Comparison of Post-Resistance Treatment Outcomes in EGFR-Mutated NSCLC Patients Following Third-Generation EGFR-TKI Therapy: A Retrospective Cohort Study

Abstract

Background

Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have become the first-line treatment for non-small cell lung cancer (NSCLC) with EGFR-sensitive mutations. The optimal treatment strategy after resistance to third-generation EGFR-TKIs still needs further exploration.

Methods

This retrospective study included patients with advanced lung adenocarcinoma who were consecutively enrolled at our hospital between January 2018 and July 2023. All patient data were fully de-identified prior to analysis, and no information that could directly or indirectly identify individual participants was included in this study. We evaluated the objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) across various treatment strategies. We also explored the tumor microenvironment (TME) in a subset of patients.

Results

A total of 206 patients were included. Chemo-antiangiogenesis (47.6%) achieved longer mPFS than chemo-immunotherapy (8.00 vs. 5.70 months, p=0.033), with higher ORR (34.7% vs. 16.7%, p=0.003). Median OS was shorter in the immunotherapy group (25.83 vs. 32.33 months, p=0.012). TME analysis (n=27) revealed an immunosuppressive profile (low PD-L1, CCL5, CD8, granzyme B; high Foxp3). In EGFR exon 21 L858R patients, EGFR-TKI continuation was inferior to chemotherapy (mPFS: 3.53 vs. 8.00 months, p=0.001; ORR: 10.5% vs. 40.3%, p=0.015; DCR: 27.4% vs. 69.2%, p=0.009). In oligoprogressive disease, EGFR-TKIs plus radiotherapy improved OS (37.23 vs. 27.77 vs. 25.83 months, p=0.045). Platelet count, LDH, D-dimer, and smoking history were independent predictors of poor prognosis.

Conclusions

Chemo-antiangiogenesis remains a key treatment option after resistance. For oligoprogressive disease, continuing EGFR-TKIs with local radiotherapy may provide superior survival benefit. Chemo-immunotherapy appears less effective, potentially due to an immunosuppressive TME. Prospective validation is warranted.

Keywords

non-small cell lung cancer (NSCLC)third-generation EGFR-TKIs resistance antiangiogenic therapy immunotherapy

Key Findings

This study compared chemo-antiangiogenesis therapy with chemo-immunotherapy in patients with EGFR-mutant non-small cell lung cancer (NSCLC) who developed resistance to third-generation EGFR-TKIs., showing superior median progression-free survival (mPFS) for the former (8.00 vs. 5.70 months, p=0.033). Oligoprogressive patients benefited more from continuing third-generation EGFR-TKIs plus local radiotherapy, achieving a longer median overall survival (mOS: 37.23 vs. 25.83 months, p=0.045). Patients with EGFR exon21 L858R mutations had lower response rates to EGFR-TKI-containing therapies versus chemotherapy-based regimens (mPFS: 3.53 vs. 8.00 months, p=0.001; ORR: 10.5% vs. 40.3%, p=0.015). Tumor microenvironment analysis revealed an immunosuppressive profile, potentially limiting immunotherapy efficacy. Elevated platelet count, LDH, D-dimer levels, and smoking history were identified as independent predictors of poor prognosis.

What is known and what is new?

The study provides real-world evidence favoring chemo-antiangiogenesis over chemo-immunotherapy post-TKI resistance due to its PFS advantage. It highlights oligoprogression as a critical subgroup benefiting from EGFR-TKI continuation combined with local radiotherapy. The presence of EGFR L858R mutation is shown to be a negative predictor for EGFR-TKI retreatment efficacy, advocating for chemotherapy-based alternatives. The study validates the role of an immunosuppressive tumor microenvironment in explaining limited responses to immunotherapy in EGFR-TKI-resistant NSCLC.

What is the implication, and what should change now?

Clinically, prioritize chemo-antiangiogenesis for systemic progression and EGFR-TKI plus radiotherapy for oligoprogression. Avoid EGFR-TKI retreatment in L858R-mutant patients. Incorporate elevated platelet count, LDH, D-dimer, and smoking history into prognostic stratification. Future research should include prospective trials to confirm these findings and explore tumor microenvironment dynamics, particularly investigating immunotherapy combinations targeting immunosuppression.

Introduction

Targeted therapy has significantly transformed the treatment landscape for patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). Third-generation EGFR-tyrosine kinase inhibitors (TKIs) are now established as the first-line treatment standard for these patient.^1-4 However, despite these advancements, the development of resistance remains an inevitable challenge.⁵ The mechanisms of resistance to EGFR-TKIs are complex and heterogeneous, generally categorized into EGFR-dependent and non-EGFR-dependent pathways.^6,7 This complexity in resistance mechanisms makes selecting subsequent treatments after EGFR-TKI resistance particularly challenging.

