Reversal of Acquired Resistance to EGFR–TKI in T790M-negative Patients With Non–small-cell Lung Cancer Using Anlotinib

: Background: Treatment options for epidermal growth factor receptor (EGFR) T790M-negative patients with non–small-cell lung cancer (NSCLC) and acquired resistance (AR) to EGFR–tyrosine kinase inhibitor (EGFR–TKI) are limited. The efficacy of EGFR–TKI and anti-angiogenic drug combination therapy in these patients is known. We investigated the effectiveness of EGFR–TKI+anlotinib combination therapy in patients with T790M-negative NSCLC. Method: We evaluated the antitumor effects of gefitinib combined with anlotinib in gefitinib-resistant lung adenocarcinoma cells. We also investigated the treatment effect and absence of adverse events of EGFR–TKI+anlotinib therapy in 22 T790M-negative patients after EGFR–TKI treatment failure between January 2018 and August 2020. Results: Anlotinib reversed gefitinib resistance in the gefitinib-resistant cell line, PC9/GR, by enhancing anti-proliferative and pro-apoptotic effects of gefitinib. The gefitinib+anlotinib treatment exerted a synergistic antitumor effect by downregulating the activation of VEGFR2 and downstream effectors, Akt and ERK. The EGFR– TKI+anlotinib therapy exhibited an objective response rate of 18.2% and a disease control rate of 95.5%. The median progression-free survival (PFS) was 11.53 ± 1.94 months, whereas the median overall survival was not reached. The median PFS was longer in patients exhibiting gradual progression (13.30 ± 1.69 months) than in patients with dramatic progression (8.60 ± 5.39 months, p = 0.041). One Grade 3 adverse event was noted (diarrhea, n = 2, 9.1%), and Grade 4 or 5 adverse events were absent. Conclusion: EGFR–TKI combined with anlotinib demonstrated powerful antitumor activity in vitro and excellent treatment effect in T790M-negative NSCLC patients after AR.


Background
Lung cancer is one of the most fatal cancer worldwide, and non-small-cell lung cancer (NSCLC) is the most common histological type accounting for up to 85% of lung cancer cases [1,2] . The investigation of lung cancer pathogenesis and identification of various therapeutic molecular targets has increased the significance of targeted therapy in NSCLC. The most widely used targeted agents, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), significantly prolonged the survival in EGFR-mutant patients with NSCLC [3,4] . However, the clinical benefits of EGFR-TKI therapies are challenged by acquired resistance (AR). EGFR-T790M mutation is the most common mechanism of AR [5] . Third-generation EGFR-TKI such as osimertinib have been reported to produce promising responses in T790M-positive patients [6] .
However, the treatment options for T790M-negative patients are limited.
Vascular endothelial growth factor receptor (VEGFR) is another significant target for the treatment of NSCLC. Molecular research exhibits that VEGFR and EGFR share common signaling pathways such as PI3K/Akt and mitogen-activated protein kinase signaling pathways. EGFR inhibition can downregulate VEGF expression by hypoxiainducible factor (HIF)-1-independent and HIF-1-dependent mechanisms [7] . Locally secreted VEGF promotes angiogenesis in the tumor microenvironment and is involved in EGFR-TKI resistance [8] . Based on these findings, we speculated that the dual targeting of EGFR and VEGFR may be theoretically effective in patients with acquired EGFR-TKI resistance. Numerous recent clinical trials support our hypothesis. Afatinib plus bevacizumab (a potent monoclonal antibody targeting vascular endothelial growth factor A) have reported positive treatment effect in patients with NSCLC after AR to EGFR-TKI [9] . Apatinib, a small-molecule TKI that blocks VEGFR2, has also been reported to enhance the antitumor activity of EGFR-TKI in NSCLC with AR [10] .
Anlotinib is a novel oral multi-targeted small-molecular TKI approved for the treatment of third-line advanced NSCLC. Anlotinib mainly exerts its anti-angiogenic and broad-spectrum antitumor effects by the highly potent and specific suppression of VEGFR2 [11] . A subgroup analysis of the ALTER0303 trial demonstrated that patients previously treated with gefitinib exhibited better progression-free survival (PFS) and overall survival (OS) after treatment with anlotinib [12] . However, a recent study conversely reported that anlotinib exhibited an inferior treatment effect compared with chemotherapy in T790M-negative patients with NSCLC after first-line EGFR-TKI treatment failure [13] .
Therefore, the present study investigated whether combined anlotinib and gefitinib treatment could reverse gefitinib resistance in vitro and whether T790M-negative patients with NSCLC could benefit from this combination therapy after EGFR-TKI treatment failure.

Cell viability assay
Cell viability was measured by CCK8 assay (cell counting kit-8, Selleck, Shanghai, China). PC9 and PC9/GR cells were plated in 96-well plates at a density of 3 × 10 3 /well and incubated overnight. Subsequently, the cells were exposed to increasing
Subsequently, the cells were exposed to DMSO, 0. Experiments were repeated in triplicate, and an average was obtained.

