Real‐world clinical analysis in 190 advanced NSCLC patients with uncommon EGFR mutations: A multi‐center study

Abstract Differently from epidermal growth factor receptor (EGFR) 19Del and L858R mutations, the panoramic description of uncommon EGFR mutations is far from mature. Our understanding of its population characteristics, treatment response, and drug resistance mechanisms needs urgent expansion and deepening. Our study enrolled 437 patients with non–small‐cell lung cancer from four clinical centers and who had uncommon EGFR mutations. The clinical characteristics of all patients and the treatment outcomes of 190 advanced patients who received pharmacotherapy were analyzed. Moreover, the acquired resistance mechanisms were explored based on 53 tissue or liquid re‐biopsy data in 45 patients. Patients with EGFR 20ins had a shorter survival time compared with patients with non‐20ins mutations. In total, 149 cases had received EGFR‐tyrosine kinase inhibitors (TKI); afatinib was significantly superior to other EGFR‐TKIs both in ORR and mPFS in all uncommon mutations and especially in the L861Q group. The most common acquired drug resistance mechanism was MET amplification, followed by EGFR T790M, which was significantly different from common EGFR mutations.

437 patients with non-small-cell lung cancer from four clinical centers and who had uncommon EGFR mutations. The clinical characteristics of all patients and the treatment outcomes of 190 advanced patients who received pharmacotherapy were analyzed. Moreover, the acquired resistance mechanisms were explored based on 53 tissue or liquid re-biopsy data in 45 patients. Patients with EGFR 20ins had a shorter survival time compared with patients with non-20ins mutations. In total, 149 cases had received EGFR-tyrosine kinase inhibitors (TKI); afatinib was significantly superior to other EGFR-TKIs both in ORR and mPFS in all uncommon mutations and especially in the L861Q group. The most common acquired drug resistance mechanism was MET amplification, followed by EGFR T790M, which was significantly different from common EGFR mutations.

K E Y W O R D S
clinical outcomes, EGFR uncommon mutation, EGFR-TKI, NSCLC, resistance mechanism 50% and a median PFS (mPFS) of 8.2 months in a phase II clinical trial (KCSG-LU15-09) with 37 patients, 8 and dacomitinib obtained an ORR of 56.3% and an mPFS of 10.3 months in 32 subjects. 9 Even though some studies have supported the fact that EGFR-TKIs also have efficacy for uncommon EGFR mutations, very few studies have compared the outcomes between different targeted drugs or different mutation subtypes. Moreover, the acquired resistance mechanism for EGFR-TKIs in this population is far from clear. Some small sample studies have considered the EGFR T790M mutation as the main acquired resistance mechanism, while c-MET amplification was detected at a high frequency in the real world. [10][11][12] Further research is needed to evaluate the treatment options considering mutation specificity and revealing resistance mechanisms in more detail.
In this study, 437 patients with EGFR uncommon mutations were enrolled to analyze their clinical characteristics and sensitivity to different anti-tumor therapies. In addition, we explored potential drug resistance mechanisms on a large scale of 53 re-biopsy data in 45 patients harboring uncommon EGFR mutations.

| PATIENTS AND ME THODS
As seen in Figure 1 Categorical variables were reported as frequency and percentage, and compared using chi-squared test and Fisher's exact probability method. Continuous variables such as age was reported as mean, minimum, and maximum using an independent-sample t-test.
Kaplan-Meier survival analysis was used to estimate median overall survival (mOS) and mPFS, and compared using log-rank test. A pvalue less than 0.05 was considered statistically significant.

| Baseline characteristics
As shown in Table S1, the demographic data for all 437 patients revealed that women and non-smokers occupied a slightly higher proportion than men and previous smokers for all patients, which was similar to the characteristics for EGFR classical mutations. Overall, F I G U R E 1 A flowchart of the patient cohort. EGFR-TKI, epidermal growth factor receptor tyrosine kinase inhibitor; NSCLC, non-small-cell lung cancer; firstgeneration drugs, gefitinib, erlotinib and icotinib were included. 96.1% of patients were diagnosed with adenocarcinoma and 14.9% of patients had a family history of cancer.
Mutation categories are shown in Figure 2. The simplex mutation accounted for 76% (n = 332) of all mutation types, including 20ins, G719X, E709K, S768I, and L861Q. Here, 20ins was the most frequent mutation type, accounting for 24.5% (n = 107). G719X and L861Q also were a large proportion at 24.3% (n = 106) and 22.4% (n = 98), respectively. Only 4.6% of patients were diagnosed with simplex S768I. The occurrence of the E709K mutation was very rare; 24% (n = 105) of patients had more than one mutation. It seems that there were no obvious rules in the detailed distribution of various combinations, as shown in Figure 2A,B. G719X was the most common mutation among those compound factors, and the most frequent combination was G719X + S768I, accounting for 41.0% of all compound mutations, followed by G719X + L861Q ( Figure 2C).

