Possibility of brigatinib‐based therapy, or chemotherapy plus anti‐angiogenic treatment after resistance of osimertinib harboring EGFR T790M‐cis‐C797S mutations in lung adenocarcinoma patients

Abstract Background There was no standard treatment for patients who acquired resistance to osimertinib mediated by epidermal growth factor receptor (EGFR) T790M‐cis‐C797S. The aim of this study was to investigate the association between different therapeutic strategies and survival outcomes among these patients. Methods This retrospective cohort study analyzed 46 patients with metastatic lung adenocarcinoma and EGFR T790M‐cis‐C797S after osimertinib progression from January 1, 2017 to October 31, 2020. Among them, 13 patients received brigatinib‐based therapy, 23 patients received chemotherapy in combination of anti‐angiogenics or not, and 10 patients received other targeted treatments like dacomtinib, bevacizumab, or a combined therapy of osimertinib and other targeted drugs. Results Compared to other targeted therapy, brigatinib‐based therapy (median progression‐free survival [mPFS]: 4.40 vs. 1.63 months, hazard ratio [HR] = 0.39, 95% confidence interval [CI]: 0.21–0.73, p = 0.001) and chemotherapy‐based treatment (mPFS: 4.70 vs. 1.63 months, HR = 0.18, 95% CI: 0.06–0.50, p < 0.001) presented a better survival outcome and there was no significant difference between brigatinib‐based therapy and chemotherapy‐based treatment (mPFS: 4.40 vs. 4.70 months, HR = 1.24, 95% CI: 0.57–2.67, p = 0.58). Chemotherapy combined with anti‐angiogenics achieved a better efficacy than only chemotherapy (mPFS: 5.50 vs. 1.03 months, HR = 0.30, 95% CI: 0.11–0.83, p = 0.02). Patients carrying EGFR exon 19 deletion mutation had a longer PFS than those who harboring EGFR exon 21 p.L858R mutation (4.57 vs. 1.03 months, HR = 0.18, 95% CI: 0.06–0.54, p = 0.001), no matter they received brigatinib‐based therapy (mPFS: 5.00 vs. 3.23 months, HR = 0.19, 95% CI: 0.01–0.96, p = 0.05) or chemotherapy‐based treatment (mPFS: 7.23 vs. 1.03 months, HR = 0.05, 95% CI 0.01–0.49, p < 0.001). Conclusion Brigatinib‐based therapy and chemotherapy plus anti‐angiogenics could be considered beyond progression from osimertinib therapy. For patients harboring EGFR exon 19 deletion/T790M/cis‐C797S mutation, the clinical efficacy was superior to patients harboring EGFR exon 21 p.L858R/T790M/cis‐C797S mutation.


| INTRODUCTION
In the Chinese population, non-small cell lung cancer (NSCLC) harboring an epidermal growth factor receptor (EGFR)-activating mutation accounts for 40% to 65%. 1 Despite potent efficacy with first-line EGFR tyrosine kinase inhibitors (EGFR-TKIs), a majority of patients eventually developed resistance after around 1 year. [2][3][4] Approximately 60% of patients are found to have a EGFR exon 20 p.T790M (EGFR T790M) mutation in the gene coding EGFR at the time of progression and osimertinib was substantially effective to target T790M. 5 However, it is inevitable to encounter resistance generated by various mechanisms and the most common is the emergence of EGFR C797S mutation. 6,7 Depending on the allelic relationship with T790M, C797S is defined as cis-C797S or trans-C797S 8 which have clinical implications that T790M-trans-C797S maintains sensitivity to the combination of first-and third-generation of EGFR TKIs, whereas T790M-cis-C797S mediate resistance to osimertinib and non-response to first, second, or third generation of EGFR-TKIs. Up to now, there is not a standard therapy guideline for patients harboring EGFR T790M-cis-C797S and as a result of lack of appropriate targeted therapy, the most common treatment is chemotherapy. However, the treatment efficacy was not clear yet.
To substantially promote survival outcomes of these patients, we urgently need to investigate novel therapeutic strategies. Brigatinib (AP26113) was a TKI targeted either anaplastic lymphoma kinase (ALK) or EGFR and cetuximab, an antibody that blocks EGFR dimerization, hampering the kinase uniformly susceptible to the allosteric agent, both are reported to overcome resistance in preclinical research. 9,10 Moreover, several clinical studies have observed benefits from the treatment of brigatinib/ cetuximab regimen. 11,12 Furthermore, in a retrospective research, five patients who received brigatinb combination with cetuximab achieved a favorable outcome that progression-free survival (PFS) reached 14 months and 60% of objective response rate. 13 In conclusion, previous studies have introduced potential clinical response strategies for patients harboring EGFR T790M-cis-C797S mutation. Albeit, the existing studies were small-sample-sized and needed more powerful validation to identify clinical efficacy in these patients. Our study aimed to illustrate the landscape of various therapeutic strategies in a real-world setting. Then we dedicated to comparing their efficacy to bring reference evidence for clinical practice.

