Introduction
Lung cancer is a leading cause of cancer-related deaths worldwide and number of lung cancer deaths is estimated to increase by 86% by 2035 (1,2).
For years, standard treatments for the patients with advanced metastatic non-small cell lung cancer (NSCLC) included cytotoxic chemotherapy and specific inhibitors for NSCLC subtypes, harboring epidermal growth factor receptor (EGFR) mutations or chromosomal rearrangements of anaplastic lymphoma kinase (ALK) (3). As a result, the patient with advanced NSCLC had a median survival of approximately 1 year after chemotherapy and 2-3 years after the treatment with EGFR tyrosine kinase inhibitors (4,5).
Recently, immunotherapy with immune checkpoint inhibitors has demonstrated durable responses and significantly improved survival rate in the patients with NSCLC. Programmed death protein 1 (PD-1) and its ligand (PD-L1) are the first introduced checkpoints being targeted in NSCLC and according to several clinical trials, antibodies against PD-1 and PD-L1 have significant efficacy in both, the first line and the second line treatment of metastatic NSCLC (6-9). Despite of demonstrated successes, response to the immunotherapy interventions is seen only in a subset (≤5%) of patients (10). Therefore, a substantial amount of current research is focused on improved performance of immunotherapies with novel combinations (including cytotoxic agents) together with biomarker optimization (11,12). In fact, the immunotherapy and chemotherapy combinations that are already proven to be more effective are now entered into clinical practice of metastatic NSCLC management (9,13).
To date, the most advanced biomarker for NSCLC is an immunohistochemical expression level of PD-L1 on the tumor cells. In different NSCLC cell line models and between patients, the expression levels of PD-L1 may vary significantly (14), whereas several phase-III clinical studies have reported better treatment responses in patients with higher tumor proportion score of PD-L1 (7-9). Previous reports additionally showed that the treatment efficacy of immune checkpoint inhibitors also correlated with the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations that all lead to high mutational burden (15,16). Furthermore, it has been reported that pathogenic DNA damage response (DDR) and repair mutations are associated with improved treatment response rate, progression-free survival, and overall survival in patients with NSCLC treated with programmed death ligand 1 [PD-(L)1] inhibitor therapy (17). Therefore, it is suggested that combining DDR inhibitors with DNA damaging agents, or PD-1 blockade can enhance the overall DNA damage and induce a more sustained upregulation of the cGAS-STING pathway and consequently the production of Th1 cytokines (18).
Changes in DNA repair machinery are especially important, when combinations of immune checkpoint inhibitors and conventional cytotoxic anti-cancer therapies (chemotherapy, radiotherapy) are in consideration. It has been demonstrated that an ability of cancer cells to repair therapeutically induced DNA damage has extensive impacts on anti-cancer therapeutic efficacy (19). Moreover, since DDR inhibition can also induce and amplify DNA damage in cancer cells, combining DDR inhibitors with immune checkpoint inhibitors and/or cytotoxic therapies represents an attractive strategy to improve patient outcomes (18).
DNA-dependent protein kinase catalytic subunit (DNA-PKcs), encoded by PRKDC/XRCC7 gene, is a pivotal DNA damage response (DDR) player (20). Along with Ku heterodimer, DNA-PKcs forms the DNA-PK holoenzyme complex. Of note, Ku heterodimer consists of Ku70 (XRCC6) and Ku80 (XRCC5) subunits. DNA-PK holoenzyme complex is a core component of non-homologous end joining (NHEJ) machinery, which alongside homologous recombination (HR), comprise the two major canonical pathways for DNA double strand breaks (DSBs) repair (21,22). Expression and functioning of DNA-PK in several NSCLC cell lines has been reported (23), whereas the inhibition of DNA-PK was shown to enhance chemosensitivity as well as radiosensitivity in these cells (24,25). Despite of that, interactions between PD-1/PD-L1 axis and DNA-PK are not fully explored.
The aim of our retrospective study was to describe the relationship between the expression of immune checkpoints PD-1 and PD-L1 and an enzyme DNA-PK, which is a part of key pathway for a repair of cancer therapy induced damage. For that, we carried out the immunohistochemical staining of the proteins of interest in the biopsy specimen obtained from patients thus providing a unique insight into the tumor and its microenvironment which includes the components of immune system.