Cell apoptosis detection
PC9/GR cells were seeded overnight in 6-well plates at 1 × 10 5 cells/well. They

Protein expression by Western blotting
The total cellular protein lysates of PC9 and PC9/GR cells were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated with specific antibodies against EGFR, p-EGFR, VEGFR2, p-VEGFR2, ERK1/2, p-ERK1/2, Akt, p-Akt, and cleaved-caspase 3 overnight at 4℃. Glyceraldehyde 3phosphate dehydrogenase was used as an internal control for EGFR, p-EGFR, VEGFR2, p-VEGFR2, ERK1/2, p-ERK1/2, Akt, and p-Akt, whereas β-actin was used as an internal control for cleaved-caspase 3. The immunoreactive bands were visualized with enhanced chemiluminescence (ECL) using an ECL detection system. The band densities were quantified using ImageJ (NIH). Experiments were performed in triplicate, and an average was obtained.

Study population
The present retrospective study was conducted in the department of Medical Oncology at the First Affiliated Hospital of Nanjing Medical University between January 2018 and August 2020. This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. Patients with cytologically or histologically confirmed NSCLC with EGFR-sensitive mutation, exhibiting AR to first-or second-generation EGFR-TKI [according to the criteria by Jackman et al [14] ], confirmed T790M negative by rebiopsy after AR, and those on a combination therapy of EGFR-TKI and anlotinib (Chia Tai Tianqing Pharmaceutical Group Co., Ltd., Jiangsu, China) as subsequent therapy were included in the study. As for rebiopsy, after the patients tested EGFR-positive by first-time biopsy and exhibited resistance to targeted therapy, they were rebiopsied to evaluate T790M mutation. Only T790Mnegative patients were included in the study. Patients lost to follow-up or those with a follow-up period (between the first treatment of anlotinib and the latest follow-up) of less than 2 months were excluded from the study. EGFR-TKI was continued, and anlotinib (one cycle of 10 mg p.o. daily for 14 days, discontinued for 7 days, and repeated every 21 days) was added until disease progression or intolerable toxicity. The dosage adjustments (increase or decrease) were decided by the attending physician according to clinical response and adverse events. The medical records of these patients were collected to study related characteristics such as age, sex, histologic type, EGFR mutation type, and prior EGFR-TKI treatment.

Assessment of treatment effect and adverse events
Tumor assessment was performed using radiographical data according to the  [15] . PFS was defined as the time from the initiation of combination therapy till the first observed progression. OS was defined as the time from the initiation of combination therapy till death. The adverse events were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0).

Statistical analysis
Statistical analysis was performed using SPSS version 23.0 (IBM, Armonk, USA) and

Resensitization of PC9/GR cells to gefitinib by anlotinib
To demonstrate the resistance of PC9/GR cells to gefitinib, we treated the PC9 and  Figure 2).

Reversal of the EGFR-TKI resistance in patients by anlotinib
The A confirmed partial response was observed in 4 (18.2%) patients after combination therapy, whereas 17 (77.3%) patients exhibited stable disease ( Figure 5A, B).
Unfortunately, 1 patient (4.5%) was insensitive to this combination strategy and exhibited disease progression in his first assessment, resulting in an objective response rate (ORR) of 18.2% and disease control rate (DCR) of 95.5%. The median follow-up time was 8.39 months. The median PFS was 11.53 ± 1.94 months ( Figure 5C), whereas the median OS was not reached.
Subgroup analysis was performed according to type of EGFR mutations. The median PFS appeared to be longer in the Del-19 group (not reached) than in the L858R (8.60 ± 1.79 months) and rare mutation (9.63 ± 6.23 months) groups, although the difference was not statistically significant (P = 0.186, Figure 5D). Subgroup analysis according to the mode of EGFR-TKI failure was then performed. The median PFS in patients with gradual progression (13.30 ± 1.69 months) was much longer than that in patients with dramatic progression (8.60 ± 5.39 months, p = 0.041; Figure 5 E).
The adverse events are presented in Table 2. The most common adverse events were rash (n = 9, 40.9%) and diarrhea (n = 6, 27.3%) related to EGFR-TKI followed by transaminase elevation (n = 4, 18.2%). Only one adverse event was graded as Grade 3 (diarrhea, n = 2, 9.1%), and no adverse event was graded as Grade 4 or 5. Other adverse events such as paronychia, thrombosis, bleeding, proteinuria, and leukopenia were rarely seen.