In recent years, immunotherapy has emerged as a promising treatment strategy, garnering considerable attention, especially in the context of NSCLC. However, the efficacy of immune checkpoint inhibitors (ICIs) as monotherapy in patients with EGFR-mutated NSCLC is suboptimal.^8-11 On the other hand, some studies suggest that combining immunotherapy with chemotherapy may yield synergistic effects.¹² For example, a retrospective study from China showed that patients who received combination immunotherapy after EGFR-TKI resistance had longer progression-free survival (PFS) than those who received single-agent immunotherapy.¹³ However, not all studies have drawn the same conclusion; some studies have shown that immunotherapy combined with chemotherapy did not improve the prognosis of EGFR-TKI-resistant NSCLC patients and had limited benefits.^14-16

In this context, novel combination therapy approaches have attracted great interest, particularly the combination of ICIs with anti-angiogenic agents in conjunction with chemotherapy. Anti-angiogenic agents enhance the activity of the immune system by reducing immunosuppressive cells in the tumor microenvironment (TME) and normalize tumor vasculature, providing a better pathway for the action of ICIs.^17-19 Major clinical trials such as IMpower-150 and ORIENT-31 have demonstrated potential clinical benefits of combining ICIs with chemotherapy and anti-angiogenic therapy in advanced EGFR-mutated NSCLC patients who have failed EGFR-TKIs treatment.^20-25 Nevertheless, there are still some challenges associated with the extensive clinical application of the chemotherapy combined with immunotherapy and anti-angiogenic therapy regimen. A prospective study found that the feasibility and outcomes of the quadruple regimen were not as good as those of the IMpower-150 study, and its safety required active management.²⁶ Thus, while these studies offer hope, further clinical research is still warranted to validate the optimal treatment approach.

Despite progress in targeted and combination therapies, data specifically addressing treatment strategies after resistance to third-generation EGFR-TKIs remain limited. Accordingly, this study aimed to evaluate the efficacy of different post-resistance treatment approaches and to characterize tumor microenvironment features in patients with third-generation EGFR-TKI resistance, with the goal of informing more effective therapeutic decision-making.

Methods

Ethics Statement

The study protocol was reviewed and approved by the Institutional Review Board of the Medical Development Foundation. The reporting of this study conforms to the STROBE guidelines.²⁷ All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.The requirement for informed consent was waived by the Ethics Committee of the Medical Development Foundation due to the retrospective nature of the study and the use of de-identified patient data.

Patients

This retrospective study included patients with advanced lung adenocarcinoma treated at our Hospital between January 2018 and July 2023. Eligible patients were those who developed resistance to third-generation EGFR-TKIs. Clinical data, including gender and serum marker levels within one week prior to the initiation of the subsequent treatment regimen, were collected. Inclusion criteria were:(I) confirmed diagnosis of NSCLC with EGFR-sensitive mutations; (II) age between 18 and 85 years; (III) progression after first- or second-line third-generation EGFR-TKI treatment; (IV) presence of at least one measurable extracranial lesion; and (V) availability of complete clinical, imaging, and survival follow-up records. Exclusion criteria were as follows: (I) Unclear pathological diagnosis; (II) Received chemotherapy or immunotherapy prior to third-generation EGFR-TKI treatment; (III).

Patients with a complex next-line treatment profile after resistance to third-generation EGFR-TKIs, such as those receiving two or more antitumor treatment regimens in the form of local radiotherapy, double-dose targeted therapy, or chemotherapy combination therapy; (IV) Tissue transformation after resistance to third-generation EGFR-TKIs; (V) Patients with a combination of other primary tumors that have not yet been clinically cured; (VI) Patients with a combination of other serious organic diseases. This study was approved by the Ethics Committee of our Hospital and conducted in accordance with the Declaration of Helsinki. Informed consent was waived due to the retrospective nature of the analysis.The patient selection process is illustrated in Figure 1.

Figure 1.

Flowchart of patient selection

Immunohistochemistry (IHC)

Tumor tissue samples from 27 patients with EGFR mutation-positive advanced NSCLC resistant to third-generation EGFR-TKIs were analyzed. Formalin-fixed paraffin-embedded (FFPE) sections, 4-5 µm thick, were used for IHC. PD-L1 expression was measured using the PD-L1 Clone 22C3 pharmDx kit and the Dako Automated Link 48 platform. CD8 (clone 70306S, diluted at 1:100, CST, Massachusetts, USA), CCL5 (clone sc365826, diluted at 1:200, Santa Cruz, Texas, USA), Foxp3 (clone ab22510, diluted at 1:100, Abcam, Cambridge, UK) and Granzyme-B (clone ab255598, diluted at 1:3000, Abcam, Cambridge, UK) expression in T cells was evaluated.

