Anlotinib combined with PD‑1 blockade for the treatment of lung cancer: a real‑world retrospective study in China
Xiangyu Zhang1 · Liang Zeng1 · Yizhi Li1 · Qinqin Xu2 · Haiyan Yang1 · Analyn Lizaso3 · Xinru Mao3 · Ren’an Jin4 · Yu Zeng5 · Qinglin Li6 · Jianbo Wang7 · Yang Li8 · Yongchang Zhang1,9 · Nong Yang1,9

Received: 31 August 2020 / Accepted: 18 January 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021

Background This study evaluated the efficacy and safety of anlotinib combined with programmed cell death protein 1 (PD-1) blockade for the treatment of small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC).
Patients and methods SCLC (n = 28) and NSCLC (n = 177) patients who received treatment at Hunan Cancer Hospital between June 1, 2019, and July 1, 2020, were retrospectively analyzed. Progression-free survival (PFS) and treatment responses were compared among patients who received combination therapy of anlotinib plus PD-1 inhibitor, or monotherapy of either chemotherapy or PD-1 inhibitor. Independent prognostic factors were identified by Cox regression analysis.
Results Patients with relapsed SCLC who received anlotinib plus PD-1 inhibitor as a ≥ second-line therapy (n = 14) had a
significantly longer PFS than those who received PD-1 inhibitor alone (n = 14, 5.0 vs. 3.0 months; P = 0.005). For patients with previously untreated wild-type NSCLC, the combination therapy in the first-line setting (n = 6) provided a marginally longer PFS than mono-chemotherapy (n = 6, 8.0 vs. 3.0 months; P = 0.075). For patients with relapsed NSCLC, the combi- nation therapy in the ≥ second-line setting (n = 62) resulted in significantly higher objective response rate (19.3 vs. 5.0 vs. 2.4%; P = 0.013) and longer PFS (8.0 vs. 2.0 vs. 2.0 months; P <0.001) as compared to monotherapy of either chemotherapy (n = 41) or PD-1 inhibitor (n = 62). Anlotinib and PD-1 blockade combination therapy was an independent predictive factor of longer PFS (P <0.001).
Conclusion The combination of anlotinib and PD-1 inhibitor has promising efficacy and manageable toxicity as a second- or
later-line treatment of relapsed NSCLC and possibly for relapsed SCLC.
Keywords Anlotinib · Immunotherapy · Programmed death 1 inhibitor · Lung cancer

 Yongchang Zhang [email protected]
 Nong Yang [email protected]
Jianbo Wang [email protected]
1 Department of Medical Oncology, Lung Cancer
and Gastrointestinal Unit, Hunan Cancer Hospital/The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China
2 Qinghai Provincial People’s Hospital, Xining 810000, China
3 Burning Rock Biotech, Guangzhou 510300, China

4 Sir Run Run Shaw Hospital, Zhejiang University, 3 East Qingchun Road, Hangzhou 310016, Zhejiang, China
5 Department of Pathology, Tongji Hospital, Tongji University School of Medicine, Shanghai, China
6 Cancer Hospital, University of Chinese Academy of Sciences, Hangzhou 310022, Zhejiang, China
7 Department of Radiotherapy, Qilu Hospital of Shandong University, Jinan City, Shandong, China
8 Department of Genetics, School of Life Science, Anhui Medical University, Hefei, China
9 Graduate School, University of South China, Hengyang 421001, Hunan, China