| Patients' selection and data collection
This study retrospectively reviewed a total of 46 postoperative recurrent or metastatic adenocarcinoma patients who carried cis-C797S mutation when progressed beyond osimertinib treatment. All the patients received treatment in National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College and Beijing Chest Hospital, Capital Medical University from January 1, 2017 to October 31, 2020 when the presence of C797S mutation. The presence of measurable lesions according to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1). Next-generation sequencing was used to detect EGFR cis-C797S mutation. Patients with brain metastases or meningeal metastases were eligible at baseline. The following chart of this study was illustrated in Table S1.

| Treatment assessment and definitions
Depending on the treatment, we divided patients into three groups: brigatinib-based therapy, chemotherapybased treatment combined with anti-angiogenics or not, and other targeted therapies like dacomtinib, Conclusion: Brigatinib-based therapy and chemotherapy plus anti-angiogenics could be considered beyond progression from osimertinib therapy. For patients harboring EGFR exon 19 deletion/T790M/cis-C797S mutation, the clinical efficacy was superior to patients harboring EGFR exon 21 p.L858R/T790M/cis-C797S mutation.

K E Y W O R D S
cis-C797S, EGFR, non-small cell lung cancer, resistance, treatment bevacizumab, or a combined therapy of osimertinib and other targeted drugs. Patients in the brigatinibbased therapy group received brigatinib orally once daily at an initial dose of 90 mg for 7 days and increased to 180 mg from day 8 onward if tolerated and when cetuximab used at a dose of 500 mg/m 2 , administrated intravenously on days 1 and 8 of a 21-day cycle. Patients received chemotherapy and targeted therapy at a standard dose according to the China Society of Clinical Oncology guideline or National Comprehensive Cancer Network (NCCN) guideline. Imaging examination at baseline was confirmed with measurable target lesions documented by computed tomography images of the chest and abdomen, brain magnetic resonance imaging, and whole-bone scans. Whether the response presented a complete response (CR), partial response (PR), stable disease (SD) or progressive disease has been evaluated every 2 months according to the RECIST version 1.1.
The primary endpoint was PFS which was defined as the duration from the initiation of therapy to the date of disease progression or death. Overall survival (OS) was the period from the date of treatment to death or last follow-up that August 1st, 2021. Disease control rate (DCR) was defined as the percentage of CR, PR, and SD. Smokers were defined as current or former smokers and non-smokers were the people who smoke less than 100 cigarettes in their lifetime. All clinical data were collected from electronic records. As an observational study, this research was exempted from obtaining patients' informed consent without therapeutic intervention, and this research was approved by the Research Ethics Board of Cancer Hospital and conducted in accordance with the Declaration of Helsinki as well.

| Tissue processing and organoid culture
Lung cancer organoids were derived from pleural effusion of one lung cancer patient who carried EGFR T790M-cis-C797S mutation at the National Cancer Center/National Clinical Research Center for Cancer/ Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. On arrival, tumor tissues were washed with cold phosphatebuffered saline, cut into small pieces, washed with Advanced DMEM/F12 (Thermo Fisher Scientific; containing 1× Glutamax, 10 mM HEPES and antibiotics) and digested with collagenase (Sigma-Aldrich; Cat. No. C9407, 2 mg/ml) for 1-2 h at 37°C. After washing twice with fresh medium (2% fetal calf serum) and centrifugation (17.8 g, 4 min), dissociated cells were seeded into growth factor-reduced matrigel (Corning, Inc.) with the presence of Advanced DMEM/F12 at 37°C for 30 min. Next, the surface of a solidified mixture of cell suspension/Matrigel was sealed with complete human organoid medium (500 μl), which was comprised of advanced DMEM/F12 supplemented with series additives as described by Lampis et al., 14 with the replacement of every 3 days. When the organoids ranged up to 200-500 μm in diameter (about 1 week), organoids were dissociated and passaged weekly using TrypLE Express (Gibco). The patient-derived tumor organoids (PDTO, 2 × 10 6 cells/tube, p3) were conducted using the Recovery Cell Culture Freezing Medium (Gibco) and stored at −80°C before a drug screening.

