Lung cancer remains one of the most common and one of the most lethal cancers worldwide [1]. Throughout the last decade distinct molecular factors were identified as driving tumor growth and spread and/or as being prognostic in non-small cell lung cancer (NSCLC). Several attempts followed to specifically target these factors and thereby influence the clinical course of disease. Molecular based therapies targeting epidermal growth factor receptor (EGFR) mutants [2,3] or ALK rearrangements [4] were shown to improve the outcome within well-defined subgroups of non-squamous cell carcinoma patients. So far, only for a minority of all patients, targetable genetic alterations have been identified. NSCLC patients with progressive disease and without targetable alterations are treated with traditional chemotherapies. The majority of them suffers from chemotherapy-associated toxicities and poor overall survival due to chemotherapy resistance. The recognition of cancer by the immune system and mechanisms of cancer to escape the immune control are areas of increasing research interest. To enlarge our therapeutic armamentarium, new potent antigens need to be identified. In the future, novel and modified immunotherapeutic concepts might improve cancer cell recognition for effective tumor control.

Although an increased CD4⁺/CD8⁺ cell infiltration of the tumor stroma has previously been shown to represent a favorable prognostic factor in NSCLC [5], ineffective therapeutic approaches with IL-2 [6] and interferon [7] have led to the conclusion that NSCLC is non-immunogenic. Recent gains of information in the field of tumor immunology include identification of key regulators of immune responses with broad impact on natural and therapeutic antitumor immune responses. The best-characterized immunological checkpoints with a major impact on both cancer growth and cancer therapy are cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed death receptor 1 (PD-1) and their respective ligands. Both receptors (i.e. CTLA-4 and PD-1) inhibit T cell activation through distinct and potentially synergistic mechanisms. While CTLA-4 fails to downregulate the survival gene Bcl-xl, PD-1 engagement is suggested to induce T cell apoptosis [8]. The PD-1 receptor is expressed by CD4⁺ and CD8⁺ lymphocytes, regulatory T cells (Tregs) and B lymphocytes [9]. Upregulation of PD-1 modulates peritumoral inflammatory processes [10]. Consequently, binding of PD-1 by the two major ligands PD-L1 (CD274) or PD-L2 (CD 273) inhibits cytokine production. Inflammatory cytokines are reported to induce PD-L1 expression in tumor cells. PD-L1 interacts with PD-1 on T cells and downregulates T cell effector functions. This mechanism can enable cancer cells to evade host immune surveillance. Indeed, in several tumor types, increased PD-1 levels were found in tumor infiltrating lymphocytes (TILs) [11]. Besides adaptive PD-L1 upregulation in an inflammatory cytokine milieu, tumors can have innate potential to inactivate PD-1 by oncogene driven PD-L1 tumor expression [12]. Antibodies against the PD-1-pathway have been successfully applied to reverse T cell tolerance of malignant cells and induce tumor regression [12]. In first clinical studies, anti-PD-1 antibodies have shown activity in some NSCLC patients [13]. While these antibodies are undergoing clinical evaluation in lung cancer and other malignancies, the concrete biological significance of PD-1 and PD-L1 expression in cancer remains unclear. PD-L1-positivity was found to be associated with an inflammatory tumor microenvironment in lung cancers with squamous cell carcinoma [14] or adenocarcinoma histology [15]. Recently, one study revealed high PD-L1 expression by tumor cells to predict complete response as evaluated by histopathology to pre-operative chemotherapy in breast cancer [16]. In the context of the emerging PD-1 pathway inhibitors, the particular characteristics of patients with expected therapeutic response to these agents still need to be defined.

The present study aims at the evaluation of the prognostic significance of PD-1 expression in TILs and PD-L1 expression in NSCLC tumor cells in a large study collective of NSCLC using immunohistochemistry, with a particular emphasis on clinicopathological parameters.

Blockade of inhibitory immune checkpoints is currently arising as a potential immunological option for tumor therapy. Targeting the PD-1/PD-L1 pathway in lung cancer has shown promise to positively affect prognosis in first clinical studies [13,21]. A more detailed understanding of the significance of the PD-1/PD-L1 pathway in this cancer is important to advance this promising treatment modality to its full potential. In order to evaluate the prognostic impact of tumor infiltration by PD-1 positive lymphocytes and PD-L1 expression by tumor cells, we performed a systematic study in a well-defined collective of 321 NSCLC patients undergoing primary tumor resection without preceding neoadjuvant therapy. Only completely resected, non-metastatic patients with a clear NSCLC histology were included for the statistical evaluation.

Tumor infiltration by PD-1 positive lymphocytes was detected in 22% of the tumor samples and 24% of the tumors displayed positivity for PD-L1 with 16 samples (5%) showing synchronous positivity for both. The finding concerning PD-L1 corresponds well to other published studies, which report immunohistochemical expression rates of 25–65% for PD-L1 in tumors of NSCLC patients [16,22–26]. Differences might be due to variabilities of the tumor microenvironment and to non-static expression at a single point of time [27]. To our knowledge, in contrast to reports on PD-L1 expression in tumor cells, no comparable data regarding the infiltration levels with PD-1 positive cells have been published, yet. Moreover, no mechanistic relation between the extent of PD-1 positive lymphocytic infiltration and PD-L1 positivity of the tumor cells has been shown so far. Hence, we did not find any association between PD-1 positive TILs and PD-L1 positive tumor cells.

