Bevacizumab Reduces S100A9-Positive MDSCs Linked to Intracranial Control in Patients with EGFR-Mutant Lung Adenocarcinoma

ORIGINAL ARTICLE

Bevacizumab Reduces S100A9-Positive MDSCs Linked to Intracranial Control in Patients with EGFR-Mutant Lung Adenocarcinoma Po-Hao Feng, MD, PhD,a,b,c Kuan-Yuan Chen, MD,a,d Yu-Chen Huang, MD,e Ching-Shan Luo, MD,a Shen Ming Wu, PhD,a Tzu-Tao Chen, MD,a Chun-Nin Lee, MD,a Chi-Tai Yeh, PhD,f Hsiao-Chi Chuang, PhD,g Chia-Li Han, PhD,h Chiou-Feng Lin, PhD,i Wei-Hwa Lee, MD,j Chih-Hsi Kuo, MD,e Kang-Yun Lee, MD, PhDa,b,* a

Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, Taipei, Republic of China b Division of Pulmonary Medicine, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Republic of China c Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD d Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Republic of China e Division of Pulmonary Medicine, Department of Internal Medicine, Chang Gung Medical Foundation, Linko Branch, Taoyuan, Republic of China f Department of Medical Research and Education, Shuang Ho Hospital, Taipei Medical University, Taipei, Republic of China g School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Republic of China h Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, College of Pharmacy, Taipei Medical University, Taipei, Republic of China i Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Republic of China j Department of Pathology, Shuang Ho Hospital, Taipei Medical University, Taipei, Republic of China Received 7 March 2018; revised 27 March 2018; accepted 28 March 2018 Available online - 20 April 2018

ABSTRACT Introduction: In vitro models have demonstrated immunemodulating effects of bevacizumab (BEV). Combinations of an EGFR tyrosine kinase inhibitor (TKI) with BEV improve progression-free survival (PFS) in patients with EGFR-mutated lung adenocarcinoma. How BEV confers this clinical effect and the underlying mechanisms of its effect are not clear. Methods: A total of 55 patients with stage 4 EGFR-mutated lung adenocarcinoma were enrolled. Myeloid-derived suppressor cells (MDSCs), type 1 and type 2 helper T cells, and cytotoxic T lymphocytes were analyzed by flow cytometry. Clinical data were collected for analysis. Result: In all, 25 patients received EGFR TKI and BEV combination therapy (the BEV/TKI group) and 30 patients received EGFR TKI monotherapy (the TKI-only group). The BEV/TKI group had longer PFS (23.0 versus 8.6 months [p ¼ 0.001]) and, in particular, better intracranial control rates (80.0% versus 43.0% [p ¼ 0.03]), a longer time to intracranial progression (49.1 versus 12.9 months [p ¼ 0.002]), and fewer new brain metastases (38.0% versus 71.0% [p ¼ 0.03]) than the TKI-only group did. The BEV/ TKI group had a lower percentage of circulating MDSCs (20.4% ± 6.5% before treatment versus 12.8% ± 6.6% after

treatment, respectively [p ¼ 0.02]), and higher percentages of type 1 helper T cells (22.9% ± 15.3% versus 33.2% ± 15.6% [p < 0.01]) and cytotoxic T lymphocytes (15.5% ± 7.2% versus 21.2% ± 5.6% [p < 0.01]) after treatment, changes that were not seen in the TKI-only group. Pretreatment percentage of MDSCs was correlated with PFS, with this correlation attenuated after BEV/TKI treatment. Percentage of MDSCs was also associated with shorter time to intracranial progression. Conclusion: Combining a EGFR TKI with BEV extended PFS and protected against brain metastasis. Those effects were probably due to the reduction of circulating S100A9-positive

*Corresponding author. Drs. Kuo and Lee contributed equally to this work. Disclosure: The authors declare no conflict of interest. Address for correspondence: Kang-Yun Lee, MD, PhD, No. 291, Zhongzheng Rd., Zhonghe District, New Taipei City, 23561, Taiwan. E-mail: [email protected] ª 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. ISSN: 1556-0864 https://doi.org/10.1016/j.jtho.2018.03.032