AR development is a major challenge in the EGFR-TKI treatment of NSCLC.
More than 50% of all AR to EGFR-TKI is attributed to the development of the EGFR T790M mutation [5] . T790M-positive patients often respond to the third-generation EGFR-TKI, exhibiting better prognosis than T790M-negative patients [6] . Thus, an effective therapeutic strategy must be investigated for these T790M-negative patients.
The present study observed that gefitinib combined with anlotinib exhibited a significant antitumor effect on gefitinib-resistant lung adenocarcinoma cells in vitro and that the combination therapy of EGFR-TKI and anlotinib demonstrated promising treatment effects and negligible adverse events in T790M-negative patients with NSCLC. To the best of our knowledge, this is the first study to evaluate the effect of the combination therapy of EGFR-TKI and anlotinib on T790M-negative patients after EGFR-TKI treatment failure.
Anlotinib is a multi-targeted small-molecular TKI that inhibits a group of kinases such as VEGFR1/2/3, c-Kit, and PDGFRβ [11] . In China, this drug has been approved as third-line treatment of locally advanced or metastatic cases of NSCLC with progression or recurrence [16] . The ALTER 0303 clinical trial demonstrated favorable outcomes in patients with advanced NSCLC receiving anlotinib as a third-line or further therapy [17] .
Molecular research has shown that by targeting VEGFR2, anlotinib plays antiangiogenesis and antitumor roles in NSCLC [11] . The present study observed that Several studies have demonstrated that EGFR-TKI combined with anti-angiogenic drugs such as bevacizumab [9] , apatinib [18] , and ramucirumab [19]  The treatment effect of EGFR-TKI varies among different EGFR mutations.
Therefore, we performed subgroup analysis according to the type of EGFR mutations.
The estimated median PFS was longer in the Del-19 group (not reached) than in the L858R (8.60 ± 1.79 months) and rare mutation (9.63 ± 6.23 months) groups, although this difference was not statistically significant (P = 0.186). A larger sample size could demonstrate more credible results.
Yang et al divided the diversity of EGFR-TKI failure into three modes according to specific criteria derived from clinical factors, namely dramatic progression, gradual progression, and local progression [15] . In our study, 19 patients (86.4%) experienced gradual progression, whereas the other 3 patients (13.6%) experienced dramatic progression. These patients received a combination therapy of EGFR-TKI and anlotinib due to refusal to chemotherapy. We further performed subgroup analysis according to EGFR-TKI failure mode and discovered that patients exhibiting gradual progression (median PFS, 13.30 ± 1.69 months) are more likely to benefit from this combination strategy than the those exhibiting dramatic progression (8.60 ± 5.39 months, P = 0.041). This finding is concurrent with Yang et al's study [15] , which reported that patients in the dramatic progression group demonstrated a better survival when switching to chemotherapeutic regimens, whereas continuation of EGFR-TKI achieved significantly longer OS in the patients exhibiting gradual progression. Our findings are consistent with a previous subgroup analysis in the ALTER 0303 trial, which reported that patients could benefit from anlotinib after prior gefitinib therapy [12] . In contrast, another recent research noted that anlotinib is less effective than chemotherapy in T790M-negative patients after first-line EGFR-TKI treatment [13] . The lack of data regarding EGFR-TKI failure modes in these studies may explain this difference in results. The ABC clinical trial of the combination of afatinib and bevacizumab after AR to EGFR-TKI in EGFR-mutant NSCLC exhibited a median PFS of 6.3 months [9] . A retrospective analysis of EGFR-TKI combined with apatinib after EGFR-TKI failure exhibited a median PFS of 4.6 months [10] . The present study suggested a much longer median PFS of 11.5 months, which indicated a strong synergistic effect of the EGFR-TKI and anlotinib combination therapy.
Despite the significant findings, the present study has several limitations. It was a single-center retrospective study with a small sample size. Prospective multicenter studies with a larger sample size are required to further strengthen our finding.          Treatment with ge tinib plus anlotinib synergistically inhibited proliferation of PC9/GR cells. A and B, EdU proliferation assays were performed 48 h after treatment with ge tinib, anlotinib, and ge nib plus anlotinib. C and D, Colony-formation assays were performed to analyze the colony-formation e ciency of PC9/GR cells in the different treatment groups. Results exhibited as mean ± SD of three independent experiments performed in triplicates. Signi cance levels determined using the t test are indicated (ns: not signi cant compared with the control group. *P < 0.01, **P < 0.001 compared with the control group. #P < 0.01, ##P < 0.001 compared with the ge tinib group).

Figure 3
Treatment with ge tinib plus anlotinib synergistically promoted apoptosis of PC9/GR cells. A and C, After treatment with ge tinib, anlotinib, and ge nib plus anlotinib for 48 h, annexin V-FITC/PI staining was used to determine the apoptosis rate of PC9/GR cells. B and D, The apoptotic marker protein, cleavedcaspase 3, was analyzed by western blot analysis. Signi cance levels determined by the t test are indicated (ns: not signi cant compared with the control group. *P < 0.05, **P < 0.01 compared with the control group. #P < 0.05, ##P < 0.01 compared with the ge tinib group).

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