In the present study, it was found that serum exosomal PD-L1 levels were useful for predicting anti-PD-1 therapies for recurrent NSCLC, and that they tended to be associated with survival in patients with early-stage NSCLC. Although serum exosomal PD-L1 expression was significantly associated with CD8 + TILs and tumor PD-L1 levels, no correlation was observed between the expression and pathological stage, lymph node status, or EGFR mutation status. When comparing tumor PD-L1 and serum exosomal PD-L1 expression, there were several conflicting recurrent cases showing low tumor PD-L1, and high serum PD-L1, and vice versa.
Antibodies targeting PD-1/PD-L1 pathways have emerged as the gold-standard treatment for first- or second-line treatment of stage IV and recurrent NSCLC1–5. PD-L1 expression assessed by IHC staining has been widely evaluated in clinical trials as a predictive biomarker1–6. Patients using pembrolizumab monotherapy for previously treated PD-L1-positive NSCLC experienced have been found to have prolonged overall survival (OS) compared to those treated with docetaxel2. Pembrolizumab also significantly improved progression-free survival and OS compared with platinum-based chemotherapy in patients with chemo-naïve advanced NSCLC who had a PD-L1 TPS of 50 % or greater and did not have EGFR mutation or anaplastic lymphoma kinase (ALK) rearrangement 3, 5. Moreover, the Keynote-042 trial showed that the therapeutic effect of pembrolizumab remained significant in those with a TPS of 1–49 %4. However, PD-L1 expression is known to be highly heterogeneous with a low interobserver and inter-assay reproducibility, and discordance due to different antibodies, limited specificity, different platforms, and different thresholds also exists22–25. Because of this, there is a need to identify and develop other effective markers for clinical use.
Tumor mutation burden (TMB), which refers to the number of somatic gene mutations per coding area of a tumor genome, is an emerging predictive biomarker for ICI efficacy in treating various solid tumors such as melanoma, bladder cancer, and NSCLC23, 26, 27. High TMB is associated with lasting clinical benefit from ICIs in patients with advanced NSCLC, while PD-L1 expression and TMB are not significantly correlated within most cancer subtypes1, 2, 6, 27. The U.S. Food and Drug Administration has approved TMB as a companion diagnostic biomarker for pembrolizumab, however, the cut-off values for TMB vary according to study methodology and assay platforms, and therefore further investigations are needed to confirm the optimal cut-off value in different tumors6.
TILs are reportedly associated with treatment effects of ICIs8, 25, 28, 29. In particular, CD8 + TILs are thought to play a pivotal role in directly killing tumor cells as well as maintaining immune surveillance; these functions could be prevented by the signaling produced by the PD-1/PD-L1 axis25, 30. Many studies have also demonstrated that CD8 + TIL levels are a significant prognostic factor in advanced and early-stage lung cancer7–9, 31, 32. Previous studies demonstrated that the frequency and prognostic impact of TILs differed according to tumor histology and staging, and that the presence of TILs was associated with improved survival exclusively in non-adenocarcinomas31, 33. In the present study, no significant differences were found in RFS and anti-PD-1 responses according to the expression of CD8 + TILs. This could be because of a large proportion of adenocarcinoma cases and/or a highly heterogeneous population in terms of pathological stage.
In the current study, tumor PD-L1 expression status was significantly associated with prognosis in pathological stage I and stage I-III NSCLC. Numerous published studies have demonstrated the prognostic significance of PD-L1 expression in NSCLC34–39. However, some studies have shown that high PD-L1 expression in tumors is a favorable prognostic biomarker for survival, whereas currently no tumor PD-L1 status is a factor associated with favorable outcomes34–42. D'Arcangelo et al. state that a possible reason for this discrepancy was patient ethnicity, pointing out that the only studies with a negative prognostic impact of PD-L1 expression were those conducted in the Asian population38. The observed high frequency of EGFR mutations in Asian patients compared with Caucasian populations is a well-known, and NSCLC harboring EGFR mutations or ALK rearrangements are reported to be associated with low efficacy of ICIs41, 43–46. Different antibodies used for IHC staining, different experimental platforms, different PD-L1 thresholds, and different tumor biology according to ethnicity might be responsible for these conflicting prognostic results.
PD-L1 is expressed on tumor cells, immune cells, and other cells in the tumor microenvironment, as well as being found in extracellular forms such as exosomes18–21. Recent studies have shown that PD-L1 expressing exosomes can inhibit antitumor immune responses either locally and systemically, depending on the target cell's location18, 19, 21. In patients with metastatic melanoma, circulating exosomal PD-L1 was shown predict the clinical outcomes of anti-PD-1 therapy, while exosomal PD-L1 has been shown to be a poor prognostic marker in patients with gastric cancer and head and neck cancer; few publications have reported whether exosomal PD-L1 is a predictive marker for ICIs21, 47, 48. In the present study, six of eight recurrent NSCLC patients with a serum exosomal PD-L1 level of ≥ 200 pg/mL demonstrated a partial response for anti-PD-1 therapies. Further research is warranted to determine whether serum exosomal PD-L1 together with tumor PD-L1 status or other blood biomarkers can be used as a more accurate method for predicting the efficacy of anti-PD-1/PD-L1 therapies.
Despites its insights, this study is limited by its retrospective nature and potential biases. The cut-off value of serum exosomal PD-L1 of 200 pg/mL, which dichotomized the high and low groups is somewhat arbitrary.The number of patients in this study and the number of recurrent NSCLC cases were also too small to provide strong statistical power for the drawn conclusions. There was also heterogeneity in the type of anti-PD-1 therapies used for recurrent diseases as well as in the order of regimens, and the isolation and characterization methods used for exosome study are also still a matter of debate. Further studies are required before this liquid biopsy approach for patients who require ICIs can be proven effective and successfully applied in daily clinical practice.
In the present study, our findings demonstrated that the measurement of serum exosomal PD-L1 as a quantitative complementary factor together with tumor PD-L1 status might help predict anti-PD-1 response and assess clinical outcomes in patients with NSCLC. This blood-based liquid biopsy approach can contribute to the appropriate ICI treatment decision-making process in both advanced and recurrent lung cancer when there is a limit to the amount of tissue that can be harvested.