PD-L1 expression was quantified using the tumor proportion score (TPS), defined as the percentage of tumor cells showing partial or complete membrane staining. A TPS of ≥1% was considered positive. Staining intensity and the percentage of positively stained cells were used to assess CD8, CCL5, Foxp3, and Granzyme B expression. Two pathologists independently reviewed the immunohistochemically stained sections.

Efficacy Assessment and Prognosis Analysis Indicators

Efficacy was assessed using RECIST 1.1 criteria, including complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD).²⁸ PFS is defined as progression-free survival from the start of next-line treatment after progression on a third-generation EGFR TKI to the development of resistance. OS is defined as the time from initiation of next-line treatment after progression on a third-generation EGFR-TKIs to death from any cause. ORR is defined as the sum of complete response rate and partial response rate, while DCR is defined as the sum of complete response rate, partial response rate and stable disease rate. Oligoprogression was defined as disease progression limited to up to three organs with five or fewer lesions after maintaining overall disease control with third-generation EGFR TKIs.²⁹ Based on progression patterns, patients were systematically stratified into oligoprogressive and systemic progression groups for subsequent analyses.Primary resistance was the lack of objective tumor response within three months of treatment with third-generation EGFR TKIs, with clear disease progression.³⁰ Follow-up was until November 3, 2023, or until death. Imaging data were assessed by an experienced thoracic oncologist.

Statistical Analysis

Statistical analyses were performed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA), X-tile version 3.6.1, and GraphPad Prism version 9.5.0.Continuous variables were summarized as medians with ranges, and categorical variables were presented as frequencies and percentages. Comparisons of categorical variables, including objective response rate (ORR) and disease control rate (DCR), were performed using the chi-square test or Fisher’s exact test, as appropriate.

Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan–Meier method, and survival differences between groups were compared using the log-rank test.

Univariate Cox proportional hazards regression analysis was conducted to evaluate potential prognostic factors associated with PFS and OS. Variables with a P value <0.10 in univariate analysis were subsequently entered into multivariate Cox regression models to identify independent prognostic factors, with hazard ratios (HRs) and 95% confidence intervals (CIs) reported.

Optimal cutoff values for continuous variables were determined using X-tile software. All statistical tests were two-sided, and a P value <0.05 was considered statistically significant.Cutoff values and scoring criteria were predefined to ensure reproducibility.

Given the retrospective and exploratory design of this study, no formal a priori sample size calculation was performed, and the sample size was determined by the number of eligible patients available during the study period.

Results

Patient Characteristics

After screening for eligibility and applying the predefined inclusion and exclusion criteria, the final study population was established (Figure 1). This study included 206 patients with a median follow-up of 14.5 months and a median age of 61 years (range: 33-84 years), of whom 38.35% (n=79) were male. The mutation types before initiating third-generation EGFR-TKI treatment were EGFR 19del (58.25%, n=120) and L858R (41.75%, n=86), with 51.3% (n=98) of patients concurrently positive for the EGFR T790M mutation. A total of 41.26% (n=85) of patients received third-generation EGFR-TKIs as first-line therapy, while the remaining 58.74% (n=121) received them as second-line therapy following the emergence of the T790M mutation after first- or second-generation EGFR-TKI treatment. Primary resistance to third-generation EGFR-TKIs was observed in 11.17% (n=23) of patients, and 21.36% (n=44) experienced oligoprogression (Table 1). As shown in Table 1, although no statistically significant differences were observed in measured baseline characteristics, potential imbalances in unmeasured factors and post-baseline variables cannot be excluded.

Table 1.

Baseline Clinical Characteristics of Overall Patients Stratified by Treatment Strategy After Third-Generation EGFR-TKI Resistance (n = 206)