Lung cancer is the most commonly diagnosed cancer accounting for 20% of cancer-related mortality worldwide [1]. In China, an estimated 610,200 patients died from lung cancer in 2015, accounting for 22% of cancer-related mor- tality, with most patients presented with locally advanced or metastatic disease at the time of diagnosis [2, 3]. In the recent years, the advances in novel drugs and personal- ized treatment strategies have led to an improvement in the prognosis of patients with advanced lung cancer, with most patients tended to receive multiple lines of therapy to manage their disease throughout their lifetime [4].
Immune checkpoint inhibitors targeting the pro- grammed cell death protein 1 (PD-1) and its ligand pro- grammed death-ligand 1 (PD-L1) have revolutionized the therapeutic landscape of advanced non-small-cell lung cancer (NSCLC), especially NSCLCs without driver gene mutations. Checkpoint inhibitors are approved as first-line therapy of advanced NSCLC with PD-L1 expression of
≥% or in combination with chemotherapy in patients with previously treated advanced NSCLC [16–19]. The addition of PD-L1 inhibitor to chemotherapy for the first-line treat- ment of extensive-stage small-cell lung cancer (SCLC) has resulted in improved progression-free survival (PFS) and overall survival (OS) compared to chemotherapy alone [20].
Anlotinib is a novel multi-target tyrosine kinase inhib- itor that inhibits tumor angiogenesis and proliferative signaling [5]. Anlotinib has shown efficacy in advanced NSCLC and SCLC via suppressing the activation of pro- angiogenic signals from receptor tyrosine kinases includ- ing vascular endothelial growth factor (VEGF) recep- tor types 2 and 3, fibroblast growth factor receptor 1, platelet-derived growth factor receptor β, and stem cell factor receptor [5–9]. In patients with relapsed NSCLC, anlotinib monotherapy as a third-line and beyond treat- ment provided significant PFS benefits than placebo as shown by both the ALTER0302 and ALTER0303 stud- ies [6, 10, 11], which led to its approval by the China National Medical Products Administration. Consistently, in patients with relapsed SCLC, anlotinib as a third-line and beyond treatment provided significantly better disease control rate (DCR), and longer PFS and OS, as compared to placebo in the ALTER1202 study [12, 13]. The effi- cacy and safety of anlotinib monotherapy as third-line or beyond treatment of patients with advanced lung cancer have also been demonstrated in the real-world setting [14, 15]. Anti-angiogenic inhibitor-containing combination therapies, by normalizing tumor vasculature, are a new paradigm in the treatment of various solid tumors includ- ing lung cancer [21]. At least two preclinical studies have

demonstrated that anlotinib monotherapy can ameliorate the tumor immune microenvironment by downregulat- ing PD-L1 expression to inhibit tumor growth [22]. In a lung cancer mouse model, the combination of anlotinib and PD-1 inhibitor promoted the infiltration of the innate immune cells and conferred potentially synergistic anti- tumor activity [23]. The synergistic anti-tumor activ- ity of combined inhibition of angiogenesis and immune checkpoint blockade has been demonstrated clinically by the improved efficacy of the addition of PD-L1 inhibitor atezolizumab and anti-angiogenic monoclonal antibody bevacizumab to chemotherapy in the first-line treatment of non-squamous NSCLC in the IMPOWER150 study, which led to the FDA approval of this treatment regimen [24]. PD-1 inhibitor sintilimab combined with anlotinib had shown promising efficacy and safety in the first-line treatment of patients with NSCLC [25].
In this real-world study, we investigated the efficacy and safety of the combination of anlotinib and PD-1 inhibitor in relapsed SCLC as second-line and later-line regimen and previously untreated and relapsed NSCLC as front-line or later-line regimen, respectively.

Patients and methods
Study population

Consecutive patients with SCLC and NSCLC (stage IIIA/B or IV) confirmed by histological and cytological examina- tion, who were admitted between June 1, 2019 and July 1, 2020, were retrospectively screened. Among the 1,596 patients screened, 28 patients with relapsed SCLC and 177 patients with treatment-naïve and relapsed NSCLC were included in the study. Patients with relapsed SCLC who received second- or later-line treatment of anlotinib combined with PD-1 inhibitor were grouped as cohort A (n = 14) and those who received chemotherapy were grouped as cohort B (n = 14). Patients with treatment-naïve NSCLC who received first-line therapy of anlotinib combined with PD-1 inhibitor were grouped as cohort C (n = 6), while those who received PD-1 inhibitor monotherapy were grouped as cohort D (n = 6). Patients with relapsed NSCLC who received second- or later-line treatment of anlotinib com- bined with PD-1 inhibitor were grouped as cohort E (n = 62), those who received chemotherapy monotherapy as cohort F (n = 62), and those who received PD-1 inhibitor mono- therapy as cohort G (n = 41). For relapsed patients, anlotinib and PD-1 inhibitor combination therapy was given after dis- ease progression following at least one line of chemotherapy. Patients with brain metastases at baseline and pretreated with immunotherapy were included. Gene mutation status

Fig. 1 Study flowchart

was determined by next-generation sequencing. The study flowchart is displayed in Fig. 1.