Discussion
Blockade of inhibitory immune checkpoints PD-1/PD-L1 has become a valid treatment option for several tumors including advanced NSCLC (7,8,30). Moreover, immunotherapy is increasingly used in combination with cytotoxic treatments, such as chemotherapy (9,13). Although the combined treatments in NSCLC are more effective, the underling mechanisms that lead to higher antitumor activity are not fully understood. Therefore, the aim of our retrospective study was to describe the relationship between the expression of immune checkpoints (PD-1, PD-L1) and DNA-PK, which is a part of key pathway involved in repairing cytotoxic cancer therapy induced damage.
Till today, immunohistochemical expression level of PD-L1 on tumor cells is the most advanced biomarker in NSCLC. Several phase-III clinical studies have reported better treatment responses in the patients with higher tumor proportion score of PD-L1. The latter has been clearly shown in monotherapy studies in both, first- and second-line treatment of NSCLC patients (7,8,30). Similarly, in the recently published study on lung adenocarcinoma patients, a positive relation was found between higher PD-L1 expression and longer overall survival wherein pembrolizumab plus pemetrexed and a platinum-based drug combination was used (9). Nevertheless, in patients with squamous cell carcinoma, the relationship between higher PD-L1 expression and longer overall survival was not detected (13). The latter indicates that the clinical usefulness of PD-L1 as a biomarker in patients receiving immunotherapy with cytotoxic agents may be less clear given that the combination treatments improved outcomes over chemotherapy across all categories of PD-L1 tumor proportion scores (9,13). Therefore, the development of biomarkers for anti-PD-1/PD-L1 and chemotherapy combinations requires better understanding of PD-1 and PD-L1 expression profiles that is complemented with the markers, which are important in cytotoxic cancer therapy.
In our retrospective study, we first analyzed the expression of PD-1, PD-L1 and DNA-PK according to the clinical parameters (male vs. female, current vs. non-smoker), histological form (adenocarcinoma vs. squamous cell cancer) and the extent of inflammation. In all the evaluated parameters, we detected significant differences for PD-1 and PD-L1. Significantly higher number of positive PD-1 cells were seen in the tumor tissues from males, which supported the observations in the previous studies, wherein higher PD-1 scores were found in males (31). Indeed, it was shown that immunotherapy tends to be more effective in male cancer patients, when compared to female patients, whereas higher expression of PD-1 and PD-L1 was suggested as one of the reasons for the increased efficacy (32,33).
Our study also revealed that NSCLC tissues with no or trivial visual inflammation had higher number of PD-1 positive cells. It is well known that there are key histological differences between acute and chronic inflammation, especially in terms of leukocyte types that are predominantly present in the tissue (polymorphonuclear neutrophils vs. macrophages and lymphocytes) (34). Additionally, numerous studies showed that PD-1 expression can be found in multiple immune cell types, including T cells, B cells, macrophages as well as dendritic cells (35). In tumors, chronic inflammation leads to permanent stimulation of T cells, which promotes T cell exhaustion that finally results in upregulation of co-inhibitory receptors such as PD-1(36). Therefore, it is reasonable to believe that higher number of PD-1 positive cells in the tumor's tissues with no or trivial inflammation as seen in our study was mainly due to inhibitory tumor microenvironment.
Recent studies have raised questions about analytical and clinical comparability of different PD-L1 assays used in clinical trials and practice because the assays utilize various IHC antibodies, staining platforms and may have their own scoring systems (26,28,29). Here, we showed that the results of different IHC PD-L1 antibodies do not correlate. Therefore, it is very important to use clinically relevant antibodies even in retrospective studies.
PD-L1 immunohistochemistry with clinically validated antibody also revealed that the expression of PD-L1 was significantly higher in the NSCLC patients with ‘current smokers’ status. Several meta-analyses and other studies showed that high PD-L1 expression was positively correlated with different clinical and pathological features in lung cancer patients, such as male gender, smoking status, higher histological grade, larger tumor size, positive lymph nodal metastasis and TNM stage (37-39). Our study did not find any association between PD-L1 and other clinical and the selected pathological characteristics. It could be due to several reasons; first, our patient groups were not well balanced. Additionally, we were unable to rule out tumor heterogeneity, which was showed influential for PD-L1 expression in NSCLC as reported recently (40).