Clinical Characteristic	Total (n= 206)	EGFR-TKIs+LR (n= 28)	EGFR-TKIs+DDT (n = 34)	Chemo+Ant (n = 98)	Chemo+ICIs (n = 46)	P value
Gender, n (%)						0.998
Male	79 (38.35)	10 (35.71)	11 (32.35)	38 (38.78)	20 (43.48)
Female	127 (61.65)	18 (64.29)	23 (67.65)	60 (61.22)	26 (56.52)
Age, n (%)						0.871
<52 years	33 (16.02)	4 (14.29)	3 (8.82)	18 (18.37)	8 (17.39)
≥52 years	173 (83.98)	24 (85.71)	31 (91.18)	80 (81.63)	38 (82.61)
Smoke, n (%)						0.109
Never	168 (81.55)	23 (82.14)	30 (88.24)	79 (80.61)	36 (78.26)
Current/former	38 (18.45)	5 (17.86)	4 (11.76)	19 (19.39)	10 (21.74)
ECOG, n (%)						0.642
0-2	197 (95.63)	27 (96.43)	28 (82.35)	96 (97.96)	46 (100.00)
≥2	9 (4.37)	1 (3.57)	6 (17.65)	2 (2.04)	0 (0.00)
Brain-metastasis, n (%)						0.741
No	134 (65.05)	14 (50.00)	19 (55.88)	66 (67.35)	35 (76.09)
Yes	72 (34.95)	14 (50.00)	15 (44.12)	32 (32.65)	11 (23.91)
Liver-metastasis, n (%)						0.343
No	190 (92.23)	28 (100.00)	29 (85.29)	90 (91.84)	43 (93.48)
Yes	16 (7.77)	0 (0.00)	5 (14.71)	8 (8.16)	3 (6.52)
Progression pattern, n (%)						<0.001
Oligoprogression	44 (21.36)	26 (100.0)	1 (2.9)	11 (10.1)	5 (13.9)
Systemic progression	162 (78.64)	0 (0.0)	33 (97.1)	98 (89.9)	31 (86.1)
EGFR, n (%)						0.452
19 Del	120 (58.25)	14 (50.00)	19 (55.88)	57 (58.16)	30 (65.22)
L858R	86 (41.75)	14 (50.00)	15 (44.12)	41 (41.84)	16 (34.78)
Line of EGFR-TKITherapy, n (%)						0.585
1	85 (41.26)	11 (39.29)	13 (38.24)	36 (36.73)	25 (54.35)
2	121 (58.74)	17 (60.71)	21 (61.76)	62 (63.27)	21 (45.65)
EGFR-TKI primary resistance, n (%)						0.264
No	183 (88.83)	26 (92.86)	34 (100.00)	84 (85.71)	39 (84.78)
Yes	23 (11.17)	2 (7.14)	0 (0.00)	14 (14.29)	7 (15.22)

EGFR: epidermal growth factor receptor; NSCLC: non-small cell lung cancer; TKIs: tyrosine kinase inhibitors; LR: local radiotherapy; DDT: doubling dose therapy; Chemo: chemotherapy; Ant: antiangiogenetic; ICIs: Immune checkpoint inhibitors;ex 19del: exon 19 deletion

Overall Efficacy of Subsequent Therapy After Resistance to Third-Generation EGFR-TKIs

Following resistance to third-generation EGFR-TKIs, 47.6% (n=98) of patients received chemo-antiangiogenesis therapy, 22.3% (n=46) received chemo-immunotherapy, 13.6% (n=28) underwent local radiotherapy combined with third-generation EGFR-TKIs, and 16.5% (n=34) received double-dose third-generation EGFR-TKIs. None of the patients achieved a CR, while 25.2% (n=52) achieved a PR, 64.6% (n=133) had SD, and 10.2% (n=21) had PD. The ORR was 25.2%, and the DCR was 89.8% (Figure 2). The mPFS was 6.7 months (Figure 3A), and the mOS was 30.1 months (Figure 3B).

Figure 2.

Evaluation of best efficacy stratified by treatment strategy after third-generation EGFR-TKIs resistance

Figure 3.

Clinical efficacy of NSCLC patients with EGFR-sensitive mutation after third-generation EGFR-TKIs resistance for each of the four treatment regimens. (A) Progression-free survival (PFS); (B) overall survival (OS)

Efficacy of EGFR-TKIs-Containing Therapies After Resistance

After progression on third-generation EGFR-TKIs, 30.1% (n=62) of patients continued receiving EGFR-TKI-containing therapies, including third-generation EGFR-TKIs combined with local radiotherapy and double-dose EGFR-TKIs. For those receiving double-dose third-generation EGFR-TKIs, the ORR was 8.8%, with a DCR of 82.4% and an mPFS of 5.50 months (95% CI: 3.20-7.80). Among patients with oligoprogression, those receiving third-generation EGFR-TKIs combined with local radiotherapy exhibited an ORR of 10.7%, a DCR of 82.1%, and an mPFS of 4.67 months. Notably, patients treated with third-generation EGFR-TKIs combined with local radiotherapy had a significantly prolonged mOS compared to those treated with chemo-antiangiogenesis or chemo-immunotherapy (37.23 vs. 27.77 vs. 25.83 months, p=0.045) (Figure 4A), though the mPFS did not differ significantly (4.67 vs. 8.00 vs. 5.70 months, p=0.067) (Figure 4B).

