Osimertinib plus Bevacizumab Improve the Outcome of Leptomeningeal Metastasis Patients with EGFR Mutant Non-Small-Cell Lung Cancer

2. Materials And Methods

2.1. Patients

We retrospectively reviewed the charts of 27 patients diagnosed with LM from EGFR-mutant NSCLC who received osimertinib with or without bevacizumab at the Second Affiliated Hospital of Nanchang. The date of LM diagnosis was defined as the date of first CSF cytology examination revealing malignant cells or the date of first MRI (brain or spine) demonstrating LM. The assessment of LM response was based on the modified RANO LM radiological criteria, and the CNS and extra-CNS response was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1: comprehensive evaluation according to the improvement of clinical symptoms, the performance of MRI and the clearance of CSF tumor cells[14]. Given that lumbar puncture is an invasive procedure, the response criteria of LM were judged according to the improvement of clinical symptoms and the performance of MRI in our study. All LM patients underwent 1.5T whole brain and spinal cord enhanced MRI scan at baseline, and the MRI scan thickness was 1mm, bravo and cube sequence having a high sensitivity and specificity in the diagnosis of LM. The Overall survival (OS) was defined as the time between the initiation of diagnosis to the date of death or last follow-up by November 5, 2020. The intracranial Progression-free Survival (iPFS) was defined as the time from the diagnosis of LM to the disease progression or death. Four weeks after the initiation of Osimertinib and bevacizumab, neurological evaluations, brain MRI and chest/abdominal computed tomography were routinely performed and were then performed every 1 months. The main endpoint of this study was iPFS.

The eligibility criteria were as follows: (i) age 18-80 years; (ii) histologically or cytologically confirmed NSCLC; (iii) the detection of an EGFR mutation, with EGFR status identified from primary lung tumors using the amplification refractory mutation system (ARMS) or next-generation sequencing (NGS) analysis; (iv) LM defined by CSF positivity for malignant cells and/or focal or diffuse enhancement of leptomeninges, nerve roots or the ependymal surface diagnosed by magnetic resonance imaging (MRI) with gadolinium contrast; (v) Patients receiving osimertinib plus bevacizumab or osimertinib. (vi) Patients without history of treatment with osimertinib before a diagnosis of LM. Patients who did not meet these criteria were excluded. Clinical outcomes were compared with the Kaplan-Meier log-rank test. This study was approved by the Institutional Review Board of the Second Affiliated Hospital of Nanchang University.

2.2. Cell Culture

H1975 cells (EGFR exon20 T790M and exon21 L858R) were purchased from Pro-cell in 2020 (Wuhan, China, was identified by STR) and maintained in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum and 1% antibiotic solution in a humidified incubator with 5% CO₂. All reference compounds were purchased from Gibco.

H1975 cells were stably transfected with a GV260 (GeneChem, Shanghai, China) vector containing luciferase, and bioluminescence signals were measured by an in vivo imaging system. H1975-luc tumor cells were prepared for injection after trypsinization and washing with PBS. A viable cell count was performed with trypan blue to adjust the cell concentration to 2 × 10⁶ cells in PBS for each injection.

2.3. Animal model of LM

BALB/C nude female mice (6-8 weeks) were obtained from institutional animal breeding services (the Nanchang Royo Biotech Co., Ltd.). The animals were group housed five a cage in a temperature-controlled room on a 12-h/12-h light/dark cycle with unlimited access to food and water. All animal procedures in this study were approved by the Animal Care and Use Committee of Nanchang University, and ethical approval was obtained from the Institutional Review Board of the Second Affiliated Hospital of Nanchang University.

Mice were anesthetized with 4% isoflurane. The skull was exposed with a skin incision (1 cm) under sterile conditions (ethanol skin wipe) to locate the bregma. A Hamilton syringe needle with H1975-luc human NSCLC cells was injected into the right lateral ventricle (anteroposterior 2.0 mm from the bregma; lateral 0.2 mm to the right; and dorsoventral 4 mm) at 2 µl per min. A total volume of 5 µl of cell suspension was injected [15, 16]. The tumor burden of intracranial lesions and the tumor size in the pia mater were measured using a BLI technique with an in vivo imaging system, MRI and H&E staining.