Anlotinib and PD‑1 inhibitor regimens
Anlotinib was taken orally once daily (12 mg) on days 1–14 of a 21-day cycle. PD-1 inhibitors of either pembrolizumab (200 mg) or toripalimab (240 mg) were intravenously admin- istered on day 1 of a 21-day cycle. Treatment was continued until disease progression, intolerable toxicity, or death.

Efficacy and safety evaluation
The data cutoff was on July 20, 2020. Efficacy was assessed according to the Response Evaluation Criteria in Solid Tumors version 1.1. PD-L1 tumor proportion score (TPS) was defined as the percentage of tumor cells with membrane PD-L1 expression. Some specimens were unevaluable for PD-L1 expression due to inadequate number or lack of tumor cells. For stratification purposes, patients with unevaluable PD-L1 expression were included in the subgroup with a TPS of less than 1%. These patients were excluded from PD-L1 TPS-based analyses of efficacy. The primary study endpoint was PFS, which was defined as the time from initiation of anlotinib treatment to disease progression or death. The secondary endpoints were OS,

objective response rate (ORR), and DCR. OS was defined as the duration from the beginning of anlotinib treatment to patient death. Safety was evaluated by the incidence and severity of adverse events (AEs), graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.02.

Ethics statement
This retrospective study was approved by the Ethics Committee of Hunan Cancer Hospital (approval number: 2017YYQ-SSB-126), and was conducted according to the principles of the Declaration of Helsinki as revised in 2013. Informed consent was waived by the ethics commit- tee due to the retrospective nature of the study.

Statistical analyses
Survival analyses were performed using the Kaplan–Meier method and the log-rank test. Multivariate Cox regres- sion was used to identify significantly different factors in univariate analysis with a P <0.1. All statistical analyses were performed using SPSS version 22.0 (IBM, New York, USA). Statistical significance was defined as P <0.05.

Baseline characteristics

Among the patients with relapsed SCLC, 23 (82.1%) patients were males and 5 (17.9%) were never-smokers. Four patients (16.7%) had brain metastasis before receiv- ing treatment (baseline). One patient was diagnosed with limited-stage SCLC and 27 had extensive-stage SCLC. Baseline characteristics were similar between cohorts A and B. Among the patients with previously untreated wild- type NSCLC who received first-line treatment (cohorts C and D, total n = 12), 7 (58.3%) patients were males and 5 (41.6%) were never-smokers. Patients with squa- mous carcinoma and adenocarcinoma accounted for 50% each. Brain metastasis at baseline was found in 2 patients (16.7%). PD-L1 TPS of 50%, 1–50%, and ≤ % was found in 3, 3, and 2 patients, respectively. Four patients had unknown PD-L1 expression. Three patients in cohort C had PD-L1 expression >50%, while no patient in cohort D had > 50% PD-L1 expression (P = 0.03). EGFR/ALK/ROS1 mutations were not detected in cohorts C and D. Among the patients with relapsed NSCLC who received sec- ond- and later-line treatment (cohorts E, F, and G, total n = 165), no statistical difference was observed among the three cohort in terms of gender, smoking history, East- ern Cooperative Oncology Group (ECOG) performance status, brain metastasis status at baseline, and PD-L1 expression level. Eleven patients (18.0%) in cohort E had EGFR/ALK/ROS1 mutations (EGFR mutation: n = 10, ALK mutation: n = 1), while 10 patients (18.0%) in cohort F had EGFR-mutated NSCLC and one (1.6%) was ALK-positive. The baseline clinical characteristics of the whole study population are exhibited in Table 1.

