Significance of inflammatory indexes in atezolizumab monotherapy outcomes in previously treated non-small-cell lung cancer patients

Cancer immunotherapy, including atezolizumab monotherapy, is a promising alternative strategy for patients with advanced non-small-cell lung cancer (NSCLC). Several inflammatory indices have been reported as potential biomarkers regarding the effectiveness of various treatments. This study aimed to analyze the efficacy of atezolizumab monotherapy using baseline inflammatory markers in NSCLC patients. We retrospectively enrolled 81 NSCLC patients who received atezolizumab monotherapy at six different medical institutions in Japan. The Cox proportional hazards model was used to assess the impact of the clinical variables, including inflammatory indexes, on clinical outcomes. Median progression-free survival (PFS) and overall survival (OS) were 60 days and 252 days, respectively. The objective response rate was 7.4%, and the disease control rate was 54.3%. Patients with high neutrophil to lymphocyte ratio (NLR), low lymphocyte to monocyte ratio (LMR), and/or high platelet to lymphocyte ratio (PLR), at baseline, demonstrated substantially shorter PFS and OS compared to those with a low NLR, high LMR, and/or low PLR. The multivariate analysis demonstrated that a high baseline NLR was substantially associated with short PFS and short OS. Our retrospective observations suggest that inflammatory indices may be a potential negative prognostic factor of atezolizumab monotherapy outcomes in NSCLC patients.


Scientific Reports
| (2020) 10:17495 | https://doi.org/10.1038/s41598-020-74573-0 www.nature.com/scientificreports/ Atezolizumab is a humanized, engineered monoclonal antibody that targets PD-L1, and contributes to preventing the interaction between PD-L1 and B7.1 receptor. The OAK study, a randomized phase 3 trial, demonstrated that the atezolizumab treatment group had a median OS of 13.8 months, which was substantially higher than the 9.9 months observed for the docetaxel group. Moreover, atezolizumab monotherapy showed tolerability with a more favorable safety profile than docetaxel 3,4 . PD-L1 expression in tumors has been used clinically as a positive biomarker for effective ICI treatment in NSCLC 5 . However, the anti-PD-L1 antibody clone SP142, which was utilized for clinical trials with atezolizumab, was relatively less concordant in PD-L1 expression than other antibodies, such as 28-8, 22C3, and SP263 in patients with NSCLC 6,7 . Furthermore, several recent studies have reported potential ICI biomarkers in the host, such as preexisting autoimmune antibodies 8 , steroid use 9 , microbiome 10 , white blood cell count 11 , sarcopenia 12 , and body mass index (BMI) [13][14][15] . Several inflammatory indices, such as the NLR, LMR, and PLR, which are recognized as important markers of inflammatory processes, have also been reported as potential predictors of the effectiveness of anti-PD-1 antibody therapy [16][17][18] . However, little is known regarding the subpopulation of NSCLC patients who exhibit clinical outcomes that require treatment with atezolizumab monotherapy. In this retrospective study, we analyzed the efficacy of atezolizumab monotherapy, using the baseline values of specific inflammatory markers, in 81 patients previously treated for NSCLC.