2.4. In Vivo Pharmacodynamic Study

After confirming tumor formation, the mice were randomly divided into 4 groups (n=9 per group): the control(0.9% normal saline, daily, oral), osimertinib (25 mg/kg, daily, oral)[17], and bevacizumab (5 mg/kg twice a week, i.p.)[18], and osimertinib plus bevacizumab. Brain tissues were collected at 1, 6, 12, and 24 h after a single dose of treatment to determine the penetration of osimertinib in the brain with a validated liquid chromatography-tandem mass spectrometry method(n=3). Furthermore, tumor tissues were collected in formalin or frozen at the endpoint of experiments, EGFR downstream signals were evaluated by immunoblotting, and an angiogenesis marker (CD31) was evaluated by immunofluorescence. Once the nude mice lost more than 20% of their weight, the experiment reached the endpoint, and the mice were euthanized by intraperitoneal injection of pentobarbital sodium(150mg/kg). Each experimental group has at least 9 mice, and a total of 40 mice are included.

2.5. Formaldehyde perfusion of the brain

To keep the mouse brain tissue as intact as possible, this experiment used formaldehyde perfusion to fix the brain. The mice were anesthetized by the intraperitoneal injection of 1% pentobarbital sodium(50 mg/kg), the chest skin was cut open with scissors, the heart was exposed, a syringe needle was inserted into the left ventricle from the left apex towards the aorta, the needle was fixed with hemostatic forceps, physiological saline was quickly perfused until the liver tissue color turned gray, and then paraformaldehyde was slowly perfused. The mouse limbs twitched, and the whole body became stiff. After successful perfusion, the mouse brain tissue was removed and fixed with paraformaldehyde for subsequent experiments.

2.6. Histology

2.6.1. H&E staining and immunofluorescence

Mouse brain tissues were quickly excised, immersed in 10% paraformaldehyde and embedded in paraffin. Brain tissues were cut into 5-µm thick sections. Slides were stained with hematoxylin and eosin (H&E). Pictures were obtained with ×10 and ×40 objective respectively, the pathology diagnosis was completed by two independent pathology doctors. Tumor microvessel density (TMD) was measured by staining for CD31 (Proteintech, Wuhan, China), as previously reported[19]. Briefly, TMD was assessed in hot spots of brain tissue cross-sections identified by light microscopy. Five equal areas were then photographed with a 40x objective (400x magnification). The staining was scored by two independent and experienced pathologists and calculated as the product of the staining intensity. The areas and integrated optical density (IOD) of the slides were analyzed by Image-Pro Plus 6.0 software.

2.6.2. Western blotting

Western blotting was performed according to standard methods, as described previously[19]. The tumor sections in brain tissue were snap-frozen in liquid nitrogen for protein isolation, and then tumors were homogenized with a mortar and pestle and lysed in RIPA buffer containing Halt protease & phosphatase inhibitor cocktail. Soluble proteins were quantified by a BCA protein level detection kit and then subjected to SDS-PAGE followed by immunoblotting. Antibody incubation was conducted overnight at 4°C. Antibodies included EGFR(1:1000, Servicebio, GB11084-2), AKT (1:1000, Servicebio, GB11689), S473 p-AKT (1:1000, Affinity, AF0832), ERK (1/2) (1:1000, Servicebio, GB11560), p-ERK (1/2) (1:1000, Cell Signaling Technology, #4370), E-cadherin (1:1000, Servicebio, GB14076), HIF-1α (1:1000, Servicebio, GB111339), ACTIN (1:3000, Servicebio, GB12001). Secondary antibodies were diluted 1:5000, and immunoreactive proteins were visualized using a Western blotting machine (Thermo) and analyzed with ImageJ software.