| Statistical analysis
SPSS version 23.0 (SPSS, Inc.) was used for statistical analysis. Univariate analysis was performed using the logrank test, and multi-variants analysis using a Cox proportional hazard regression model. PFS was estimated using Kaplan-Meier analysis, and the log-rank test was utilized to compare the differences in survival distributions between groups. All statistical tests with two-sided p < 0.05 were considered statistically significant. Variables included age, gender, smoking history, Eastern Cooperative Oncology Group (ECOG) scores, metastatic sites, mutation landscape, and treatment regimen. Graphpad 5.0 was used to present survival curves.

| Treatment and outcomes
This study further analyzed all patients who have received therapy after cis-C797S presence. A total of 23 patients received chemotherapy-based therapy (50.0%), 13 patients (28.3%) applied brigatinib-based treatment and 10 patients (21.7%) received other targeted treatments, like dacomtinib, bevacizumab, or a combined therapy of osimertinib, and other targeted drugs. The most common chemotherapy regimens were pemetrexed-based regimens (n = 15). Seventeen patients tended to combine with anti-angiogenics. The details of therapy regimens were illustrated in Tables (Figure 2A). However, for the OS, we have not discovered a similar tendency. The median OS of brigatinb-based therapy, chemotherapy-based therapy, and other targeted therapy were 9.93, 18.90, and 4.00 months, respectively. Although there was a numerical difference, the statistical significance did not reach between the three groups ( Figure S1). In patients whose therapy efficacy could be evaluated, the DCR in patients who received brigatinib-based therapy, chemotherapybased therapy, and other targeted therapy were 75.0% (9/12), 91.3% (21/23), and 30.0% (3/10), respectively ( Table 2).
We also conducted several subgroup analyses according to different variants. Considering the type of chemotherapy, the patients who underwent pemetrexed-based treatment achieved a mPFS of 4.70 months, and in patients who received non-pemetrexed treatment, the mPFS was 4.10 months (HR = 0.78, 95% CI: 0.31-1.99, p = 0.60). The median OS was 18.90 and 11.13 months in the two groups (HR = 0.56, 95% CI: 0.17-1.82, p = 0.33). Based on whether combining with anti-angiogenic treatment, we divided the chemotherapy group into two subgroups. The patients who underwent anti-angiogenic  Figure S2). Furthermore, considering the comparable efficacy of brigatinib-based treatment and chemotherapy and varied efficacy due to whether chemotherapy combines with anti-angiogenic treatment, we compared the efficacy of brigatinib-based therapy with chemotherapy alone. The median PFS of the two groups was 4.40 and 1.03 months (HR = 0.56, 95% CI: 0.20-1.59, p = 0.27).

| Co-mutation type and survival outcomes
We analyzed the relationship between co-mutation gene type and treatment efficacy. There were eight patients carrying EGFR 21L858R mutation and 29 patients carrying EGFR 19del mutation. In all patients carrying EGFR 19del mutation and EGFR 21L858R mutation, there was a significant difference on PFS of 4.57 and 1.

| PDTO model and drugsensitivity test
The PDTO model technique is a cutting-edge technology for in vitro three-dimensional culture of tumor precision medicine. 14,15 The PDTO model can replicate the tissue complexity and genetic heterogeneity of tumors. In this model, we conducted a drug sensitivity test and brigatinibbased therapy displayed a satisfied inhibitory activity. The brigatinib monotherapy, brigatinib combined with cetuximab, brigatinib combined with osimertinib achieved an inhibition rate of 81%, 88%, and 87%, respectively. However, the inhibition rate of chemotherapy, which was around 24%-30%, was unsatisfactory, and osimertinib had a 32% inhibition rate ( Figure 4). Regretfully, due to the limitation of the technique, the test could not be conducted on chemotherapy combined with anti-angiogenic therapy.