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MDSCs by BEV, which leads to restoration of effective antitumor immunity. Our data also support the rationale for a BEV–immune checkpoint inhibitor combination.  2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. Keywords: EGFR; Angiogenesis; Myeloid-derived suppressor cells; Lung cancer

Introduction EGFR tyrosine kinase inhibitors (TKIs) have great efficacy and improve quality of life in patients with lung adenocarcinoma harboring activating EGFR mutations.1,2 However, the disease eventually progresses, primarily or distantly, by developing acquired resistance. In the brain, disease control might also be dampened by low central nervous system (CNS) concentrations of EGFR TKIs (hemodynamic failure). Brain metastasis is common in patients with advanced lung cancer (with the incidence being about 20% to 40%) and is a poor prognostic factor for survival.3 For asymptomatic brain metastasis, EGFR TKIs had an intracranial control rate of 60% to 70%.4,5 Although the combination of whole brain radiotherapy (WBRT) and an EGFR TKI improved intracranial response rate and prolonged progression-free survival (PFS), it did not prolong overall survival (OS).6 To reduce unwanted neurotoxic side effects, WBRT deferred until intracranial progression might be sufficient in closely monitored patients with asymptomatic brain metastasis treated with EGFR TKIs.7 Although EGFR TKIs have shown much better CNS efficacy than conventional chemotherapy has, the brain is still a frequent site of disease recurrence.8 Bevacizumab (BEV), which is a humanized immunoglobulin G1 monoclonal antibody to the vascular endothelial growth factor (VEGF), has been demonstrated to provide a survival benefit when used in combination with platinum-based doublet chemotherapy in patients with nonsquamous NSCLC.9 Adding BEV to chemotherapy robustly increases both extracranial and intracranial efficacy in EGFR wild-type nonsquamous NSCLC and probably delays the onset of brain metastasis.10–12 In EGFR-mutant lung adenocarcinoma, combination of BEV with EGFR TKI, albeit similarly in OS comparing to EGFRTKI alone, did improve response rate and PFS.13 However, neither the clinical details of the combination of BEV and an EGFR TKI nor its underlying mechanism have been clearly demonstrated in a clinical setting. Myeloid-derived suppressor cells (MDSCs) are known to play important roles in tumor immune evasion and to contribute to tumor metastasis. Tumor-infiltrating monocytic MDSCs facilitate tumor cell dissemination by inducing the epithelial-

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mesenchymal transition phenotype.14 MDSCs expressed both VEGF receptor (VEGFR) 1 and VEGFR 2. In a tumorbearing mouse model, infusion of VEGF could result in accumulation of granulocyte-differentiation antigen-1– positive MDSCs and inhibition of dendritic cell maturation.15,16 In patients with ovarian cancers, VEGF was demonstrated to induce MDSCs, as confirmed by a mouse model.17 We thus hypothesized that anti-VEGF monoclonal antibody might be able to reduce MDSC level. We have previously reported that the level of circulating monocytic S100A9-positive MDSCs correlated with treatment response and PFS with first-line chemotherapy in patients with EGFR wild-type lung adenocarcinoma.18 Our unpublished observation showed a similar correlation in EGFR-mutant EGFR TKI–treated lung adenocarcinoma. A growing body of preclinical evidence has supported an immune-modulating role of BEV, including a reduction in MDSC level; however, evidence in humans and clinical relevance have not been well elucidated. In this study, through recruiting patients with EGFRmutated lung adenocarcinoma treated with an EGFR TKI alone or in combination with BEV, we were able to conduct a sophisticated analysis of clinical outcomes and immune profiles before and after treatment and thereby evaluate the immune-modulating effects of BEV, focusing on its clinical relevance.