2.6.3 IHC

Tumor-bearing brain tissue were collected from mice of each group. Tissues were fixed in 10% buffered formalin for 24 hours following standard procedure for processing, paraffin-embedding, and sectioning to assess PCNA (1:200, Servicebio, GB11010), E-cadherin (1:200, Servicebio, GB14076) by IHC assay. The reaction was visualized using the Servicebio image analysis system, the staining was scored by two independent and experienced pathologists and calculated as the product of the staining intensity. first divides the positive grade: negative without staining, score 0, weak positive light yellow, score 1, medium positive brown, score 2, strong positive brown, score 3 points. Then analyze and calculate the area of weak, medium, and strong positive in the measurement area, the tissue area of the measurement area, the cumulative optical density value of the positive and the positive area. PCNA and E-cadherin quantification for each sample was determined and molecular data using modified H-scores ([{% of weak staining} × 1] + [{% of moderate staining} × 2] + [{% of strong staining} × 3]), to determine the overall percentage of PCNA and E-cad positivity across the entire stained sample, yielding a range from 0-300[20, 21].

2.7. Statistics

Preclinical data are presented as the mean ± SD. Data were analyzed by one-way ANOVA, followed by Dunnett’s test or Student’s t-test. Survival analysis of animal was performed using a Kaplan-Meier survival curve and a log-rank test. The Kaplan-Meier method was used to estimate and graphically present OS and iPFS. All analyses were performed using GraphPad Prism 7.0 (GraphPad Prism Software, Inc) or IBM SPSS statistics 22. Experiments were repeated independently at least three times. p <0.05 was considered statistically significant.

3. Results

3.1. Osimertinib plus bevacizumab improved the efficacy of LM patients with EGFR-mutant NSCLC.

A total of 70 patients diagnosed with lung cancer and LM in at the Second Affiliated Hospital of Nanchang University from October 2017 to March 2020 were collected. 27 patients with EGFR mutation-positive NSCLC with LM were selected according to the inclusion criteria (Table 1). Enrolled patients received osimertinib 80 mg orally daily with or without bevacizumab 7.5 mg/kg intravenously every three weeks. Treatment continued until the disease progressed, unacceptable adverse events occurred, or the patient withdrew consent. The pathological type of all patients is lung adenocarcinoma. In these patients, EGFR mutations included L858R (51.8%) (1 T790M), exon 19 deletion (48%) (2 T790M). The median follow-up time was 19.1 months. In osimertinib group, Of the 11 total patients, 81.8% (9) had clinical response, 3 of patients achieved partial response (PR) (27.3%),6 of patients had stable disease (SD), 2 of patients had progressive disease (PD). In osimertinib plus bevacizumab group, Of the 16 total patients, 81.3% (13) had a clinical response, 6 of patients achieved PR (37.5%),7 of patients had SD, 3 of patients had PD. The assessments of response to osimertinib or osimertinib plus bevacizumab in 27 LM patients were shown in supplementary table 1 and 2. The Swimmer plot was accordance with treatment duration of 27 LM patients (Fig. 1A). There is an increasing of median OS of the patients who received osimertinib and bevacizumab (n = 16, 18.0 months) as compared with patients with Osimertinib treatment (n=11, 13.7 months) (log-rank test, p=0.046, HR=2.867, 95%CI:1.007-8.162). The median iPFS is 10.6 months versus 5.5 months (log-rank test, p=0.037, HR=3.401, 95%CI:1.079-10.720) (Fig. 1B and C). These data suggested that osimertinib plus bevacizumab improve the survival time of patients with LM from EGFR mutant NSCLC.