Treatment history
Treatment information before enrollment is provided in Table 2. Six patients in cohort A relapsed from first- line PD-1 inhibitor combined with chemotherapy before receiving anlotinib combined with PD-1 inhibitor. All 14 patients in cohort B had no history of immunotherapy use and were only pretreated with chemotherapy. Before receiving anlotinib and PD-1 inhibitor combination therapy, 30 (48.4%) patients in cohort E received prior immunotherapy (monotherapy: n = 12, combined with chemotherapy: n = 18), with 16 patients received immu- notherapy in the first-line setting, 12 in the second-line setting, and 2 in the later-line setting. Patients in cohorts F and G had no history of immunotherapy before receiving

mono-chemotherapy and mono-immunotherapy, respec- tively. In cohort F, 53 and 9 patients received mono-chem- otherapy as second- and third-line treatment, respectively. In cohort G, mono-immunotherapy was administered in 32 patients in the second-line setting, 7 in the third-line setting, and 2 in the > third-line setting.

Efficacy comparison in SCLC cohorts
In cohort A, 2 (14.3%) patients achieved partial response (PR), 9 (64.3%) had stable disease (SD), and 3 (21.4%) had progressive disease (PD), resulting in an ORR of 14.3% and DCR of 78.6% (Table 3).. Cohort B had a numeri- cally lower DCR (42.9%) as compared with the cohort A (P = 0.335). The PFS in cohort A was significantly longer than that in cohort B (5.0 vs. 3.0 months; P = 0.005, Fig. 2a). No difference in PFS was observed between patients with (n = 6) and without (n = 8) history of immunotherapy (8.0 vs
5.0 months; P = 0.52; Fig. S1A).

Efficacy comparison in the previously untreated wild‑type NSCLC cohorts
Cohort C had an ORR of 16.7% and a DCR of 83.3% with front-line anlotinib and pembrolizumab combination ther- apy. For patients in cohort D, none had CR or PR, and 2 patients had SD, resulting in a DCR of 33.3% with front- line pembrolizumab monotherapy, which was numerically lower than cohort C (P = 0.362, Table 3). A marginally longer PFS was observed in cohort C than in cohort D (8.0 vs. 3.0 months, P = 0.075, Fig. 2b). The detailed information for cohort C and D is summarized in Table S1.

Efficacy comparison in the relapsed NSCLC cohorts
In cohort E, the ORR and DCR were 19.3 and 85.5%, respectively. Cohort E had significantly higher ORR (19.3 vs. 5.0 vs. 2.4%; P = 0.013) and numerically higher
DCR (85.5 vs. 50.0 vs. 58.5%; P = 0.153) as compared with cohort F and cohort G (Table 3). Among the patients who had mutation-positive relapsed NSCLCs, those who received anlotinib and PD-1 inhibitor combination as second-line or later-line therapy (cohort E; n = 11) had a significantly longer PFS than those who received mono- chemotherapy (cohort F; n = 11) (5.0 vs. 2.0 months: P = 0.0012, Fig. 2c). Consistent with mutation-positive patients, patients with wild-type relapsed NSCLC who received anlotinib and PD-1 inhibitor combination as second-line or later-line therapy (cohort E; n = 51) had

Table 1 Baseline clinical characteristics

(n = 41)

years (range)

carcinoma noma


ECOG Eastern Cooperative Oncology Group, PD-L1 Programmed death ligand, EGFR Epidermal growth factor receptor, ALK Anaplastic lymphoma kinase, ROS1 c-ros oncogene 1

Table 2 Prior treatment
Treatment his- SCLC; n (%) NSCLC; n (%)
tory ≥Second-line First-line ≥Second-line
Total Cohort A (n = 14) Cohort B (n = 14) P Total Cohort C (n = 6) Cohort D(n = 6) P Total Cohort Cohort F Cohort G P
E(n = 62) (n = 62) (n = 41)
Previous treatment lines
0 0 (0.0) 0 (0.0) 0 (0.0) 0.000 12 (100.0) 6 (100.0) 6 (100.0) 1.000 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0.000
1 17 (60.7) 3 (21.4) 14 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 110 (66.7) 25 (40.3) 53 (85.5) 32 (78.0)
2 7 (25.0) 7 (50.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 43 (26.1) 27 (43.5) 9 (14.5) 7 (17.1)
≥3 4 (14.2) 4 (28.6) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 12 (7.3) 10 (16.1) 0 (0.0) 2 (4.9)
Previous immunotherapy
Yes 6 (21.4) 6 (42.9) 0 (0.0) 0.006 0 (0.0) 0 (0.0) 0 (0.0) 1.000 30 (18.2) 30 (48.4) 0 (0.0) 1 (2.4) 0.000
No 22 (78.6) 8 (57.1) 14 (100.0) 0 (0.0) 0 (0.0) 0 (0.0) 134 (81.2) 32 (51.6) 62 (100.0) 40 (97.6)
Previous immunotherapy lines
0 22 (78.6) 8 (57.1) 14 (100.0) 0.000 12 (100.0) 6 (100.0) 6 (100.0) 1.000 32 (51.6) 32 (51.6) 0 (0.0) 41 (100.0) /
1 6 (21.4) 6 (42.9) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 16 (25.0) 16 (25.0) 0 (0.0) 0 (0.0)
2 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 12 (18.8) 12 (18.8) 0 (0.0) 0 (0.0)
≥3 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (0.4) 2 (0.4) 0 (0.0) 0 (0.0)
Previous immunotherapy strategy