Subjects and Methods Subjects This study was approved by both the Taipei Medical University Joint Institutional Review Board (TMU-JIRB No. 201402046) and the Chang Gung Medical Foundation Institutional Review Board (IRB 102-3377B), and informed consent was obtained from all subjects. All subjects had stage IV lung adenocarcinoma harboring an activating EGFR mutation. Thirteen patients were recruited from Chang Gung Medical Foundation–Linko Branch, and 42 patients were recruited from Shuang Ho Hospital. In all, 25 patients received combination therapy consisting of an EGFR TKI and BEV, and 30 patients received first-line EGFR TKI treatment only. EGFR TKI treatment included erlotinib, 150 mg, or gefitinib, 250 mg, once daily. BEV was given in a dose of 7.5 to 15 mg/kg at the physician’s decision every 3 weeks. Tumor response was evaluated by computed tomography according to the Response Evaluation Criteria in Solid Tumors, version 1.1, criteria.19 The site of image-confirmed first tumor progression and new brain metastasis or progress of known brain metastasis were both documented. PFS was defined as the interval from the start of first-line treatment until imagedocumented progress of the lesion or death, and OS was defined as the interval from the start of first-line of

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treatment until death or the cutoff date of December 31, 2017. Intracranial control rate was defined as the number of patients with no progress of their brain metastasis and no new brain metastasis divided by the total number of patients. Extracranial control rate was defined as the number of patients without new extracranial metastasis or progression of their primary lesion divided by the total number of patients. Time to progression (TTP) of the intracranial and extracranial lesions was defined as the interval from the start of first-line treatment until progression of the intracranial and extracranial lesions.

Flow Cytometry Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll Paque density gradient medium (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). PBMCs were stained according to the manufacturer’s recommendations. CD11b-positive CD14-positive S100A9-positive monocytic MDSCs (S100A9-positive MDSCs) were stained as previous described.18 To measure T-cell subsets in PBMCs, T-cell expression of CD4, CD8, interleukin-4 (IL-4), and interferon gamma (IFN- g) were measured. Briefly, 2  105 PBMC cells were permeabilized with fluorescence-activated cell sorting permeabilization buffer (BD Biosciences, San Jose, CA) and then stained with mouse antihuman CD4 (BD Biosciences), CD8 (BD Biosciences), IFN- g (eBioscience), and IL-4 (eBioscience) according to the manufacturers’ recommendations and incubated for 30 minutes at 4 C. The data from10,000 events in live cells were analyzed with FlowJo software (TreeStar, Inc., Ashland, OR). To understand the influence of the percentage change of lymphocytes in PBMCs, lymphocytes were gated and transformed into the percentage of lymphocytes.

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EGFR TKI and BEV (the BEV/TKI group) and 30 patients in the group treated with a EGFR TKI alone (the TKI-only group). Baseline characteristics were summarized in Table 1. The median follow-up time was 31 months. Both groups were in similar terms of distribution according to sex, age, Eastern Cooperative Oncology Group performance status, smoking status, EGFR mutation type, metastatic sites, and brain metastasis. Both groups had a high prevalence of brain metastasis (64% and 50% in the BEV/TKI and TKI-only groups, respectively [p ¼ 0.41]) at diagnosis, with most being asymptomatic. Most of the patients in both groups received erlotinib. The average BEV dose was 10.8 mg/kg given every 3 weeks. Each group included six patients who had received WBRT for their baseline brain metastasis (see Table 1). Table 1. Baseline Characteristics Characteristic

Combination EGFR TKI Group (n ¼ 26) (n ¼ 30)

p Value

Baseline Characteristics

Sex, n (%) Male Female Mean age, y (range) ECOG performance status, n (%) 0 or 1 2 Smoking, n (%) Never-smoker Smoker EGFR mutation status, n (%) 19 Del L858R Site of metastasis at diagnosis, n (%) Contralateral lung Brain Bone Liver Adrenal gland Pleural effusion/ pleural seeding Brain metastasis type, n (% of brain metastases) Oligometastasis Multiple Asymptomatic Whole brain radiation therapy, n (% of brain metastases) EGFR TKI, n (%) Gefitinib Erlotinib Afatinib

In total, 55 patients were enrolled in this study: 25 patients in the group treated with a combination of an

TKI, tyrosine kinase inhibitor; ECOG, Eastern Cooperative Oncology Group; Del, deletion.