Figure 4.

Kaplan-Meier survival curves of PFS and OS of EGFR-mutated advanced NSCLC patients treated with different therapies after resistance to third-generation EGFR-TKIs. (A) PFS of patients for the third-generation EGFR-TKIs in combination with local radiotherapy, chemo-antiangiogenesis therapy and chemo-immunotherapy. (B) OS of patients for the third-generation EGFR-TKIs in combination with local radiotherapy, chemo-antiangiogenesis therapy and chemo-immunotherapy. (C) PFS of patients with EGFR 21 exon L858R mutation for EGFR TKI-containing and non-EGFR TKI-targeted therapies. (D) OS of patients with EGFR 21 exon L858R mutation for EGFR TKI-containing and non-EGFR TKI-targeted therapies

Patients with the L858R mutation treated with EGFR-TKI-containing therapies showed significantly poorer outcomes compared to those receiving non-EGFR-TKI-targeted therapies, with an mPFS of 3.53 vs. 8.00 months (p=0.001) (Figure 4C), an ORR of 10.5% vs. 40.3% (p=0.015), and a DCR of 27.4% vs. 69.2% (p=0.009), respectively. However, mOS did not significantly differ between the groups (27.77 vs. 37.23 months, p=0.981) (Figure 4D).

Efficacy of Chemotherapy Combination Therapy

Chemo-antiangiogenesis therapy showed a significantly longer mPFS compared to chemo-immunotherapy (8.00 vs. 5.70 months, p=0.033) (Figure 5A), though mOS was similar between the two groups (27.77 vs. 25.83 months, p=0.067) (Figure 5B). No significant differences in ORR and DCR were observed between the two groups (ORR: 34.3% vs. 26.7%, p=0.36; DCR: 93.9% vs. 91.1%, p=0.791).

Figure 5.

Kaplan-Meier survival curves of PFS and OS of EGFR-mutated advanced NSCLC patients treated with chemotherapy combination after resistance to third-generation EGFR-TKIs. (A) PFS of chemo-antiangiogenesis therapy and chemo-immunotherapy; (B) OS of chemo-antiangiogenesis therapy and chemo-immunotherapy; (C) PFS of anti-angiogenic regimen-containing and anti-angiogenic regimen-absent regimen; (D) OS of anti-angiogenic regimen-containing and anti-angiogenic regimen-absent regimen; (E) Immunotherapy-containing regimen and PFS without immunotherapy regimen; (F) OS with immunotherapy regimen and OS without immunotherapy regimen

Further analysis comparing patients who received anti-angiogenic therapy with those who did not revealed a higher mPFS in the anti-angiogenic therapy group (8.00 vs. 5.40 months, p=0.044) (Figure 5C) and a higher ORR (34.7% vs. 16.7%, p=0.003). Importantly, patients with oligoprogression were not systematically allocated to the non–anti-angiogenic therapy group. Treatment selection was based on clinical judgment and disease progression patterns, rather than anti-angiogenic use alone. However, there were no significant differences in mOS and DCR between the groups (mOS: 27.77 vs. 30.13 months, p=0.778; DCR: 93.9% vs. 86.1%, p=0.066).

Patients were also divided into two groups based on whether they received immunotherapy. Those who did not receive immunotherapy had a superior mOS compared to the immunotherapy group (32.33 vs. 25.83 months, p=0.012) (Figure 5D). However, mPFS was similar between the groups (5.80 vs. 6.97 months, p=0.431) (Figure 5E), with no significant differences in ORR (33.3% vs. 23.5%, p=0.219) or DCR (91.7% vs. 89.4%, p=0.918) (Figure 5F).

Survival Analysis Stratified by Progression Pattern

Kaplan–Meier survival analysis was performed to evaluate the impact of progression patterns on clinical outcomes. Patients with oligoprogression demonstrated significantly longer progression-free survival (PFS) and overall survival (OS) compared with those with systemic progression (Figure 6A–B).

Figure 6.

Kaplan–Meier survival curves stratified by progression pattern. (A) Progression-free survival (PFS) in patients with oligoprogression vs systemic progression. (B) Overall survival (OS) in patients with oligoprogression vs systemic progression

Tumor Microenvironment Markers After Resistance

Among the 27 analyzed tumor tissue specimens, 22.2% (n=6) were PD-L1 positive (TPS≥1%), and only 14.8% (n=4) demonstrated high PD-L1 expression (TPS≥50%). CD8 positivity was observed in 29.6% (n=8) of cases, CCL5 positivity in 11.1% (n=3), and Foxp3 positivity in 33.3% (n=9). None of the cases showed positive expression of Granzyme-B (Figure 7).