Table 1

Patient characteristics (n=27)
Characteristics	Osimertinib (n=11) %	Osimertinib and bevacizumab (n=16) %
Age (median)	47-70(56)	46-75(60)
Gender Female Male	4(36.4) 7(63.6)	8(50.0) 8(50.0)
History of tobacco exposure Yes No	6(54.5) 5(45.5)	7(43.8) 9(56.2)
ECOG performance status score ≥2 <2	9(81.8) 2(18.2)	11(68.8) 5(31.2)
EGFR mutation Exon 21 L858R Exon 19 deletion	6(54.5) (1 T790M) 5(45.5) (1 T790M)	8(50.0) 8(50.0) (1 T790M)
Neurological symptoms Yes No	10(90.9) 1(9.1)	15(93.8) 1(6.2)
Exclusively diagnosed LM by CSF cytology Yes No	5(45.5) 6(54.5)	8(50.0) 8(50.0)
Exclusively diagnosed LM by MRI Positive Negative	9(81.8) 2(18.2)	12(75.0) 4(25.0)
Both positive for MRI and CSF cytology Yes No	5(45.5) 6(54.5)	5(31.2) 11(68.8)
Systemic therapy before LM diagnosis First generation TKIs Yes No Chemotherapy Yes No Brain radiotherapy Yes No	10(90.9) 1(9.1) 1(9.1) 10(90.9) 2(18.2) 9(81.8)	14(87.5) 2(12.5) 3(18.7) 13(81.3) 2(12.5) 14(87.5)
Subsequent treatments after osi±beva Osimertinib + ITC Osimertinib +WBRT Chemotherapy Osimertinib 160mg Continued prior treatments	1(9.1) 0(0.0) 2(18.2) 1(9.1) 7(63.6)	3(18.8) 1(6.2) 1(6.2) 0(0.0) 11(68.8)
CSF, cerebrospinal fluid; ECOG, Eastern Cooperative Oncology Group; EGFR, epidermal growth factor receptor; LM, leptomeningeal metastasis; MRI, magnetic resonance imaging; TKIs, tyrosine kinase inhibitors. osi±beva, osimertinib with or without bevacizumab; ITC, intrathecal chemotherapy; WBRT, whole-brain radiotherapy.

3.2. Osimertinib plus bevacizumab demonstrated impressive CNS penetration in vivo

To determine the efficacy of osimertinib plus bevacizumab in LM in EGFR-mutant NSCLC, we constructed a model of LM with lateral ventricle injection (Fig. 2A), and the lung cancer LM xenograft model was confirmed by IVIS imaging, MRI and H&E (Fig. 2B, C and E). Mass spectrometry analysis showed that the average concentration of osimertinib in the brain tissue in combined group was higher than osimertinib group [ 803.2 vs 606.5 ng/ml (1 hour after treatments), 359.6 vs 193.8 ng/ml (6 hour), 91.8 vs 31.2 ng/ml (12 hour), 3.8 vs 1.0 ng/ml (24 hour) (n=3 per group, p<0.05)] (Fig. 2D). These results suggested that when osimertinib is combined with bevacizumab, the concentration of osimertinib in the mouse brain was increased.

3.3. Osimertinib plus bevacizumab effectively inhibits the growth of EGFR-mutant LM xenografts in nude mice

The effects of osimertinib plus bevacizumab in the LM xenograft model were validated. Within 2 weeks of treatment, the three groups had different degrees of tumor regression except for the progression of the control group compared to baseline (Fig. 3A and B). The body weight change and survival curves implied that the tumor regression of the combined group was superior to that of the osimertinib group after 3 weeks of treatment. The mice in the bevacizumab group lost a significant amount of weight and died quickly, similar to mice in the control group. (n=6 per group, p<0.05) (Fig. 3C and F). Compared with that in the osimertinib group, the tumors in the osimertinib plus bevacizumab group were significantly regressed (Fig. 3D and G). These results suggested that the combination effectively inhibits the growth of EGFR-mutant LM tumors in vivo.

3.4. Osimertinib plus bevacizumab suppresses the EGFR downstream signaling pathway and modulates E-cadherin levels in EGFR-mutant LM model mice

The EGFR downstream signaling pathway was examined in tumor tissues by Western blotting. The results showed that p-AKT and EGFR were significantly decreased in the osimertinib plus bevacizumab group compared with the other groups. (n=3, p<0.05) (Fig. 4A and B (a-e)). Meanwhile, our data showed that TMD was significantly decreased in the osimertinib and bevacizumab group compared with that in the other groups (n=3, p<0.05) (Fig. 4C and B (f)). These results further verified that osimertinib and bevacizumab could play a synergistic effect in EGFR-mutant LM model. To understand the potentially mechanism of the combination treatment, we assessed PCNA, E-cadherin, ADAM9 and HIF-1α levels in xenograft tumors receiving the treatments. The combination did not affect the levels of ADAM9 and HIF-1α, but the E-cad levels was more significantly decreased in combination group compared with osimertinib group (n=3, p<0.05) (Fig. 5). Hence, we demonstrated the in vivo modulation of E-cadherin by the combination of osimertinib and bevacizumab.