Statistics Quantitative variables were assessed by the MannWhitney test or paired t tests for continuous variables, and the categorical variances between groups were assessed by Kruskal-Wallis analysis. The relationships between two parameters were investigated by using the Spearman rank correlation test. Survival curves were estimated by the Kaplan-Meier method, and the log-rank test was used to compare the patient survival times per group. GraphPad Prism software (version 6.0, GraphPad Software, San Diego, CA) was used for all statistical analyses, and statistical significance was defined as a p value less than 0.05. The significance level was set at p less than 0.05.

Results

0.27 7 (28) 13 (43) 18 (72) 17 (57) 62 ± 10.7 (42–84) 68 ± 11.6 (53–89) 0.06 0.44 23 (92) 2 (8)

25 (83) 5 (17)

22 (88) 3 (12)

24 (80) 6 (20)

0.49

1.00 10 (40) 15 (60)

11 (37) 19 (63)

10 (40) 16 (64) 12 (48) 3 (12) 1 (4) 14 (56)

11 (37) 15 (50) 14 (47) 3 (10) 2 (7) 14 (47)

1.00 0.41 1.00 1.00 1.00 0.59

4 (25) 12 (75) 7 (44) 6 (38)

4 (27) 11 (73) 8 (53) 6 (40)

1.00 1.00 0.72 1.00

3 (12) 16 (64) 6 (24)

6 (21) 21 (70) 3 (10)

0.33

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Treatment Response and Pattern of Failure Both groups responded well to the treatments, with objective response rates of 72% and 70% in the BEV/ TKI and TKI-only groups, respectively (p ¼ 1.00). The BEV/TKI group had longer PFS and OS times than the TKI-only group (median PFS 23.0 versus 8.6 months [p < 0.001] and median OS 49.1 versus 15.7 months [p ¼ 0.01]) (Fig. 1A and B and Table 2). The intracranial control rate was higher in the BEV/TKI group than in the TKI-only group (80% versus 43% [p ¼ 0.03]) (see Table 2). The BEV/TKI group demonstrated a trend of higher (albeit not significantly so) extracranial control rate (64% versus 30% [p ¼ 0.06]). The TTP of both intracranial and extracranial lesions was longer in BEV/ TKI group (intracranial 49.1 versus 12.9 months [p ¼ 0.002] and extracranial 24.6 versus 10.7 months [p ¼ 0.002]) (see Table 2 and Fig. 1C and D). To clarify whether better intracranial control rate was related to treatment, we evaluated the site of first progression and the disease progression pattern. Both groups had a similar pattern of disease progression, except that the TKI-only group had more brain metastasis as the first progression site than the BEV/TKI group did (38% versus 71% [p ¼ 0.03]) (see Table 2). These data indicate that BEV had a particular effect on protection against brain metastasis.

Immune-Modulating Effects of BEV To understand the potential underlying mechanisms mediating the clinical benefit of BEV, we analyzed

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S100A9-positive peripheral blood MDSCs from the same patients before and after treatment. The baseline percentage of S100A9-positive MDSCs of the two groups was similar (20.4% ± 6.5% versus 16.9% ± 9.0% [p ¼ 0.31]) (Fig. 2A). With similar tumor response rates, we found that the percentage of S100A9-positive MDSCs decreased only after BEV/TKI treatment and not in TKIonly group (see Fig. 2A and B) (in the BEV/TKI group: 20.4% ± 6.5% versus 12.8% ± 6.6% of PBMCs before and after treatment, respectively [p ¼ 0.02]; TKI-only group: 16.4% ± 9.5% versus 14.3% ± 10.3%, [p > 0.05]). MDSC level decreased in every BEV-treated patient. There was no difference between the patients receiving the two different doses of BEV. In the 7.5-mg/ kg group, the MDSC levels before and after treatment were 20.6% ± 6.5% and 14.8% ± 7.7%, respectively, (p < 0.01); in the 15-mg/kg group, the respective levels were 20.2% ± 7.6% versus 10.3% ± 4.7% (p < 0.01). To see whether the change in MDSC level is associated with clinical benefit of BEV, we tested its correlation with PFS. MDSC levels were well correlated with PFS in both groups before treatment; however, in the BEV/TKI group, the correlation was not significant after treatment (Fig. 2C). These data imply that MDSC level is related to PFS and that the reduction of MDSC level by BEV is linked to the favorable PFS. Importantly, the effect on MDSC level is not due to tumor shrinkage, as shown in the TKI-only group. BEV might have changed tumor biology or the tumor microenvironment, leading to a more effective immunity.

