Clinical Characteristics of Advanced Non-Small Cell Lung Cancer with EGFR Exon 20 Insertions

Background: Epidermal growth factor receptor (EGFR) exon 20 insertion mutations account for 4%–10% of all EGFR mutations in patients with non-small cell lung cancer (NSCLC). However, data on the differences in clinical characteristics between patients with EGFR exon 20 insertion mutations and major mutations (exon 19 deletion and L858R) are limited. Methods: We retrospectively reviewed advanced NSCLC patients with EGFR mutations, including EGFR exon 20 insertions and major mutations, who were treated with systemic therapy between January 2011 and December 2019. Results: We identied 23 patients with EGFR exon 20 insertions and 534 patients with EGFR mutations. Among patients with exon 20 insertion, the median age was 60 years (range: 27–88 years), and females and never smokers were predominant. The clinical characteristics of patients with exon 20 insertions were similar to those with major EGFR mutations. Regarding the clinical outcomes in patients with exon 20 insertions, 17 patients received platinum doublet as rst-line therapy, and the overall response rate (ORR) and median progression-free survival (mPFS) were 11.8% and 8.9%. Additionally, eight patients received anti-PD-1 antibodies and seven patients received EGFR-tyrosine kinase inhibitors (TKIs) in any-line therapy, and their ORR and mPFS were 0%, 25% and 2.2, 3.1 months, respectively. Overall survival was signicantly shorter in patients with exon 20 insertions than in those with EGFR major mutations (29.3 vs. 43.4 months, p=0.04). Conclusions: The clinical outcomes in patients with exon 20 insertions were not satisfactory compared to those in patients with major EGFR mutations.


Introduction
Lung cancer is the leading cause of cancer-related death worldwide, with non-small lung cancer (NSCLC) accounting for approximately 80% of all lung cancer deaths [1]. The frequency of epidermal growth factor receptor (EGFR) mutations has been reported to be 47.9% in adenocarcinoma and 4.6% in squamous cell carcinoma among Asian populations and 19.2% in adenocarcinoma and 3.3% in squamous cell carcinoma among Western populations [2]. EGFR mutations mainly occur between exons 18 and 21, and the most common genetic mutation is the deletion of exon 19 and L858R in exon 21, which accounts for 85-95% of all EGFR mutations [3]. EGFR mutations are commonly found in never smokers, women, and patients with adenocarcinoma [4,5]. Most patients with these EGFR mutations respond to treatment with EGFR tyrosine kinase inhibitors (TKIs) such as ge tinib, erlotinib, afatinib, and osimertinib, with median progression-free survivals (mPFS) of 9.2-18.9 months[6-10]. [11] Exon 20 insertion mutations account for about 4-10% of all EGFR mutations, and is associated with a lack of sensitivity to the aforementioned EGFR-TKIs [3,[12][13][14]. The standard treatment for patients with exon 20 insertion is systemic chemotherapy, which is similar to the treatment of other NSCLC cases without driver mutations [15,16]. However, novel targeted therapies against NSCLC with exon 20 insertion mutations, such as poziotinib [17], mobocertinib (TAK-788) [18], and amivantamab (JNJ-61186372) [19] have been developed.
Few studies have focused on the differences in clinical characteristics between patients with exon 20 insertions and those with EGFR major mutations. Our study therefore aimed to clarify the clinical characteristics and outcomes, including the e cacy of systemic treatment in patients with exon 20 insertion mutations, compared with those with major EGFR mutations.

Subjects
We retrospectively reviewed the records of advanced NSCLC patients with exon 20 insertion mutations treated with systemic chemotherapy, and those with EGFR major mutations (e.g., deletion in exon 19 and L858R in exon 21) treated with EGFR-TKIs as initial treatment at the National Cancer Center Hospital in Japan between January 2011 and December 2019. We collected data on patient characteristics, variants of exon 20 insertion, and clinical outcomes.

Detection of EGFR mutation including exon 20 insertion mutations
The diagnosis of EGFR mutation including exon 20 insertion was performed based on PCR-based methods (Scorpion-Arms methods and Cobas® EGFR Mutation Test v2; Roche Diagnostics, Basel, Switzerland) and next-generation sequencing (NGS), such as OncoGuide NCC Oncopanel System (NCC Oncopanel, Sysmex, Kobe, Japan).

Statistical Analysis
To evaluate the differences in clinical characteristics between the patients, Fisher's exact test was performed. The treatment effect was evaluated based on the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) [20]. The overall response rate (ORR) was de ned as the percentage of patients with the best overall response of complete response (CR) or partial response (PR). We also used the Kaplan-Meier method to investigate PFS and overall survival (OS). OS was de ned as the time from the date of diagnosis to death. PFS was de ned as the time from the start of treatment to disease progression or death and was censored on the date the patient was last known as progression-free. All statistical analyses were performed using the EZR ver. 1.41 [21]. This study was approved by the Ethics Committee of the National Cancer Center Hospital (2015 − 355).