4. Discussion

Due to the unclear mechanism of LM and the existence of the blood-brain barrier, it is difficult for drugs to reach an effective intracranial concentration. The treatment of LM in lung cancer is still a complicated problem. This is the first study to compare the effects of osimertinib with or without bevacizumab in LM of EGFR-mutant NSCLC. The efficacy is compared through a combination of basic research and clinical analysis and its possible mechanism is explored.

Studies have shown that EGFR-TKIs (erlotinib) and angiogenesis inhibitors that target endothelial growth factor receptor (anti-VEGFR) (bevacizumab) (A+T) achieved superior PFS and acceptable safety in NSCLC patients with intracranial metastasis[22]. Our study retrospectively analyzed 27 LM patients with EGFR-mutant lung cancer who received osimertinib with or without bevacizumab, the median OS of osimertinib plus bevacizumab group (n=16) compared osimertinib group (n=11) was 18.0 months versus 13.7 months (log-rank test, p=0.046, HR=2.867, 95%CI:1.007-8.162). The median iPFS is 10.6 months versus 5.5 months (log-rank test, p=0.037, HR=3.401, 95%CI:1.079-10.720). Then we investigated the antitumor effects of osimertinib and bevacizumab in an EGFR-mutant LM model. We found that osimertinib plus bevacizumab significantly improved the concentration of osimertinib in mouse brain tissue.

A series of studies have supported that A+ T therapy can result in improved survival benefits[23]. The JO25567 recommended combined erlotinib and bevacizumab as a first-line regimen in EGFR mutation-positive NSCLC[24, 25]. The NEJ026 also suggested that TKIs combined with bevacizumab extended PFS 3.6 months compared to erlotinib in EGFR-mutant NSCLC[11]. The PFS of NSCLC patients with pleural or pericardial effusion is expected to be prolonged with the combined use of osimertinib plus bevacizumab and to demonstrate their safety[26]. Consistent with the above studies, our preclinical experiments and retrospective analysis indicated that osimertinib and bevacizumab improve the survival of LM patients with EGFR mutant NSCLC. In contrast, some reports showed that TKIs combined with angiogenesis inhibitors did not improve PFS in EGFR-mutant NSCLC[27]. Although the overall response rate (ORR) was better with osimertinib plus bevacizumab than osimertinib alone (68% vs 54%), median PFS was not longer with osimertinib plus bevacizumab (9.4 months vs 13.5 months)[28]. Dr. Toi reported that compared with osimertinib alone (n=40), the combination of osimertinib and bevacizumab (n=41) did not increase the PFS of NSCLC patients. Although the ORR of the combined group was higher, there was no difference in OS between the two groups[28]. Recent studies have found that 160 mg of osimertinib could have better efficacy than 80 mg in controlling LM[8, 29, 30]. Further investigation to compare the efficacy of osimertinib 80 to 160 mg in patients with LM is warranted.

VEGF is a key regulator of angiogenesis and a validated target for NSCLC[31]. The biologically synergistic antitumor activity of EGFR inhibition in combination with VEGF/VEGFR pathway blockade have been demonstrated in preclinical studies[32]. In EGFR-mutant NSCLCs, up-regulated EGFR signaling increases VEGF through hypoxia-independent mechanisms, and elevated VEGF, in turn, contributes to the emergence of resistance to EGFR tyrosine kinase inhibitors (TKIs) [33, 34], and EGFR, similar to VEGFR-2, can be expressed on tumor-associated endothelial cells[23, 32]. The inhibitory effects of afatinib on EMT and tumorigenesis may be associated with the ERK‑VEGF/MMP9 signaling pathway[35]. The TMD has been regarded as one important indicator for quantitatively analyzing tumor angiogenesis, which can clearly reflect the intra-tumoral blood vessels state and tumor-induced angiogenesis ability[36, 37]. Tumor vascularization is critical to the pathogenesis of solid tumors, and TMD is related to tumor invasiveness and metastasis formation which could be used as a potential predictive marker for bevacizumab benefit[38]. Bevacizumab prunes vessels while normalizing those remaining[39]. Osimertinib is a small molecule targeted drug, which is easier to penetrate the blood-brain barrier than the first and second-generation targeted drugs[17]. These studies suggest that dual blockade of the VEGF and EGFR pathways would be more effective. Our results showed that the combination group has a more significant reduction in TMD. Perhaps it is the reason that bevacizumab improved the concentrations of osimertinib in the mouse brain, which further indicated that osimertinib plus bevacizumab have a synergistic effect. The further mechanism still needs to be investigated. Considering, our study found that the combined treatment significantly increased the effective intracranial concentration of osimertinib, modulated the level of E-cadherin and downregulated the levels of EGFR and downstream signaling pathways including p-AKT and reduced TMD, indicated that combined osimertinib with bevacizumab could play a synergistic effect in EGFR-mutant LM model possibly by modulating the level of E-cadherin. In preclinically, osimertinib combined with bevacizumab is a more ideal and optimized treatment plan for EGFR mutant NSCLC with LM.