B TKI+ Avastin, n=25 TKI, n=30 Log-Rank test, p<0.001 Median PFS= 23.0 vs 8.6 m

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Figure 1. Kaplan-Meier survival curve of progression-free survival (PFS) (A), overall survival (OS) (B), intracranial time to progression (TTP) (C), and extracranial lesion TTP (D). TKI, tyrosine kinase inhibitor.

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Table 2. Treatment Response and Tumor Progress Pattern

Characteristic Clinical response, n (%) Complete response Partial response Stable disease Progressive disease Objective response rate, n (%) Intracranial control rate, n (%) Disease progression after first-line treatment First site of progression, n (% of disease progression) Lung primary site progression, Brain metastasis progression New brain metastasis Previous brain metastasis progression Others metastasis progression Disease progression pattern, n (% of disease progression) Only primary site progression Only distant site progression New brain metastasis Original brain metastasis progression Other metastasis progression Both primary and distant progression New brain metastasis Original brain metastasis progression Other metastasis progression Time to progression, intracranial, median (95% CI)

Combination Group (n ¼ 25)

EGFR TKI (n ¼ 30)

p Value 0.97

0 (0) 18 (72) 6 (24) 1 (4) 18 (72)

0 (0) 21 (70) 8 (27) 1 (3) 21 (70)

1.00

20 (80)

13 (43)

0.03

16 (64)

28 (93)

0.02

8 (50)

19 (67)

0.21

6 (38)

17 (71)

0.03

2 (13)

7 (29)

0.46

4 (25)

10 (42)

0.73

4 (25)

7 (30)

1.00

5 (25)

6 (22)

0.49

1 (7)

4 (16)

0.64

3 (19)

2 (8)

0.34

3 (19)

2 (8)

0.34

1 (7)

3 (13)

1.00

1 (7)

8 (33)

0.12

2 (7)

7 (29)

0.45

49.1 (not reached)

12.9 (2.5–23.2)

0.002

5

Table 2. Continued

Characteristic Time to progression, extracranial, median (95% CI) Progression-free survival, median, (95% CI) Overall survival, median, (95% CI)

Combination Group (n ¼ 25)

EGFR TKI (n ¼ 30)

p Value

24.6 (18.4–30.8) 10.7 (7.2–14.2)

0.002

23.0 (12.3–-33.6) 8.6 (6.7–10.5)

<0.001

49.1 (not reached)

17.1 (10.2–24.0) 0.01

TKI, tyrosine kinase inhibitor; CI, confidence interval.

To see whether the reduction in percentage of MDSCs is really associated with more effective immunity, we analyzed type 1 helper T (Th1) cells (CD4-positive IFNg–positive), type 2 helper T (Th2) cells (CD4-positive IL-4–positive), and cytotoxic T lymphocytes (CTLs) (CD8-positive IFN-g–positive) before and after treatment. The percentages of both effector Th1 cells and CTLs in the BEV/TKI group were increased after treatment (Fig. 3) (percentage of Th1 cells 22.9% ± 15.3% versus 33.2% ± 15.6% before and after treatment, respectively, [p < 0.01] and percentage of CTLs 15.5% ± 7.2% versus 21.2% ± 5.6%, respectively, [p < 0.01]); however, they were not increased in the TKI-only group. In contrast, BEV did not seem to have any effect on Th2 cells. These data confirmed an immune-modulating effect of BEV in a real human setting.

