Real-World Efficacy and Safety of Anlotinib With and Without Immunotherapy in Advanced Non-Small Cell Lung Cancer

Aims Combination of anti-angiogenesis therapy and immunotherapy has showed synergistic effects in non-small cell lung cancer (NSCLC). The aim of this retrospective study was to investigate the efficacy and safety of anlotinib with and without immunotherapy in NSCLC. Methods Pathologically confirmed NSCLC patients (stage IIIB-IV) receiving anlotinib between November 2018 and February 2020 were enrolled for retrospective analysis. The outcomes and safety of overall patients were evaluated, and the efficacies of anlotinib plus immunotherapy and anlotinib alone was compared. The primary endpoint was progression-free survival (PFS). Results A total of 80 patients (median age: 62 years, range: 29-86 years) were included. Overall median PFS was 4.3 months (95% confidence interval (CI): 2.7-5.9 months). In univariate analysis, patients without EGFR mutation, previous EGFR target therapy, and brain metastasis had significantly longer PFS. Cox regression analysis showed that only brain metastasis was an independent predictor of PFS. The median PFS of patients receiving anlotinib plus immunotherapy was slightly longer than that of patients receiving anlotinib alone (4.2 vs 3.1 months); however, the difference was not statistically significant. A tendency of longer median PFS was observed in patients with adenocarcinoma, EGFR wild type, stage IV, no liver metastasis, former smoker, ≥2 previous treatment lines, no previous VEGF or EGFR target therapies in anlotinib plus immunotherapy group. Treatments with anlotinib alone or anlotinib plus immunotherapy were well tolerable. The most common adverse events were fatigue, decreased hemoglobin count, hypertension, hand-foot syndrome, oral mucositis and hoarseness. Conclusion Anlotinib is well tolerable and effective in advanced NSCLC patients. Brain metastasis is an independent predictor of PFS in NSCLC patients receiving anlotinib. Future prospective studies with larger sample size and extended follow-up are needed to confirm the clinical benefit in NSCLC patients treated with anlotinib combined with immunotherapy.


INTRODUCTION
Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related mortality worldwide (1,2). About 80%-85% of patients have non-small cell lung cancers (NSCLC), and most have locally advanced or metastatic disease at diagnosis and therefore poor prognosis. Target therapy and immunotherapy have remarkably improved the overall survival (OS) and quality of life of these patients (3)(4)(5)(6)(7); however, for those who fail frontline treatment but remain in satisfactory physical condition, treatment options are limited. Novel effective drugs need to be identified.
Anlotinib is a tyrosine kinase inhibitor (TKI) targeting multiple tumor angiogenesis and proliferation signaling receptors, including receptor tyrosine kinases vascular endothelial growth factor receptor 1-3 (VEGFR 1-3), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor 1-4 (FGFR 1-4), platelet-derived growth factor receptor a and b (PDGFR a and b), and stem cell factor receptor (c-Kit) (8)(9)(10).The phase III ALTER 0303 study demonstrated that patients with advanced NSCLC receiving anlotinib as third or further line treatment had significantly longer OS (median OS: 9.6 vs 6.3 months) and progression-free survival (PFS) (median PFS: 5.4 vs 1.4 months) than patients receiving placebo (11). Based on these findings, anlotinib was approved as third line therapy for advanced NSCLC patients after two lines of chemotherapy by the China National Medical Products Administration in 2018. In a real-world study, Wu et al. retrospectively analyzed the efficacy of anlotinib as salvage treatment in 81 NSCLC patients and reported similar results: the median PFS of NSCLC patients treated with anlotinib was 5 months, with patients with Eastern Cooperative Oncology Group performance status (ECOG PS) 0-1 and those without brain metastasis most likely achieve longer PFS (12). In another study of 52 NSCLC patients receiving anlotinib as third-or later-line treatment, Zhang et al. found median PFS and OS to be 4.5 months and 9 months, respectively, with relatively longer survival rates in patients with ECOG PS 0-1 and patients without liver metastasis (13). In contrast to these findings, Shao et al. reported median PFS of only 3.3 months in 58 advanced lung cancer patients (50 with NSCLC) who were treated with anlotinib as third or later-line treatment (14). These inconsistent results call for more detailed analysis of the real-world efficacy of anlotinib.
Previous studies have shown that combination of antiangiogenesis therapy and other type of therapies had synergistic effects, and revealed prolonged PFS and OS in the first line and further line treatment (15)(16)(17). However, previous clinical trials failed to demonstrate a survival benefit with combination of antiangiogenesis TKIs (such as nintedanib, sorafenib, and sunitinib) with chemotherapy in NSCLC (18)(19)(20)(21). At the 20th World Conference on Lung Cancer (WCLC 2019), Han and colleagues reported the results of phase II clinical trials which showed that the combination of anlotinib and chemotherapy or target therapy as first-line treatment for advanced NSCLC increased the objective response rate (ORR) and the disease control rate (DCR): ORR was 60% for chemotherapy plus anlotinib and 92.6% for erlotinib plus anlotinib, while DCR was 96.7% for chemotherapy plus anlotinib and 100% for erlotinib plus anlotinib (22,23). In addition, in a phase I study, Han et al. reported ORR of 72.7%, DCR of 100%, and 12month PFS rate of 71.4% with the combination of sintilimab with anlotinib as first-line treatment for NSCLC (24,25). These evidences suggest that anlotinib combined with other therapies might be promising treatment for advanced NSCLC. Indeed, these therapeutic strategies have already been applied in clinical practice. Wang et al. showed that the median PFS and OS were 6.9 months and 14.5 months in 67 NSCLC patients treated with anlotinib plus anti-programmed death-1 (PD-1) antibody after previous systemic treatment (26).Thus, we performed a retrospective analysis of realworld data to investigate the efficacy of anlotinib used alone and in combination with other agents in advanced NSCLC, with special attention to the combination of anlotinib with immunotherapy.