and <6 months

a significantly longer PFS than those treated with PD-1 inhibitor monotherapy (cohort G; n = 41) or mono-chem- otherapy (cohort F; n = 51, 8.0 vs. 2.0 vs. 2.0 months; P <0.001, Fig. 2d).
We also performed subgroup analysis among the patients in cohort E with wild-type relapsed NSCLCs to investigate whether various clinical factors including best response (i.e. PR or SD), treatment line (i.e. second- line vs. later-line), history of immunotherapy use, and specific immunotherapy received (i.e. pembrolizumab or toripalimab) would impact the PFS for the combina- tion therapy of anlotinib and PD-1 inhibitor. Our analy- sis revealed no statistical difference in PFS in any of the clinical factors investigated (Table S2; Fig. 3). The median PFS in patients with PR was not yet reached (n = 11) and
8.0 months (n = 33) for patients with SD as best response (P = 0.16; Fig. 3a). For patients who received the anlotinib and PD-1 inhibitor combination therapy as second-line (n = 23) or later-line treatment (n = 28), the median PFS was 7.5 and 8.0 months, respectively (P = 0.43; Fig. 3b). For patients with history of immunotherapy (n = 28) or no history of immunotherapy use (n = 23), the median PFS was 8.0 months each (P = 0.78; Fig. 3c). When considering all patients with relapsed NSCLC including mutation-pos- itive patients with (n = 30) and without (n = 32) history of immunotherapy, the median PFS was 8.0 and 7.5 months, respectively (P = 0.30; Fig. S1B). Patients who received pembrolizumab (n = 19) or toripalimab (n = 32) had PFS of 10.0 and 8.0 months, respectively (P = 0.35; Fig. 3d).

Multivariate analysis of PFS
Patients with relapsed NSCLC (n = 143) were included in the Cox proportional hazard model for multivariate sur- vival analysis. Combination therapy of anlotinib and PD-1 inhibitor was identified as an independent predictor of bet- ter survival as compared with either mono-immunotherapy (hazard ratio [HR]: 0.262, 95% confidence intervals [CI]: 0.142–0.483; P <0.001) or mono-chemotherapy (HR: 0.167,
95% CI: 0.084–0.334; P <0.001) (Table S3).

None of the patients who received anlotinib had a reduced initial dose due to intolerable AEs, and none had discon- tinued treatment due to intolerable AEs. This study did not observe any new or unexpected AEs. Generally, hemato- logic AEs were more common than non-hematologic AEs (Table S4).

Fig. 2 Comparison of PFS in relapsed SCLC (a), previously untreated wild-type NSCLC (b), relapsed mutation-positive NSCLC (c), and relapsed wild-type NSCLC (d)