| DISCUSSION
With the wide utilization of osimertinib due to the high percentage of EGFR mutation in the Asian population, many people inevitably develop resistance to osimertinib later. Albeit, Patients who acquired resistance to osimertinib were recommended to undergo initial systemic therapy options in the NCCN guideline and the clinical efficacy of these patients was still unknown. Previous studies have reported that the most common reason inducing resistance was EGFR C797S mutation and the incidence rate was around 7%-14%. 7 In the cell lines which expressed C797S mutation, 85% of them presented the cis-C797S and it showed non-response to the EGFR-TKIs. 8 Our retrospective study provides real-world evidence to investigate the clinical efficacy of various strategies for advanced NSCLC patients harboring EGFR-T790M-cis-C797S. This study is also the largest analysis committed to metastatic lung adenocarcinoma patients with cis-C797S mutation around the world to date. Preclinical studies have detected the potent efficacy of cetuximab and brigatinib in C797S mutation cell lines. 9,16,17 However, there were limited data to report the clinical efficacy in a real-world setting so far. Two case reports elaborated on the clinical efficacy of brigatinib. In case one, the patient who received brigatinib combined with cetuximab had a desirable PFS of 9.00 months. 11 And the other patient who underwent brigatinib, bevacizumab, and osimertinib reached the efficacy of PR and the treatment was still continuing. 12 However, not all the patients who received brigatinib achieved a desirable efficacy. Another case report showed a patient who was treated by brigatinib and cetuximab progressed in only 1 month. 18 A retrospective study compared the clinical efficacy of brigatinib combination with cetuximab and chemotherapy. In this study, a total of 15 patients were enrolled in which five patients received brigatinib combination with cetuximab and the PFS was 14 and 3 months. 13 To sum up, there is still inconsistency about whether cis-C797S patients could gain survival benefit by brigatinib and a lack of abundant evidence to illustrate the superiority of brigatinib to chemotherapy. In our study, the PFS of brigatinib-based therapy, chemotherapybased therapy, and other targeted therapy were 4.40, 4.70, and 1.63 months, respectively. In addition, the PFS of chemotherapy combined with anti-angiogenics and chemotherapy alone were 5.50 and 1.03 months, respectively. Bragatinib-based therapy and chemotherapy combined with anti-angiogenic treatment presented a possibility to become treatment options beyond the resistance of osimertinib. Although brigatinib (AP26113) was approved by Food and Drug Administration in 2017 for patients who were anaplastic lymphoma kinase positive, it has not been introduced in China mainland as yet. Therefore, if brigatinib was not available to some patients, considering the comparable efficacy, chemotherapy combined with antiangiogenic therapy could be an optional treatment.
This study also analyzed the relation of co-mutation gene and treatment efficacy. The patients carrying EGFR 19del mutation had a longer PFS than patients carrying EGFR 21L858R mutation. Uchibori et al. have reported that brigatinib could inhibit EGFR-triple mutation and C797S/T790M/L858R was less potent than in 19del. 9 This conclusion was coincident with our real-world study and may have a clinical indication of treatment options for various variant patients. Especially for patients who F I G U R E 4 Drug sensitive test on patient-derived tumor organoids (PDTO) model harboring the EGFR 19del, chemotherapy-based therapy could bring a decent survival benefit as PFS reached 7.23 months.
Furthermore, we conducted a drug-sensitive test on the patient-derived tumor organoids model. This is a novel technology and can replicate the tissue complexity and genetic heterogeneity of the tumor. We cultured a model derived from one patient carrying EGFR C797S/ T790M/19del mutation and found out that brigatinib and brigatinib combined with cetuximab showed a favorable inhibition rate of the model. However, the chemotherapy presented an unsatisfied efficacy which was in accordance with our real-world study that the PFS of chemotherapy without anti-angiogenic therapy was only 1.03 months which was much shorter than brigatinib-based therapy. It is regrettable that as a result of the combination with anti-angiogenic therapy cannot be conducted on PDTO model, we couldn't verify whether combination with antiangiogenic treatment had an equal efficacy of brigatinibbased therapy.
Although our real-world study precisely investigated the treatment strategies of EGFR T790M-cis-C797S mutated patients, as well as provided a detailed description of clinical characteristics, several limitations cannot be ignored. First, this was a retrospective study that easily appeared selection bias. Second, the heterogeneity of patients in this real-world study in terms of specific treatment regimens was considered a limitation. Third, the number of patients with EGFR 21L858R was limited, which led to preventing us from providing corroborative information on the clinical efficacy of brigatinib. Last but not least, the PDTO model was derived from one patient and an anti-angiogenic regimens could not be tested on this model which might lead to the bias of drug-sensitive test. Although our sample size was the largest around the world, more clinical evidence as well as cell lines and patient-derived xenograft model are still urgently needed to correspond to our findings and further draw a clear conclusion. We believe that our study provides valuable evidence to give suggestions on clinical therapy in Chinese cis-C797S-mutated NSCLC patients to a certain extent.

| CONCLUSION
In this retrospective study, clinical characteristics and real-world clinical practice in advanced NSCLC patients harboring EGFR T790M-cis-C797S mutation were observed. Brigatinib-based therapy and chemotherapy combined with anti-angiogenic therapy are considerable after progression beyond osimertinib treatment. Patients carried EGFR 19del/T790M/cis-C797S mutation achieved a better survival benefit than patients harboring EGFR 21L858R/T790M/cis-C797S mutation.