Figure 7.

Expression of PD-L1, CD8, CCL5, Foxp3 and Granzyme B in the tumor microenvironment (TME) of EGFR-sensitive mutant advanced NSCLC after resistance to third-generation EGFR TKIs. (A) Negative expression of PD-L1; (B) positive expression of PD-L1; (C) negative expression of CD8; (D) positive expression of CD8; (E) negative expression of CCL5; (F) positive expression of CCL5; (G) negative expression of Foxp3; (H)positive expression of Foxp3

Univariate and Multifactorial Analyses of PFS and OS

Univariate and multivariate analyses identified clinical parameters predictive of PFS and OS in patients with resistance to third-generation EGFR-TKIs. Table 2 shows the optimal cut-off values for each variable derived by the x-tile software. Multivariate analysis revealed that a platelet count <214×10^9/L and LDH <241 U/L were independently associated with longer PFS (HR=1.52, p=0.033; HR=1.62, p=0.024). In contrast, smoking history and a D-dimer level ≥0.44 were independently associated with shorter OS (HR=1.97, p=0.042; HR=2.01, p=0.027) (Table 3). These findings suggest that elevated platelet count, elevated LDH, elevated D-dimer, and a history of smoking are indicators of a poorer prognosis in NSCLC patients with EGFR-sensitive mutations after resistance to third-generation EGFR-TKIs.

Table 2.

Assignment of Values for Single Factor Analysis and Multifactor Analysis

values	Post-assignment group
Age	<52 years=0	≥52years=1
Gender	Male=1	Female=2
Smoking status	Never=0	Current or former=1
Line of EGFR-TKIs therapy	First line=1	Second line=2
Brain metastasis	No=0	Yes=1
Liver metastasis	No=0	Yes=1
Primary resistance	No=0	Yes=1
Monocyte count	<0.60×10⁹/L=0	≥0.60×10⁹/L =1
Neutrophil count	<5.10×10⁹/L=0	≥5.10×10⁹/L =1
Eosinophil count	<0.15×10⁹/L=0	≥0.15×10⁹/L =1
Lymphocyte count	<0.8×10⁹/L=0	≥0.8×10⁹/L =1
Platelet count	<214×10⁹/L=0	≥214×10⁹/L =1
LDH count	<241 U/L=0	≥241 U/L=1
D-dimer count	<0.44 mg/L=0	≥0.44 mg/L=1
PLR	<105=0	≥105=1
NLR	<3.2=0	≥3.2=1
LMR	<1.57=0	≥1.57=1

The assessment of the optimal cutoff values was conducted by X-tile software; LDH: lactate dehydrogenase; PLR: platelet-to-lymphocyte ratio; NLR: neutrophil-to-lymphocyte ratio; LMR: lymphocyte-to-monocyte ratio.

Table 3.

Univariate and Multivariate Analyses of Clinical Parameters of PFS and OS in the Overall Population After Third-Generation EGFR-TKI Resistance

Factors	PFS						OS
	Univariate analysis			Multivariate analysis			Univariate analysis			Multivariate analysis
	HR	95%CI	P Value	HR	95%CI	P Value	HR	95%CI	P Value	HR	95%CI	P Value
Age^a (<52/≥52 years)	0.77	0.50-1.18	0.225				0.97	0.44-2.17	0.949
Gender(male/female)	1.02	0.73-1.43	0.914				1.08	0.62-1.87	0.788
Smoking status (current or former/never)	1.03	0.65-1.63	0.888				1.93	1.03-3.63	0.037	1.97	1.02-3.78	0.042
Line of EGFR-TKIs therapy (1/2)	1.10	0.77-1.59	0.594				1.15	0.62-2.12	0.664
Brain metastasis(yes/no)	0.96	0.68-1.36	0.823				1.39	0.80-2.42	0.236
Liver metastasis(yes/no)	1.27	0.72-2.26	0.409				2.13	0.91-5.03	0.076	1.53	0.62-3.79	0.358
Primary resistance(yes/no)	1.29	0.74-2.24	0.368				0.97	0.39-2.45	0.952
Monocyte count^a (<0.60/≥0.60×10⁹/L)	1.32	0.81-2.15	0.261				1.64	0.74-3.65	0.222
Neutrophil count^a (<5.10/≥5.10×10⁹/L)	1.35	0.90-2.02	0.137				2.18	1.17-4.06	0.012	0.53	0.27-1.04	0.065
Eosinophil count^a (<0.15/≥0.15×10⁹/L)	0.76	0.50-1.17	0.213				0.90	0.43-1.83	0.749
Lymphocyte count^a (<0.80/≥0.80×10⁹/L)	0.77	0.49-1.20	0.243				0.70	0.35-1.39	0.304
Platelet count^a (<214/≥214×10⁹/L)	1.45	0.98-2.13	0.059	1.52	1.03-2.24	0.033	1.23	0.61-2.45	0.565
LDH^a (<241/≥241 U/L)	1.42	1.01-2.01	0.044	1.62	1.14-2.30	0.024	1.48	0.86-2.56	0.154
D-dimer count^a (<0.44/≥0.44 mg/L)	1.28	0.92-1.80	0.147				1.90	1.05-3.41	0.030	2.01	1.08-3.72	0.027
PLR^a(<105/≥105)	0.77	0.52-1.14	0.185				1.17	0.61-2-23	0.641
NLR^a(<3.2/≥3.2)	0.94	0.67-1.30	0.692				1.06	0.61-1.81	0.848
LMR^a(<1.57/≥1.57)	0.62	0.36-1.07	0.084	0.52	0.30-0.91	0.063	0.39	0.18-0.83	0.011	0.49	0.22-1.10	0.083