Patients and Treatments
Pathologically confirmed NSCLC patients (stage IIIB-IV according to AJCC 8th edition cancer staging system) receiving anlotinib in General Hospital of Chinese PLA between November 2018 and February 2020 were eligible for retrospective analysis. The inclusion criteria were as follows: advanced stage (IIIB-IV) NSCLC confirmed pathologically; with at least one measurable lesion; treated with anlotinib for the first time regardless of combined therapy and treatment lines; aged over 18 years old; ECOG PS score 0-2; radiotherapy allowed for stage III patients. The exclusion criteria were as follows: previous use of anlotinib; diagnosis with concomitant other cancers other than NSCLC; patients with hemoptysis (>50 ml/d) or deep vein thrombosis or pulmonary embolism; patients participated in clinical trials of other anti-tumor drugs within four weeks; severe disfunction of heart, liver, kidney and (or) other complications that might detrimental for survival determined by investigators. The medical records of the patients were reviewed to collect data on sex, age, pathological type, stage, presence of liver/brain metastasis, ECOG PS, smoking history, previous treatments, and status of EGFR, ROS1, ALK, HER2, TP53, and KRAS.
This study was approved by Ethics Committee of the General Hospital of Chinese PLA (Medical Ethics Committee of General Hospital of Chinese PLA No. S2018-092-01) and conducted according to the principles of the Declaration of Helsinki. The requirement for informed consent was waived in view of the retrospective nature of the study.

Outcomes and Safety Evaluation
Therapeutic effect was assessed using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 by computed tomography (CT) scans every 2 cycles by two doctors independently and categorized as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). When there is disagreement on CT evaluation, a third doctor was requested to reevaluate. PFS was defined as the time from anlotinib initiation until disease progression or death of any cause before disease progression. OS was defined as the time from the beginning of anlotinib to death. Best response was used to calculate objective response rate (ORR) and disease control rate (DCR). Treatmentrelated adverse events were graded using Common Terminology Criteria for Adverse Events (CTCAE) version 5.0.

Statistical Analysis
The chi-square test and Fisher's exact test were used to compare the baseline characteristics and treatment response between two groups. Analysis of variance (ANOVA) was used to compare the difference among three or more groups. Survival curves for OS and PFS were analyzed using the Kaplan-Meier method. The log-rank test was used for univariate analysis between groups. Cox regression was used to analyze the statistically significant factors according to results of univariate analysis. Forest plots were used to depict the results of stratified analysis. Statistical analysis was performed by PRISM version 7.0 (GraphPad Software, La Jolla, CA, USA) and SPSS version 26.0 (IBM Corp., Armonk, NY, USA). P<0.05 (twosided) was defined as statistical significance.

Overall Efficacy of Treatment
Of the 80 patients, PR was achieved in 9/80 patients, SD in 49/80 patients, and PD in 22/80 patients. The overall ORR and DCR were 11.3% and 72.5%, respectively. The median follow-up duration was 6.1 months (range: 2.0-15.1 months). Treatment with anlotinib was still ongoing for 27 patients by the day of last follow-up. Overall median PFS was 4.3 months (95% confidence interval (CI): 2.7-5.9 months) ( Figure 1A). Univariate analysis revealed that patients without EGFR mutation, previous EGFR target therapy and brain metastasis had statistically significant longer PFS (Figures 2A-C and Table 1). Age, sex, pathological type, TP53 and KRAS status, stage, liver metastasis, smoking history, ECOG PS, and previous treatment lines or regimens were not associated with PFS ( Table 1). In multivariate cox regression analysis, brain metastasis was the only factor independently associated with PFS (HR: 1.816, 95%CI: 1.003-3.286, P=0.049) ( Table 2). EGFR mutation and previous EGFR target therapy were not independent risk factors for PFS. The 1-year survival rate was 92.6% in this study. Since 76 (95%) patients were still alive at last follow-up, OS was not analyzed ( Figure 1B).