To test whether PD-1 and PD-L1 correlate with the activation of apoptotic pathways [8], we analyzed associations for Bcl-2 and Bcl-xl. Here, positive associations were found for Bcl-xl expression in the tumor cells and PD-L1 expression in tumor cells. However, the observed positive correlation for Bcl-xl (Fisher’s exact test; Table 2) was not found in the prognostic subgroup analysis (log rank test; Table 3). So far, the impact of this observation is not clear and it may not be relevant. While one study group reported PD-L1 expression to be associated with adenocarcinoma histology [15] and another study reported it to be associated with squamous cell carcinoma histology [14], our correlation anaylsis (Fisher’s exact test) supports the latter observation. As shown before, we did not find any association between PD-L1 expression and presence of EGFR mutations [26].

The prognostic analysis of the full study cohort, including all NSCLC histologies, did not reveal any significant effect of tumor infiltration by PD-1 positive lymphocytes and/or PD-L1 expression of the tumor cells on overall survival (Fig 2A and 2B). However, a favorable prognosis was found for PD-L1 expression in tumor cells for patients who received adjuvant therapy, with pulmonary squamous cell carcinomas, higher T descriptor or lymph node metastasis (Fig 2C and 2D). Beyond other prognostic variables such as age or tumor stage, multivariate analyses confirmed PD-L1 expression in tumor cells to be a marker for an improved prognosis for patients with these characteristics. For infiltration by PD1 positive lymphocytes borderline prognostic effects were found in some multivariate subgroup analyses.

With respect to the recent literature, previous studies regarding the prognostic role of PD-L1 for NSCLC patients have been controversial. There are studies suggesting a negative prognostic value [15,24], whereas others did not find any prognostic impact [22,24,26]. Recently, one larger study including 340 NSCLC patients reported both, tumor PD-L1 protein and PD-L1 mRNA expression to be associated with increased local lymphocytic infiltrates and increased overall survival [14]. Our results are in agreement with this and further previous reports in other tumor types. An association between PD-L1 expression with an improved overall survival was found in metastasized malignant melanoma [28], colorectal cancer [29], and breast cancer [30]. As in our study, all reported patients did not receive anti-PD1/PD-L1 therapies.

The biology of an association between PD-L1 expression and better outcome in patients with adjuvant therapy, lymphatic metastasis, and squamous cell carcinoma is not well understood. A potential explanation is that the favorable prognostic impact of PD-L1 upregulation in these conditions may indicate the presence of a mixed immune cell infiltrate containing cytotoxic and regulatory T cells and reflect a partially dysbalanced local cellular immune response, which still contributes to antitumor immune control. In this case, a specific therapeutic interference with the PD-1/PD-L1 pathway may unleash a cytotoxic T cell response in the tumor. Thus, more detailed studies on the phenotype of infiltrating immune cells, in particular with regard to T cell subpopulations, seem to be important. Of interest, neither the proliferation marker Ki-67 nor the anti-apoptotic factors were associated with the prognostic effect of PD-L1 expression, highlighting that regulation of proliferation and apoptosis may be independent form immunologic mechanisms.

With respect to therapeutic interventions, inhibition of PD-1/PD-L1 is expected to become a powerful therapeutic alternative for NSCLC [31]. Overall, for advanced NSCLC patients, the overall response rate (ORR) for PD-1 inhibitory drugs was 24%, whereas for NSCLC patients with PD-L1 expression the ORR was 100% compared to 15% for PD-L1 negative tumors [32]. The latter study argues in favour of the evaluation of PD-L1 expression as a selective biomarker, and the analysis of PD-L1 in NSCLC could serve as predictor for response to PD-1 pathway inhibition and additionally as a prognostic marker for improved clinical outcome [33].

Our oligocentric study has several limitations, such as its retrospective nature and the potential risk of bias resulting from variable treatment protocols, regarding both surgical procedures and adjuvant therapies. The issue of representative tissue sample selection for TMAs was addressed in a previous study [18]. To reduce sampling errors, each patient’s tumor was represented by three tissue cores sampled from different tumor areas covering potential histological heterogeneity.

In conclusion, PD-L1 is a prognostic factor for NSCLC patients with squamous cell carcinoma histology, lymph node metastasis and patients treated in an adjuvant setting. It is feasible to hypothesize that patients with PD-L1 expression profit the most in an adjuvant treatment setting, however the sample size of our study is too small to answer this clinically important issue. Prospective studies are required to confirm this observation. If our observation is confirmed by further and prospective analyses, PD-L1 expression could contribute to adequate risk stratification. Beyond conventional therapies, PD-L1 expression likely represents a critical biomarker for predicting the individual probability of response to treatment with PD-1/PD-L1 pathway inhibitory agents. Prospective assessment of this parameter along with clinical trials will help to establish its significance in this context and allow selecting patients with a high likelihood to respond to various therapeutic interventions.