^athe assessment of the optimal cutoff values was conducted by X-tile software; PFS: progression-free survival; OS: overall survival; HR: hazard ratio; CI: confidence interval; EGFR: epidermal growth factor receptor; TKIs: tyrosine kinase inhibitors; LDH: lactate dehydrogenase; PLR: platelet-to-lymphocyte ratio; NLR: neutrophil-to-lymphocyte ratio; LMR: lymphocyte-to- monocyte ratio.

Discussion

The optimal next-line treatment for patients with EGFR-sensitive mutation-positive advanced NSCLC after resistance to third-generation EGFR-TKIs remains unclear. This study examined the clinical outcomes of various treatment strategies in a real-world Chinese cohort, providing valuable insights into their efficacy.Importantly, several methodological limitations inherent to this retrospective study should be acknowledged. Treatment allocation was not randomized but largely driven by clinical factors, particularly patterns of disease progression such as oligoprogression versus systemic progression. These factors are themselves strong prognostic determinants and represent post-baseline variables. Therefore, although multivariable Cox regression was performed, residual confounding, selection bias, and potential time-dependent bias cannot be fully excluded. Consequently, the observed associations should be interpreted with caution, and causal inferences cannot be established.³¹

In our cohort, 21.4% of patients experienced oligoprogression after third-generation EGFR-TKI treatment. Most followed the national comprehensive cancer network (NCCN) guidelines, which recommend continuing EGFR-TKIs combined with local therapy.²¹ Our results demonstrated that this approach significantly prolonged mOS compared to chemo-antiangiogenesis or chemo-immunotherapy. These findings align with previous studies suggesting a synergistic effect between EGFR-TKIs and local radiotherapy, which may enhance drug efficacy and improve survival outcomes by increasing the permeability of the blood-brain barrier and targeting residual disease.³⁰ However, it is important to note that oligoprogression inherently represents a more favorable prognosis compared to systemic progression, which could partly explain the improved outcomes observed in these patients. Some studies highlight that patient with oligoprogression had better survival rates when treated with local therapy in conjunction with ongoing EGFR-TKIs, further supporting this notion.^32-34

For patients with the EGFR exon21 L858R mutation, continuing EGFR-TKI-containing treatments, including local radiotherapy and double-dose third-generation EGFR-TKIs, did not lead to better clinical outcomes. This could be due to the lower differentiation and higher malignancy of tumor cells with this mutation, along with a higher prevalence of co-mutations, which may diminish the efficacy of EGFR-TKIs.^35-37 These findings suggest that chemotherapy-based regimens might be more appropriate for patients harboring this specific mutation, especially after resistance to third-generation EGFR-TKIs.

The TME in EGFR-positive NSCLC typically exhibits immune suppression, with low PD-L1 expression and reduced CD8+ T-cell infiltration.^38-40 Our immunohistochemical analysis confirmed this immune-desert phenotype in patients after resistance to third-generation EGFR-TKIs, highlighting the challenges in achieving effective responses to ICIs in this population. During EGFR-TKIs treatment, the TME of EGFR-mutant NSCLC may also undergo dynamic changes, with an increase in immune-suppressive cell numbers and a decrease in immune-activating cell numbers in resistant tumors.⁴¹ The observed low expression of CCL5, CD8, and Granzyme-B, along with high Foxp3 expression, further supports the immunosuppressive nature of the TME in these patients. Specifically, the loss of CCL5, CD8, and Granzyme-B may be associated with low immune infiltration, while Tregs expressing Foxp3 induce immune suppression in the tumor microenvironment.^41-44

Although chemo-antiangiogenesis therapy was associated with a longer progression-free survival, this benefit did not translate into an overall survival advantage. While post-progression treatments and crossover may partially explain this discrepancy, a more fundamental explanation may lie in indication bias. In real-world clinical practice, treatment selection is often closely associated with disease burden, progression pattern, and overall patient condition. Patients with less aggressive disease or more limited progression may have been more likely to receive anti-angiogenic therapy, which could inherently contribute to better PFS outcomes. Therefore, the apparent benefit of anti-angiogenesis therapy should be interpreted as an association rather than a direct therapeutic effect.