This retrospective study evaluated the efficacy and safety of anlotinib combined with PD-1 inhibitor in relapsed SCLC and treatment-naïve and relapsed advanced NSCLC. The combination of anlotinib and PD-1 inhibitor demonstrated satisfactory efficacy as compared with the monotherapy of either chemotherapy or PD-1 inhibitor. In addition, anlotinib combined with PD-1 inhibitor was well-tolerated even in the later-line settings.
There is a growing body of evidence describing a com- plex relationship between tumor angiogenesis and the tumor immune microenvironment. VEGF is one of the major reg- ulator of tumor angiogenesis and also modulates immune response by reducing T-cell infiltration into the tumor micro- environment [26]. By inhibiting the VEGF/VEGF receptor signaling pathway, anlotinib reversed the immunosuppres- sion of the tumor microenvironment through the downreg- ulation of PD-L1 expression in vascular endothelial cells

in vitro and in vivo [22]. The addition of anti-angiogenic therapy to immunotherapy could enhance the efficacy of both agents to delay tumor growth by synergistically promot- ing the activation of antigen release and recognition, which transforms the immunosuppressive tumor microenvironment to an immunosupportive one [26–29]. Several clinical evi- dences have shown the potential of different combination of anti-angiogenic inhibitor and immune checkpoint inhibitor therapy [25, 30]. A number of clinical trials are also ongo- ing to investigate the efficacy of this combination regimen in various solid tumors including lung cancer [26].
Although generally sensitive to platinum-based chemo- therapy, the treatment strategy and prognosis of SCLC have had limited progress in the past two decades due to the limited use of targeted therapies [31]. Despite the lack of actionable mutations, SCLC is considered to be immu- nogenic due to its high mutation rate [32]. The addition of PD-1 inhibitor in front-line chemotherapy regimen resulted in prolonged PFS and OS as compared to chemotherapy

Fig. 3 Subgroup analysis of PFS in patients with wild-type relapsed NSCLC (n = 51) according to best response (a), treatment line (b), history of immunotherapy (c), and PD-1 inhibitor received (d)

alone in extensive-stage SCLC [33]. Immunotherapy has made limited progress in the second- or later-line treatment of SCLC. No statistically significant improvement in OS was observed with nivolumab monotherapy versus chemo- therapy of patients with relapsed SCLC after failure of first- line platinum-based chemotherapy in the phase 3 CheckMate 331 trial. However, the late separation of survival curves and potential activity in the platinum-refractory patients indicated possible long-term benefit for some patients [20]. The IFCT-1603 trial observed an improved PFS with mono- therapy of PD-L1 inhibitor atezolizumab in patients with SCLC progressing from first-line platinum-etoposide dou- blet chemotherapy; however, no OS benefit was observed with atezolizumab monotherapy [34]. While PD-1/PD-L1 inhibitor monotherapy did not show OS benefit for SCLC in the second-line setting, combination therapy of PD-1 inhibi- tor nivolumab with CTLA-4 inhibitor ipilimumab displayed

some promising results in the CheckMate 032 trial [35]. The addition of anti-angiogenic agents to immunotherapy may benefit patients with SCLC.
In this study, we evaluated the efficacy of PD-1 inhibi- tor plus anlotinib in SCLC patients after failure from first- line chemotherapy. Our findings showed that patients with relapsed SCLC who received PD-1 inhibitor monotherapy had a PFS of 3 months. Contrastingly, the addition of anlotinib to PD-1 inhibitor therapy was associated with a 2-month extension in PFS. Our findings demonstrated the synergistic effect of anlotinib plus checkpoint inhibitor also benefit patients with relapsed SCLC. However, it should be noted that the majority of SCLC patients (24/28, 85.7%) in this study had no brain metastasis with good performance status (ECOG PS 0–1) before receiving their treatment regi- mens. In addition, no biomarkers were explored to select the candidates who could benefit from this combination therapy.