Efficacy of Anlotinib Combined With Immunotherapy
To explore whether immunotherapy could improve the efficacy of anlotinb, we compared the efficacy of anlotinib plus immunotherapy (anti-PD-1 or PD-L1 antibodies) with anlotinib alone in NSCLC. There were 30 patients receiving anlotinib combined with immunotherapy, and 24 receiving anlotinib alone. Baseline characteristics were comparable between the two groups ( Table 3).
Median PFS was longer for patients receiving anlotinib plus immunotherapy than for patients receiving anlotinib alone, although the difference was not statistically significant (4.2 months vs. 3.1 months; HR: 0.627, 95% CI: 0.312-1.260; P = 0.19) (Figure 3). The ORR (12.5% vs 10%) and DCR (70.8% vs 70%) were similar between anlotinib arm and anlotinib combined with immunotherapy arm ( Table 4). Stratified analysis was performed to identify the subgroups most likely to benefit from the addition of immunotherapy to anlotinib therapy. Although no statistically significant differences were found, median PFS tended to be longer in patients with adenocarcinoma, EGFR wild type, stage IV, no liver metastasis, former smoker, ≥2 previous treatment lines, no previous VEGF or EGFR target therapies ( Table 5 and Figures 4A-H). Figure 5 presents a forest plot of the stratified analysis.
Among patients receiving anlotinib plus immunotherapy, similar to overall patients, the five most common adverse events were fatigue (70%), hypertension (50%), hand-foot syndrome (40%), decreased hemoglobin level (36.67%), and hoarseness (36.7%). Grade 3-5 Grade 3-5 adverse events occurred in 8 patients. Adverse events were similar in patients treated with anlotinib plus immunotherapy arm and patients treated with anlotinib alone. No additional adverse event was reported in anlotinib plus immunotherapy arm compared to anlotinib arm ( Table 6).