Effect of BEV on MDSCs and Clinical Translation

(continued)

To clarify the clinical correlation of MDSC level, we divided the patients into high– and low–MDSC level groups, using the median MDSC level as the cutoff level (median percentage MDSC of PBMC ¼ 16.3%). The baseline status of the two groups was no different in terms of sex, age, Eastern Cooperative Oncology Group performance status, smoking, EGFR mutation status, metastatic sites, or treatment group (BEV/TKI treatment in high- and low-MDSC groups: 31% versus 27% [p ¼ 1.00]) (Supplementary Table 1). Both high- and lowMDSC groups had similar response patterns in each treatment arm (ORR of the BEV/TKI treatment arm: 80% versus 100% [p ¼ 1.00]; ORR of the TKI-only arm: 72% versus 64% [p ¼ 1.00]) (see Supplementary Table 1). However, the group with the higher level of MDSCs had shorter PFS, OS, and intracranial lesion TTP values (see Supplementary Table 1 and Fig. 4A, D, and G). In this group with a high percentage of MDSCs, BEV/TKI treatment prolonged PFS and intracranial lesion TTP and showed an obvious trend toward longer OS (see Supplementary Table 1 and Fig. 4B, E, and H). The

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Figure 3. Effects of bevacizumab (BEV) on effector T cells. (A) Change of type 1 helper T (Th1) cells, type 2 helper 2 (Th2) cells, and cytotoxic T cells (CTLs) before and after treatment in two groups of treatment. (B) Dot plot of Th1 cells, Th2 cells, and CTLs from a representative case from BEV plus tyrosine kinase inhibitor (TKI) group. IFNg, interferon gamma; Tx, treatment; IL4, interferon 4; IL4, interleukin 4; SSC, side scatter; FSC, forward scatter; PerCP, peridinin-chlorophyll-protein complex; FITC, fluorescein isothiocyanate; PE-Cy7, phycoerythrin-cyanine dye 7.

significance was much attenuated in the group with a low percentage of MDSCs (see Supplementary Table 1 and Fig. 4C, F, and L). These data further support a linkage of PFS to MDSC level, a reduction in which by BEV (particularly in those with a high MDSC level) could extend the tumor control time. This effect is more obvious for intracranial control.

Discussion We have demonstrated in a real human setting that BEV is able to reduce the percentage of circulating S100A9-positive MDSCs, which was linked to a clinical benefit of prolonged PFS in patients with EGFR-mutant lung adenocarcinoma. This effect was particularly evident in intracranial control. The reduction of the level of S100A9-positive MDSCs by BEV was also accompanied by a restoration of a more effective antitumor immunity. In patients with NSCLC, new brain metastasis frequently developed during treatment. BEV has been proved efficacious in reducing new brain metastasis in

patients receiving chemotherapy (mainly in those with EGFR wild-type NSCLC).11 Because of poor CNS penetration of first- and second-generation EGFR TKIs, intracranial progression is seen more frequently in patients with EGFR-mutant NSCLC. Although WBRT has been shown to provide improved intracranial control at the expense of long-term neurotoxicity, it does not provide an overall survival benefit. In this study, we have shown that the combination of BEV and a EGFR TKI robustly improved intracranial control rate and intracranial lesion TTP, possibly by reducing the level of circulating S100A9positive monocytic MDSCs. The lower than expected PFS and OS values in our real-life study in the TKI-only group might be due to a higher rate of symptomatic brain metastasis. Brain metastasis is an independent poor prognostic factor for survival. Compared with the patients in the phase III EURATC trial,2 in which 13% of patients had brain metastasis, our patients had a 40% to 50% rate of symptomatic brain metastasis, with most of patients needing to receive WBRT.

Figure 2. Effect of bevacizumab (BEV) on myeloid-derived suppressor cells (MDSCs). (A) Combination of an EGKR tyrosine kinase inhibitor (TKI) and BEV reduce peripheral blood MDSCs but not in the TKI-only group. (B) Dot plot of CD11b-positive CD14-positive S100A9-positive MDSCs of representative case from the BEV plus TKI group, before and after treatment. (C) Correlation plot of the percentage of MDSCs in peripheral blood mononuclear cells (PBMCs) and progression-free survival (PFS) of treatment of two groups. Tx, treatment; SSC, side scatter; FSC, forward scatter; APC, allophycocyanin; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

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Figure 4. Kaplan-Meier survival curve of different myeloid-derived suppressor cells (MDSCs) level and treatment. (A, B, and C) Progression-free survival (PFS). (D, E, and F) Overall survival (OS). (G, H, and I) intracranial time to progression (TTP). TKI, tyrosine kinase inhibitor.