For patients with treatment-naïve NSCLC wild-type for inhibitor-sensitizing EGFR/ALK/ROS1 mutations, our find- ings provided preliminary evidence that first-line use of anlotinib combined with immunotherapy tended to have sur- vival benefit; albeit the small sample size. Although did not reach statistically significance, the PFS had a 5-month exten- sion in patients who received anlotinib combined with PD-1 inhibitor than those in the mono-immunotherapy group.
In patients with relapsed driver gene-mutated NSCLC, the efficacy of anlotinib combined with PD-1 inhibitor was assessed in the second- and later-line setting. The PFS was 3 months longer in the combination therapy group than in the mono-chemotherapy group. Our findings were consist- ent with the reported ORR (18.8%), DCR (79.2%), and PFS (6.7 months) of anlotinib and PD-1 inhibitor combination therapy in the third-line therapy of NSCLC [23]. In the CheckMate 017 and CheckMate 057 trials, the PFS of previ- ously treated squamous and non-squamous NSCLC patients in the nivolumab group were 3.5 months and 2.8 months, respectively, and the corresponding PFS in patients who received docetaxel were 2.8 months and 4.3 months, respec- tively [36]. These results were numerically similar to the observed PFS in our monotherapy of chemotherapy and immunotherapy in cohorts C and D, respectively. In the subgroup analyses, we found that satisfactory PFS benefits with anlotinib combined with immunotherapy could be observed regardless of the treatment line, whether second- line (PFS: 8.5 months) or post second-line (PFS: 9 months). Moreover, history of immunotherapy failure did not affect the efficacy of the combination therapy. Considering the preclinical evidences of the synergistic therapeutic effects of PD-1 blockade and angiogenesis inhibition [22, 23], anlo- tinib and PD-1 inhibitor treatment regimen is promising in improving survival outcomes of patients with lung cancer. The combination therapy may be applied to patients who do not fit standard therapy. No statistical significance was observed in gender, smoking history, ECOG performance status, baseline brain metastasis, and PD-L1 expression level in different cohorts. We also found no difference in the effi- cacy of this combination therapy for domestic and imported PD-1 inhibitors.
Increased AEs are an important concern for combina-
tion therapy including immune checkpoint inhibitors. In the current study, the combination therapy was generally well tolerated, with no new or unexpected increased risk of AEs observed. While rates of treatment-related hepatic AEs were slightly higher in patients with liver metastases who received the combination therapy than in the overall study population, elevation in liver enzyme was primarily graded as 1/2.
This study had several limitations. First, it was a single- center retrospective study with a limited sample size. The cohort size was very small for SCLC who received anlo- tinib and PD-1 inhibitor as second-line or later-line therapy

(n = 14) and wild-type NSCLC who received the regimen as first-line therapy (n = 6) to generate solid conclusions; how- ever, our study presented promising efficacy of this regimen for these subset of patients that is worthy of further investi- gation. Large-scale validation studies should be conducted in the future. Second, PD-L1 expression was not detected in all patients and biomarkers for patient selection were not evalu- ated. Third, the observation duration was relatively short, and OS was not analyzed.
In conclusion, our study showed that the combination of anlotinib and PD-1 inhibitor has promising efficacy and manageable toxicity as a second-line or later-line treatment for NSCLC and possibly in SCLC. Large-scale prospective clinical trials are warranted to further confirm our findings.
Supplementary Information The online version contains supplemen- tary material available at

Author contribution YZ conceptualized, supervised and acquired funding for the study. XZ, LZ, YL curated and analyzed the clinical data and wrote the original manuscript draft. QX, HY, RJ, YZ, QL and JW contributed to the cura- tion and analysis of clinical data. AL and XM contributed to the analysis of the data and manuscript preparation. YL and NY gave critical comments and suggestions. all authors reviewed and approved the manuscript.

Funding This work was funded by the Natural Science Foundation of China (grant number: 81760529), and the Natural Science Founda- tion of Hunan Province (grant numbers: 2018RS3106, 2018SK50901, kq1801102, 2019-TJ-N04, 2019JJ50357, 2019SK4010, 2020JJ5340,
and 2020JJ3025). The funding agencies had no role in the study design, data collection, analysis, interpretation, manuscript writing, and deci- sion to submit the article for publication.

Data availability Data generated from this study are included as figures and tables in the main text and as supplementary files.

Compliance with ethical standards

Conflicts of interest Analyn Lizaso and Xinru Mao are employed by Burning Rock Biotech. All the other authors declare no conflict of in- terest.
Ethics approval This study was approved by the Institutional Review Board of the Hunan Cancer Hospital (approval number: 2017YYQ- SSB-126).Informed consent
Informed consent Informed consent was waived by the ethics commit- tee due to the retrospective nature of the study.
Consent for publication We confirm that the manuscript has been reviewed and approved by all named authors for publication.

Clinical trials registration Clinical trials registrationThe ATHENA Study, NCT04322617.

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