DISCUSSION
The ALTER 0303 study and previous real-world studies showed that anlotinib alone could improve prognosis in NSCLC patients as third or later line treatment. Furthermore, the combination of anlotinib with immunotherapy, target therapy, or chemotherapy has been shown to have synergetic efficacy (22)(23)(24)27). We retrospectively analyzed real-world data to determine the  efficacies of these therapeutic strategies in NSCLC, paying special attention to the combination of anlotinib plus immunotherapy. During the course of cancer, about 40% NSCLC patients will finally develop brain metastases, which leads to poor prognosis (28). In this study, brain metastasis was identified as an independent prognostic factor in NSCLC patients treated with anlotinib. Subgroup post hoc analysis of ALTER0303 showed similar median PFS in NSCLC patients with and without brain metastases (4.17 vs 4.53 months, P=0.69) (29). Zhang et al. also reported brain metastases had no influence on PFS of NSCLC patients treated with anlotinib alone (13). However, in line with wu et al. (12), we found significantly longer median PFS in patients without brain metastases than in patients with brain metastases, whether treated with anlotinib alone or with anlotinib plus other therapies. The differences between ours and previous studies were likely due to differences in treatment regimens and sample sizes; the difference between post hoc analysis and retrospective analysis may also contribute.
In the present study, univariate analysis showed significantly longer PFS in patients without EGFR mutation than in patients with the mutation. However, consistent with previous studies (12,13,30), cox regression analysis showed both EGFR status and previous EGFR target therapy were not independent risk factors for PFS in patients receiving anlotinib. EGFR-TKIs are standard first-line treatment for NSCLC patients with EGFR mutations. Unfortunately, patients who show response invariably develop resistance after 8-12 months. The main acquired resistance mechanisms appear to be secondary mutations of EGFR (T790M), KRAS, PIK3CA and MET amplification. A recent study suggested that anlotinib could overcome acquired resistance to EGFR-TKI via inhibiting FGFR1 (31). A preclinical study showed that anlotinib could inhibit proliferation and induce apoptosis of KRAS mutation lung cancer cells through downregulating MEK and ERK. The study also revealed prolonged survival of KRAS mutation in lung cancer animal model (32). In addition, there is a previous report of an NSCLC patient harboring KRAS mutation who had PFS of 21 months after treatment with anlotinib (33). The mechanisms described above may partially explain why patients   with acquired resistance to EGFR-TKIs could benefit from anlotinib. Overall, our results and the evidence from earlier researches indicate that patients can benefit from anlotinib treatment, regardless of EGFR status and previous EGFR target therapy. Immune checkpoint inhibitors targeting PD-1 or PD-L1 have remarkably improved survival in NSCLC patients. However, only about 20% NSCLC patients respond to anti-PD-1/PD-L1 antibody monotherapy. The combination of anti-angiogenesis agents and anti-PD-1/PD-L1 antibody has shown promising benefits in solid tumors such as hepatic carcinoma, renal cancer, and NSCLC (34). Yang et al. reported that anlotinib and anti-PD-1/PD-L1 antibody acted synergistically to provide therapeutic benefit by promoting infiltration of natural killer cells, M1-like tumor-associated macrophages (TAM), and dendritic cells, while reducing the infiltration of M2-like TAM (35). Another study showed that anlotinib reduced PD-L1 expression on vascular endothelial cells via inactivation of AKT pathway, which then leads to increase in the CD8/FoxP3 ratio in the tumor immune microenvironment (36). It appears  that anlotinib changes the immunosuppressive tumor microenvironment to an immune permissive status, and thus enhances efficacy of PD-1/PD-L1 antibody. However, in the present study, PFS was only slightly longer in patients receiving anlotinib plus anti-PD-1/PD-L1 group than in those receiving anlotinib alone, and no statistical difference was found. It is worth noting that the benefits of the combination of anlotinib and immunotherapy were most noticeable in patients with wild-type EGFR, without liver metastasis, without history of previous EGFR target therapy, and former smokers; however, the survival benefit was not statistically significant. Partially, we speculated this was because of the small sample size in our study. Previous studies showed that smoking, wild-type EGFR, and absence of liver metastasis predicted longer PFS and increased ORR in NSCLC patients treated with anti-PD-1/PD-L1 antibody (37)(38)(39), while acquired resistance to previous EGFR-TKI promoted immune escape (40). Furthermore, in patients who respond to anti-PD-1/PD-L1 antibodies, anti-PD-1/PD-L1 antibodies seem to have durable antitumor efficacy (41). In our study, the failure to demonstrate significant prolongation of PFS and OS was probably due to the small sample size and the short follow-up. In this study, treatment with anlotinib-either alone or in combination with other therapies-was generally well tolerated. Similar with previous clinical trial and real-world studies, the most common adverse were fatigue, hypertension, hand-foot syndrome and oral mucositis (11)(12)(13)(14). In addition, we indicated that hoarseness was also frequently observed in our sample. Fatal bleeding has been reported in other anti-angiogenesis drugs, such as sunitinib, sorafenib and bevacizumab (42). In our study, only 2 patients with mild alimentary tract hemorrhage or hemoptysis were reported and the bleeding stopped after anlotinib was discontinued. No additional adverse event was seen in patients receiving anlotinib plus anit-PD-1/PD-L1 antibodies. Thus, the combination of anlotinib with anit-PD-1/ PD-L1 antibodies appears to be safe in NSCLC.
This study has some limitations. First, this was a retrospective analysis of data of a small sample from a single center, and so bias is inevitable and the sample size is not sufficient to obtain statistical difference. Thus, future study with lager sample size is needed. Second, PD-L1 expression is considered as the utmost predictive biomarker for NSCLC patients treated with anti-PD-1/PD-L1 agents (43)(44)(45), while the PD-L1 status was not obtained in this study. Third, the follow-up period was short; longer follow-up is necessary to evaluate the effect of anlotinib plus immunotherapy on OS.
In conclusion, anlotinib appears to be well-tolerated and effective in patients with NSCLC. Brain metastasis was found to be an independent predictor of PFS in NSCLC patients receiving anlotinib with or without other therapies. Addition of immunotherapy to anlotinib may prolong PFS in patients with adenocarcinoma, wild-type EGFR, stage IV disease, no liver metastasis, exsmoker status, two or more previous treatment lines, and no previous VEGF or EGFR target therapies. Further large prospective studies are needed to confirm our findings. Future studies should also attempt to identify predictive biomarkers that could help identify patients likely to benefit from the combination of anlotinib plus immunotherapy.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of the General Hospital of Chinese PLA (Medical Ethics Committee of General Hospital of Chinese PLA No. S2018-092-01). The ethics committee waived the requirement of written informed consent for participation.

AUTHOR CONTRIBUTIONS
Conception/design: YH, QX; Collection of assembly of data: BQ, BY, QS, YW, SZ; CT evalutation: QX, BQ, YH; Data analysis and interpretation: QX, BQ, LX; Manuscript writing: QX LX; Final approval of manuscript: All authors. All authors contributed to the article and approved the submitted version.