Patient Characteristics
We identi ed 23 patients with EGFR exon 20 insertions and 534 patients with EGFR major mutations, including 285 patients with an exon 19 deletion and 249 patients with an L858R mutation in exon 21. Patient characteristics according to EGFR mutation status are shown in Table 1. Patients with EGFR exon 20 insertions were signi cantly younger than those with major EGFR mutations (median age: 60 vs. 66 years). There were no signi cant differences in baseline characteristics between patients with exon 20 insertions and major EGFR mutations, except for age. Regarding the metastatic spread, bone (21.6%) was the most common metastatic site in patients with EGFR exon 20 insertions, followed by the central nervous system (CNS) (13.0%), liver (17.4%). Patients with intrathoracic metastases were more common in patients with EGFR exon 20 mutations (52.2%) than in those with EGFR major mutations (35.2%), although the differences were not signi cant. Of the 23 patients with an EGFR exon 20 insertion, four were assessed for variants of EGFR exon 20 insertion (Table 2).     Table 2 and Fig. 1A). Table 3 First-line treatment regimens in patients with exon20 insertion mutation. Over the clinical course in patients with EGFR exon 20 insertions, 7 and 8 patients received EGFR-TKIs and anti-PD-1 antibody monotherapy, respectively. The differences in the ORR and PFS between exon 20 and major mutation patients in the EGFR-TKI and anti-PD-1 antibody monotherapy are shown in Table 2 and Fig. 1B

Discussion
We found that there were no signi cant differences in clinical characteristics, including the distribution of metastatic sites between patients with EGFR exon 20 insertion and major mutations. Additionally, we showed that the ORR and PFS of patients treated with platinum-based chemotherapy were 11.8% and 8.9%, respectively. The OS of patients with EGFR exon 20 insertions was signi cantly shorter than in patients with major mutations who received EGFR-TKIs as initial treatment.
Very few reports have focused on the differences in clinical characteristics between major and minor EGFR mutations. Previous studies have shown that EGFR exon 20 insertion is more likely to occur in never or light smoking patients and those with adenocarcinomas [22,23]. In our study, however, there were no differences in age, sex, smoking history, histology, or stage at diagnosis between the two groups.
EGFR exon 20 insertions are related to the intrinsic resistance to EGFR-TKIs compared with EGFR major mutations, such as exon 19 deletion and L858R in exon 21 [3,12,24]. Due to the limited e cacy of EGFR-TKIs, platinum combination chemotherapy is still the standard therapy for patients with exon 20 insertion. Previous studies have reported that mPFS was 4.2 to 6.4 months and OS was 16.4 to 29.4 months, which were similar to our data [16] [25,26]. Additionally, OS in patients with exon 20 insertion was signi cantly shorter than that in patients with major mutations.
However, the clinical e cacy of EGFR-TKIs in patients with EGFR exon 20 insertion has been reported to differ according to the variant [22]. EGFR p.A763_Y764insF-QEA is considered to be more sensitive to rst-to third-generation EGFR-TKIs than other variants in exon 20 insertion mutations [22,27,28]. In our study, only four patients had detailed information on insertion variants, and variants that are reportedly sensitive to EGFR-TKIs were not included. Although, in the current clinical procedure, we did not necessarily obtain detailed variant information, and the frequency of sensitive variants seems quite low, our results strongly support that conventional EGFR-TKIs are of little use for EGFR exon 20 insertion.
We also evaluated the e cacy of anti-PD-1/PD-L1 agents in NSCLC with EGFR exon 20 mutations. In general, anti-PD-1/PD-L1 antibodies are poorly effective in EGFR-mutated NSCLC compared with those without EGFR mutations [29][30][31]. However, recent studies have reported that patients with exon 20 insertions showed better clinical outcomes with anti-PD-1 antibody compared with those with EGFR major mutations [32]. In this study, the ORR and mPFS of the anti-PD-1 antibody were 25% and 3.1 months (95% CI: 0.7-6.0. The therapeutic effect is not su cient, and more speci c treatment for exon20 insertion is desirable. Recently, novel targeted therapies against exon 20 insertion mutations, such as poziotinib, mobocertinib, and amivantmab have been developed [17][18][19]. Poziotinib, a potent TKI against EGFR and HER2 exon 20 insertion mutations, showed an ORR of 15-44% and PFS of 4.2-5.5 months in the phase II trial [33,34]. Mobocertinib is an EGFR-TKI with potent and selective preclinical inhibitory activity against EGFR exon 20 insertions, with an ORR of 43% and PFS of 7.3 months in a phase II trial. A phase III trial comparing mobocertinib with platinum-based chemotherapy as rst-line therapy is currently ongoing (NCT04129502) [35]. Amivantamab is an anti-EGFR-MET bispeci c antibody that can target diseases driven by both EGFR and MET, and has shown therapeutic e cacy in patients with a variety of mutations, including C797S, T790M, EGFR exon20 insertion, and MET ampli cation. Amivantamab showed a response rate of 36% and a PFS of 8.3 months in a Phase II/III study [19]. A study is planned for patients with advanced exon 20 insertion mutations, with carboplatin and pemetrexed with and without amivantamab (NCT04538664).
This study has some limitations. First, it is a single-center, retrospective study with a small sample size as patients with EGFR exon 20 mutations are rare. Additionally, genetic variants of exon 20 insertion were assessable in only four patients, as PCR-based testing showed only the presence of exon 20 insertion, not variant types.
In conclusion, the effectiveness of EGFR-TKIs and anti-PD-1 antibodies is not su cient. The development of an EGFR exon 20 speci c treatment is therefore warranted to improve patient survival.

Supplementary Tables
Supplemental Table 1. Clinical outcomes according to the variants of exon20 insertions.