From a biological perspective, resistance to EGFR-TKIs has been associated with activation of alternative pathways, including VEGF signaling, which provides a rationale for anti-angiogenic therapy in this setting.^6,45,46

Finally, our analysis identified several prognostic factors associated with poor outcomes in patients with EGFR-sensitive mutant NSCLC after resistance to third-generation EGFR-TKIs, including elevated platelet count, LDH, D-dimer levels, and a history of smoking. Elevated platelet count may reflect tumor-induced platelet activation, contributing to tumor proliferation and metastasis.^47,48Increased LDH levels indicate higher tumor burden and aggressiveness, which are associated with poor outcomes.^49,50 Similarly, elevated D-dimer levels correlate with heightened coagulation activity, often seen in more invasive tumors, leading to worse prognosis.^51-53 These findings suggest that these biomarkers could serve as important indicators of prognosis in this patient population.

Our study, while offering significant insights, has several limitations. In addition, the lack of randomization and the influence of clinical decision-making on treatment selection introduce inherent indication bias, which may have affected the observed treatment outcomes.It is a single-center retrospective study, limiting its generalizability. Additionally, the lack of complete data on adverse events and the limited sample size and biomarker analysis restricts the depth of our findings. Larger prospective studies are needed to explore the dynamic changes in the immune microenvironment in this patient population and to validate our findings.

Conclusions

Chemo-antiangiogenesis therapy may represent a potential treatment option for patients with advanced NSCLC after resistance to third-generation EGFR-TKIs, particularly in terms of disease control. However, given the retrospective design and potential biases, these findings should be considered hypothesis-generating rather than practice-changing. The immunosuppressive TME may restrict the survival benefits of chemo-immunotherapy. For patients experiencing oligoprogression, continuation of third-generation EGFR-TKI therapy in conjunction with local radiotherapy offers a potential therapeutic advantage. Additionally, patients harboring EGFR exon 21 L858R mutations may derive greater benefit from a chemotherapy-based regimen.

Supplemental Material

Supplemental Material - Comparison of Post-Resistance Treatment Outcomes in EGFR-Mutated NSCLC Patients Following Third-Generation EGFR-TKI Therapy: A Retrospective Cohort Study

Supplemental Material for Comparison of Post-Resistance Treatment Outcomes in EGFR-Mutated NSCLC Patients Following Third-Generation EGFR-TKI Therapy: A Retrospective Cohort Study by Xinyue Li, Kaibo Ding, Dujiang Liu, Ruiqi Song, Zhongsheng Peng, Yanjun Xu, Yi Lu in Technology in Cancer Research & Treatment.

Footnotes

ORCID iD

Xinyue Li

Ethical Considerations

This study was approved by the Ethics Committee of the Beijing KeChuang Medical Development Foundation (Approval No. IRB-2022-268) in May 2022.

Consent to Participate

The requirement for informed consent was waived by the Institutional Review Board due to the retrospective nature of the study. All patient data were de-identified prior to analysis.

Author Contributions

Xinyue Li:Conceptualization,Data curation,Writing - Original draft preparation. Kaibo Ding:Data curation, Software. Dujiang Liu:Data curation,Software. Ruiqi Song:Formal analysis. Zhongsheng Peng:Methodology,Data curation,Formal analysis,Writing - Original draft preparation. Yanjun Xu:Conceptualization,Methodology,Resources,Funding acquisition,Writing - Review & Editing. Yi Lu:Funding acquisition,Writing - Review & Editing.

Funding

This study was supported by the Zhejiang Provincial Traditional Chinese Medicine Science and Technology Plan Project (No. 2026ZF26), the Zhejiang Cancer Foundation Research Fund (No. ZJCF-2025-1-ZD-01), and the Natural Science Foundation of Zhejiang Province, China (Grant No. LTGY23H160007).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Use of Artificial Intelligence

No generative artificial intelligence was used in the preparation of this manuscript.

Supplemental Material

Supplemental material for this article is available online.

Appendix

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