study features, patients characteristics, ICI types and methodologies of PD-L1 detection, it is inappropriate and difficult to directly translate our findings into clinical practice.The largest obstacles are the PD-L1 assay techniques and cut-off values, which greatly vary among included trials.Standardization efforts, such as the standard protocols for PD-L1 detection and quantification and the optimal and unified PD-L1 thresholds defining who will benefit from ICIs Forest plot of pre-treatment exoPD-L1 levels and dynamic changes in association with progression-free survival.treatment, are urgently warranted to improve the reliability of these markers.Subgroup analyses in our study have provided some clues.We find that high pre-treatment sPD-L1 and exoPD-L1 levels using the optimal cut-off points predict worse survivals than low levels.On the contrary, no significant association is observed between survivals and pre-treatment sPD-L1 levels using median values as cut-off.Therefore, our study suggests that thresholds for sPD-L1 and exoPD-L1 can be established using optimal cut-off points predicting treatment response rather than the median values.For CTC PD-L1, CTC enrichment method seems to be a key factor influencing the prognostic value, of which EpCAM-based enrichment, instead of size-based method, can be recommended.However, these results are obtained from studies with a relative small sample size.Future large-scale, prospective studies are needed to establish the optimal thresholds of PD-L1 and validate the prognostic value in independent cohorts and clinical practice.

Conclusion
In summary, we observed significantly worse survivals in ICItreated NSCLC patients with high pre-or post-treaement sPD-L1 levels and pre-treatment exoPD-L1 levels.Moreover, patients with pre-treatment PD-L1 + CTCs and those with increasing exoPD-L1 levels after treatment had improved survivals.Therefore, these three PD-L1 blood indicators may be utilized as minimally invasive, convenient, alternative biomarkers to conventional tissue PD-L1 for decision-making and management of ICIs treatment in NSCLC patients.

FIGURE 1 Flow
FIGURE 1Flow chart of literature search.

TABLE 1
Baseline characteristics of studies included in meta-analysis.

TABLE 2
Subgroup analysis of pre-treatment sPD-L1 in association with survival outcomes.