Discussion
Several studies have demonstrated that clinical characteristics, such as age, ECOG-PS, and smoking status, are negative biomarkers related to the clinical outcomes of anti-PD-1 antibody treatment in patients with NSCLC 16,17,19 . In contrast, current clinically useful biomarkers have not been fully identified in predicting the efficacy of anti-PD-L1 antibody atezolizumab monotherapy. A pooled cohort analysis of clinical trials involving 1,489 NSCLC patients demonstrated the significance of the lung immune prognostic index, which is derived from scoring of the baseline LDH levels and NLR, in predicting survival and response outcomes of NSCLC patients treated with atezolizumab 20 . In the current retrospective analysis of 81 NSCLC patients who received atezolizumab monotherapy, the pretreatment inflammatory indices, more specifically high NLR, low LMR, and high PLR, were substantially associated with shorter PFS and OS. Increasing evidence suggests that cancer-related inflammation plays an important role in tumor development. Peripheral blood leukocytes, including neutrophils and lymphocytes, are involved in the systemic inflammatory response, and participate in tumorigenesis and tumor progression. Previous studies have shown that high levels of neutrophils promote cancer cell proliferation, invasion, and metastasis, and induce resistance to cancer therapeutics 21,22 . Additionally, peripheral neutrophil counts reportedly correlate directly with the intratumoral neutrophil population 23 . In contrast, lymphocytes inhibit tumor growth and invasion through their cytolytic activity. In fact, the immune response to human cancer cells depends primarily on the level of total lymphocytes, which can be sharply reduced by systemic inflammation. Specifically, relative lymphocytopenia may reflect lower levels of CD4 + T cells, which impairs cancer immune surveillance and defense 21,24 .
NLR is a marker of the systemic inflammatory response and reflects the balance between neutrophils and lymphocytes 25,26 . Pretreatment NLR is associated with the clinical outcomes of several therapeutic interventions in NSCLC patients, such as the response to platinum-based first-line chemotherapy in metastatic NSCLC patients, and the prognosis in operable NSCLC patients 27,28 . Moreover, inflammatory indices, including NLR, PLR, and LMR, are potential prognostic markers for lung cancer patients [29][30][31][32] . Our multivariate analysis demonstrated that high baseline NLR is an independent factor associated with poor PFS and OS. It is; however, unclear whether a high NLR is an effective prognostic factor in NSCLC patients receiving atezolizumab monotherapy. www.nature.com/scientificreports/ Therefore, it is necessary to further evaluate the clinical role of inflammatory scores, including NLR, in future studies.
As several independent clinical factors have indicated the disadvantages of immunotherapy in NSCLC patients, we evaluated the combined scores of several clinical characteristics. Our findings revealed that patients with high NLR are substantially correlated with several other clinical characteristics, such as low BMI, low Alb, and high CRP levels, at the baseline, compared to those with a low NLR. The present study is the first to reveal that the combination of a high baseline NLR and CRP levels is a potent prognostic factor for NSCLC patients receiving atezolizumab treatment. Moreover, persistent inflammatory responses in tumors suppress anti-tumor immunity and promote cancer progress through several mechanisms, including activation of type 2 T helper responses, which recruits regulatory T cells, and activation of the chemokine system 33,34 . Subsequently, neutrophils can be induced by cancer-associated inflammatory chemokines and cytokines, such as interleukin (IL)-6 and tumor necrosis factor 35,36 . CRP is also regulated by IL-6 and IL-1β, suggesting that induction of neutrophils and CRP may occur via similar inflammatory pathways. In fact, our results highlight an important relationship between high NLR and high CRP levels and promotion of poor prognosis. Although the usefulness of these combinatorial indexes remains largely unknown, they have the potential to serve as accurate biomarkers of cancer-related inflammation; hence, further large-scale investigations are warranted.
High plasma tumor mutational burden has been identified as a pivotal biomarker for the efficacy of atezolizumab monotherapy, and is associated with superior PFS in patients with previously treated NSCLC 37 ; however, its associated cost makes in unfeasible for daily use. Alternatively, blood count analysis and CRP assessments are cost effective, form part of routine clinical practice, and reveal prognostic factors that could be useful in identifying NSCLC patients who will respond poorly to atezolizumab monotherapy, thereby assisting clinical decision-making regarding appropriate therapeutic interventions in previously treated NSCLC.
The present study has several limitations. Firstly, it is a retrospective study and the cohort had a limited sample size of 81 cases, even though treatment was administered in multiple medical institutions. Secondly, all patients in the cohort were Japanese. Thirdly, the study included several biases regarding patient conditions at commencement of atezolizumab therapy, such as the number of pretreatment regimens and the ECOG-PS of the patients. Finally, our findings revealed a substantial relationship between pre-treatment blood inflammatory markers and clinical outcomes, such as PFS and OS, in NSCLC patients treated with atezolizumab. These inflammatory markers might be a prognostic factor rather than a predictive factor for patients with this disease treated with atezolizumab. Although the current study was retrospective in nature, our novel biomarker findings In summary, our observations showed that pretreatment inflammatory indexes, including a high NLR, could be promising negative prognostic factors for atezolizumab treatment in patients with previously treated for NSCLC. Since this retrospective study was conducted on a smaller scale, further experiments are needed to validate these observations.

Methods
Patients. We enrolled 81 patients, previously treated with chemotherapy for advanced NSCLC, who initiated atezolizumab monotherapy. The patients were treated between April 2018 and November 2019 at six different medical institutions, namely University Hospital, Kyoto Prefectural University of Medicine (Kyoto, Japan), Japanese Red Cross Kyoto Daiichi Hospital (Kyoto, Japan), Japanese Red Cross Kyoto Daini Hospital (Kyoto, Japan), Uji-Tokushukai Medical Center (Kyoto, Japan), Fukuchiyama City Hospital (Kyoto, Japan), and Otsu City Hospital (Shiga, Japan).
Atezolizumab was intravenously administered to patients as a fixed dose of 1200 mg every three weeks. In general, these treatments continued until disease progression, intolerable toxicity, or patient refusal was noted.   Tumor PD-L1 analysis. PD-L1 expression was analyzed by SRL, Inc. using a PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies, Santa Clara, CA). The PD-L1 tumor proportion score (TPS) was calculated as a percentage in at least 100 viable tumor cells with complete, or partial, membrane staining. Pathologists at SRL, Inc. interpreted the TPS results.
Laboratory findings. The NLR, LMR, and PLR were defined as absolute neutrophil counts divided by absolute lymphocyte count, absolute lymphocyte count divided by absolute monocyte count, and absolute platelet count divided by absolute lymphocyte count, respectively. We measured baseline albumin (Alb), C-reactive protein (CRP), lactate dehydrogenase (LDH), as well as neutrophil, lymphocyte, monocyte, and platelet counts, and NLR, LMR, and PLR. Baseline was defined as day − 10 to 0 of the first atezolizumab administration. Cut off points of NLR = 5, LMR = 1.5, and PLR = 262 were selected based on previous studies 16,19 . The cutoff values for baseline albumin, LDH, CRP, neutrophil, lymphocyte, monocyte, and platelets, were the respective median values.
Statistical analysis. Statistical analyses were performed using EZR statistical software, version 1.30 38 . All statistical tests carried out were two-sided and p < 0.05 was regarded as statistically significant. The PFS and OS were calculated using the Kaplan-Meier method, and differences were compared using the log-rank test. Continuous variables were analyzed using the Mann-Whitney U test, while categorical variables were analyzed using Fisher's exact test. Univariate analyses were performed using the Cox proportional hazards and logistic regression models.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.