Although we did not detect significant differences in tumor cell DNA-PK expression in the selected clinical and pathological characteristics, the most intriguing finding of this study was, a significant positive correlation between PD-1/PD-L1 axis and DNA-PK expression based on individual patient values. The patients whose tumors had higher number of PD-1 positive cells, also showed higher proportion of DNA-PK positive tumor cells.
As mentioned earlier, DNA-PK, a serine/threonine protein kinase, consists of a catalytic subunit (DNA-PKcs) and Ku heterodimer that consists of Ku70 and Ku80 subunits (41). DNA-PK is involved in repairing DNA double-strand breaks (DSBs) via three main pathways: classical non-homologous end-joining NHEJ (C-NHEJ), alternative NHEJ (A-NHEJ) and homologous recombination (HR) (42). It has been widely accepted that the ability of cancer cells to repair therapeutically induced DNA damage impacts anti-cancer therapeutic efficacy to a major extent (19,43,44). In our study, more than 80% of cancer cells (on average) were seen to express DNA-PK. Accordingly, in previously published studies, significantly higher DNA-PK expression was detected in NSCLC than adjacent normal tissues (45). Moreover, DNA-PK expression level serves as a biomarker for predicting a response to cytotoxic therapy and thereby survival, since worse treatment outcome was reported in the patients with high levels of DNA-PK (45,46). Furthermore, the inhibition of DNA-PK was shown to enhance chemosensitivity as well as radiosensitivity in NSCLC (24,25).
Interactions between DNA-PK and PD-1/PD-L1 axis are not fully explored. Previously, it was showed that nuclear γH2AX (unique histone subunit, which serves as a sensor of double-stranded DNA damage) expression was positively associated with PD-L1 expression in squamous cell lung carcinoma (47). Similarly, upregulation of PD-L1 expression in cancer cells was reported in response to DSBs (48). Moreover, as per TCGA database, a positive correlation was observed in mRNA levels of DNA-PK (PRKDC) and PD-L1 (CD274) (Fig. S1) (27). These evidences suggested that DNA damage response in tumor cells generates an immune response that further upregulates PD-L1 expression in tumor cells. Higher PD-L1 expression in the tumors cells consecutively sensitizes them to anti PD-L1 therapy. According to TCGA database, there is a negative correlation between the DNA-PK (PRKDC) and PD-1 (PDCD1) (Fig. S2), yet this does not directly contradict our findings: the biopsy samples used for IHC contain several types of cells, and hence protein staining can reveal correlations that are intrinsically different to those provided by mRNA sequencing in tumor cells only. This fact underlines the ultimate strength of our study.
Here, we showed that the patients, whose tumors had higher number of PD-1 positive cells, also had a higher proportion of DNA-PK positive tumor cells. Although the most well-known role of DNA-PK is, its involvement in DNA damage response pathways, this kinase also participates in other functions, e.g., promotion of genomic stability, regulation of hypoxic response via hypoxia inducible factor (HIF) dependent and HIF-independent mechanisms. In addition, DNA-PK affects metabolism, participates in transcriptional regulation of hormone receptors, and activates innate immune system (41,42,49). Various studies have established the role of DNA-PK in transcriptional programs that operate biological processes, such as epithelial to mesenchymal transition (EMT), nuclear receptor signaling and inflammatory responses (22). However, importance of PD-1 and DNA-PK association in NSCLC is not known. Therefore, detailed studies are required regarding the mechanisms that are responsible for higher expression of PD-1 and DNA-PK in tumors and further their effect on the efficacy of combined treatments in NSCLC.
This report has several limitations, including retrospective nature of the study and absence of equally balanced study groups in terms of patient number. Furthermore, we used tumor tissue samples in our study. As opposed to NSCLC cell lines, tissue samples may contain not only cancerous cells but also other components native to the tumor microenvironment which may affect the results of the study. In spite of the limitations, the authors showed that, there is a significant positive correlation between PD-1/PD-L1 axis and DNA-PK expression in NSCLC. Clear understanding of underlying mechanisms is of high importance to develop predictive and prognostic molecular markers for the combination treatment of immune checkpoint inhibitors and cytotoxic cancer therapies. In our further studies, we will focus on the tumor side of the PD-1/PD-L1 signaling axis to investigate combined effects of immunotherapy and chemotherapy in a simplified system, represented by the PD-L1 positive vs. PD-L1 negative cell lines.