| Treatment outcomes
In total, 190 advanced or recurrent cases who had received systemic therapy with regular follow-up were included in the survival analysis ( Figure S1). Among them, 29 cases harbored EGFR 20ins and 161 cases harbored non-20ins uncommon mutations. The mutation subtypes, first-line regimens, and OS times according to specific mutation

| First-line therapies in non-20ins
For 161 patients with non-20ins, we conducted further analysis, as follows. There were 38, 51, 36, 24, and 11 patients who received first-line chemotherapy, afatinib, gefitinib, icotinib, and erlotinib, respectively, and one patient who received osimertinib. The clinical characteristics at baseline showed no significant difference grouped by different therapies (Table S2). The ORR of afatinib was significantly superior to other therapies (58.8% vs. range 12.5% ~ 36.8%). Compared with chemotherapy, first-line EGFR-TKIs had longer PFS time but similar OS time ( Figure S1D).

| Efficacy of first-TKI in non-20ins
Of the 161 non-20ins patients, in total 149 cases had received EGFR-TKI. Their mutation subtypes and first-TKI drugs are demonstrated in Figure 3A Figure 3D, Table 2). Afatinib had a better DOR than other EGFR-TKIs (mDOR 14. for other EGFR-TKIs ( Figure S3). Half of the patients in the L861Q group received afatinib as the first-TKI, and half received other TKIs.
Patients with compound mutations had numerically longer mPFS times compared with simplex mutations ( Figure S2D). When afatinib was applied as a subsequent TKI, the mPFS was shorter than when using it as the first-TKI (16.6 vs. 11.1 m, p = 0.009, Figure S2F).

| Resistant mechanisms
Of the 149 patients who had ever received EGFR-TKIs, 127 patients developed a progressed disease. Forty-five patients explored acquired resistance mechanisms using the NGS test (tumor tissue rebiopsy or liquid biopsy), and seven patients accepted two or more biopsies. The TKIs given to these patients were gefitinib (10 data), erlotinib (four data), icotinib (seven data), afatinib (27 data), and osimertinib (five data). In total, 53 drug resistance test data were collected ( Figure 4). Twenty-nine data were based on tumor tissues and 24 were based on ctDNA in blood.
As a result, 30 (56.6%) tests showed the exact acquired resistance changes and 23 times did not (which was defined as no exact mutation detected or only original mutations detected). Thirteen out of 24 tests based on ctDNA were detected with acquired resistant mutations, with a positive rate of 45.8% (11/24), and more data based on tissue re-biopsy were detected with positive outcomes, with a positive rate of 65.5% (19/29 EGFR amplification was often accompanied by other acquired mutations at the same time. Further analysis based on the different drugs given previously found that patients who had received first-generation EGFR-TKI drugs tended to develop acquired EGFR T790M mutations, accounting for 28.6% (6/21), while MET amplification only occupied 9.5% (2/21). However, the detection rate of acquired T790M mutations in the second generation drug afatinib was only 11.1% (3/27), and MET amplification was the most common resistance mechanism, with a detection rate of 25.9% (7/27). The third-generation drug osimertinib was detected in only one positive drug resistance mechanism with MET amplification at 20% (1/5), which did not help in drawing a particular conclusion.

| DISCUSS ION
In this retrospective multi-center study, we analyzed the clinical characteristics, treatment outcomes, and acquired resistance mechanisms in NSCLC patients with EGFR uncommon mutations in west China. Our findings highlighted the disparities in EGFR-TKI TA B L E 1 ORR of first EGFR-TKI in 149 non-20Ins patients who had ever received EGFR-TKI.  Similar to NSCLC patients with EGFR common mutations, 13,14 women, non-smokers and adenocarcinoma made up a higher proportion of the whole uncommon mutation cohort. The prevalence of uncommon mutation subtypes in this study was broadly in line with published estimates. [15][16][17] Ex20ins, G719X (including G719A/S/C), and L861Q mutations were the most frequent. Compound mutations accounted for 24% of the population, and G719X was the most common composition.
Different uncommon mutations had variable sensitivity to different EGFR-TKIs. 18 In summary, EGFR uncommon mutations have similarities and differences compared with common mutations. Afatinib performed with great efficacy for the majority of EGFR uncommon mutations, and is highlighted as the likely preferred recommendation for L861Q.
Compound mutation would be a favorable factor for clinical outcomes and combined common plus uncommon mutations may resemble common mutations to some extent. The incidence of acquired T790M mutations in patients with uncommon mutations was very low, which was different from EGFR common mutations. Secondary c-MET amplification should not be ignored, especially when treating with afatinib. To better understand the panorama of EGFR uncommon mutations, these results need to be further confirmed in future studies and new insights are expected to be obtained.

ACK N OWLED G M ENTS
None.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflict of interest.