Although our research is instructive for the clinical treatment of NSCLC patients with LM. Owing to the animal model and the small number of patients, this study may not be conclusive. First, strictly speaking, our LM model belongs to a local growth model not metastasis model. Currently, there is no stable disease model that metastasizes to the meninges via the primary tumor. Commonly used models including cisternal injection and lateral ventricle injection, both of which are spread to the meninges through the cerebrospinal fluid circulation route. They have not been metastasized from the primary focus; hence they are not suitable for the study of the mechanism of LM. However, due to their excellent performance stability, it is benefit for drug intervention experiments. Second, it is a small sample size retrospective study, and there are several patients on survival. It is difficult to assess the iPFS of LM, and it is currently believed that a comprehensive assessment should be based on patient neurological examination, C radiological evaluation, and cerebrospinal fluid cytology. The main challenge is to define measurable and non-measurable (target) damage, and allow assessment of response changes, perform cerebrospinal fluid cytology for assessment, due to lumbar puncture is an invasive test, most patients refuse to perform. Therefore, it is difficult and subjective to evaluate iPFS in patients with LM, and the present results must be interpreted cautiously. The data were obtained from medical files, and we cannot exclude the possibility of undefined biases and/or confounding factors. Third, the interaction mechanism of osimertinib and bevacizumab needed to be further explored. The next step of our research, the phase II study of osimertinib plus bevacizumab for LM is already ongoing (NCT04425681). We are collecting the CSF and blood of NSCLC patients with LM in osimertinib group and the combination with bevacizumab group to further explore the mechanism and find the biomarkers for prognostic.

In conclusion, the current findings demonstrated that osimertinib plus bevacizumab tend to improve the outcome than osimertinib for LM patients with EGFR-mutant NSCLC, suggesting osimertinib plus bevacizumab is an appropriate treatment option for patients with LM from EGFR mutant NSCLC.

Declarations

Ethics approval and consent to participate

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were approved by the Ethical Committee on Animal Experiments in the Second Affiliated Hospital of Nanchang University (NO.: Review [2019] No. (033)), in compliance with Chinese national or institutional guidelines for the care and use of animals. The study for retrospective experiments was approved by institutional board of The Second Affiliated Hospital of Nanchang University (NO.: Review [2019] No. (032)) and individual consent for this retrospective analysis was waived. The study was conducted in accordance with guidelines from Animal Research: Reporting of In Vivo Experiments (ARRIVE) and all methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not Applicable

Availability of data and material

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare they have no conflicts of interest relevant to this submission.

Funding

This research was supported by the National Natural Science Foundation of China [grant number 81760566, 82060577], Key Research and Development Project of Jiangxi province [grant number 20181ACG70011].

Authors’ contributions

YL: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing-original draft, Writing-review & editing. JC: Formal analysis, Validation, Visualization, Writing review & editing. PX: Formal analysis, Methodology, Validation, Visualization, Writing review & editing. LX: Validation, Visualization, Writing review & editing. ZQ: Formal analysis, Methodology, Software, Validation, Writing review & editing. ZM: Data curation, Project administration, Resources, Validation, Writing-original draft, Writing-review & editing. AW: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing review & editing. All authors have read and approved the manuscript.

Acknowledgements

We are very grateful to Dr. He Yuanqiao’s team for their technical guidance on animal experiments, and to Director Huang Xiaoning for his generous help in imaging.

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