MDSCs are a heterogeneous group of cells of myeloid origin with immunosuppressive properties. Through arginase 1, inducible nitric oxide synthase, indoleamine 2,3-dioxygenase, and reactive oxygen species, MDSCs could suppress T-cell function and proliferation in the tumor microenvironment.20 Besides their role in immune suppression, MDSCs could be recruited to a premetastatic niche through the chemokines C-X-C motif chemokine ligand 1 and S100A9 and create a “premetastatic soil,” subsequently attracting tumor metastasis, including brain metastasis.21,22 MDSCs could also secrete angiogenic factors, including bombina variegate peptide 8 (Bv8) and VEGF through up-regulated signal transducer and activator of transcription 3 and promote tumor metastasis.23,24 In a previous study, sunitinib, which is a multiple tyrosine kinase inhibitor targeting VEGFRs and other related angiogenic receptors, was shown to be able to reduce MDSC level and restore T-cell function in patients with renal cell carcinoma. Our

finding supports the role of MDSCs in mediating metastasis. The clinical benefit of BEV in PFS might be due to a reduction in MDSC level. The major immune cells in the brain microenvironment are macrophages, mostly tissue-resident microglia. Recently, monocyte-derived macrophages have gained more attraction in pathologic conditions such as brain tumor.25,26 In the brain tumor microenvironment, circulating monocytes are recruited to the brain parenchyma and give rise to monocyte-derived macrophages (e.g., tumor-associated macrophages). Tumor-associated macrophages tend to be protumorigenic and accumulate with higher tumor grade.26 Circulating S100A9-positive monocytic MDSCs might also been recruited to the normal brain microenvironment and become a metastatic niche, thus resulting in further brain metastasis.21 In patients with metastatic renal cell carcinoma, the level of circulating MDSCs expressing VEGFR 1 increases with tumor progression,27 and BEV might inhibit these MDSCs

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through the VEGFR pathway. Compared with in other organs, tumors in the brain rely more on macrophages as tumor-supporting cells. This might explain the more obvious effect of BEV on intracranial control. T cells were shown to affect the metastatic potential of a primary tumor. Increased level of tumor-infiltrating lymphocytes was shown to correlate with better prognosis in many cancers, supporting the hypothesis that tumor-infiltrating lymphocytes might reduce tumor metastasis.28 In this study, we found that besides reducing the level of MDSCs, BEV treatment could result in elevation of Th1 and CTL levels. In addition to reduce the suppression effect of MDSCs, underlying mechanisms might also be decreasing the levels of proangiogenic (VEGF, angiostatin 1, and follistatin) and inflammatory cytokines (IFN-g, IL-4, and IL-17) and improve in vivo and in vitro CTL response and dendritic cell activation.29 Our study is compatible with that conducted by Wallin et al., which showed increased gene signatures associated with CD8 effector genes, Th1 chemokines, and nature killer cells after BEV treatment.30 We have thus confirmed the immune-modulating effect of BEV. In addition to its benefit to patients receiving EGFR TKIs, our data also support a potential role of BEV in combination with current immune checkpoint inhibitors. A recent promising report on the IMpower150 study did show the beneficial effect of BEV on atezolizumab in the backbone of carboplatin plus paclitaxel.

Conclusion In combination with the great efficacy of EGFR TKI in patients with metastatic EGFR-mutant lung adenocarcinoma, add-on BEV can further extend PFS and protect from brain metastasis. The reduction of circulating S100A9-positive MDSCs and restoration of effector Th1 cells and CTLs as a result of administration of BEV might be one of its underlying mechanisms. Our data also support the rationale for a BEV–immune checkpoint inhibitor combination.

Acknowledgment This study was supported by National Science Council (NSC 102-2314-B-038-048), Ministry of Science and Technology (MOST 103-2314-B-038 -056 and 104-2314-B-038-070), and Taipei Medical University (TMU103-AE1-B31, TMU103-AE2-I01-3, 103TMU-SHH-01-2, 105TMU-SHH02-2) to Dr. Feng.

Supplementary Data Note: To access the supplementary material accompanying this article, visit the online version of the Journal of Thoracic Oncology at www.jto.org and at https://doi. org/10.1016/j.jtho.2018.03.032.

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