Exon-level alterations and PD-L1 expression identified by pretreatment NGS stratify survival in -mutant non-small cell lung cancer treated with first-line osimertinib.

Introduction
Non-small cell lung cancer (NSCLC) remains one of the leading causes of cancer-related deaths. Epidermal growth factor receptor (EGFR)-activating mutations are the most common driver oncogene detected in 30–40% of NSCLC patients (1). Tyrosine kinase inhibitors (TKIs) targeting EGFR sensitizing, T790M, and uncommon EGFR mutations have caused a pivotal change in the treatment landscape of NSCLC (2-4). Indeed, the FLAURA trial, a randomized phase III trial comparing osimertinib and standard first-line EGFR TKIs (erlotinib or gefitinib), demonstrated superior progression-free survival (PFS) and overall survival (OS) benefits following first-line osimertinib treatment (5). Subsequently, osimertinib has become the standard first-line treatment for patients with EGFR-sensitizing mutations. However, despite notable PFS benefits, individual patients have exhibited variable response durations to osimertinib (from 13.8 to 22.0 months) (5). Moreover, predictive biomarkers related to the response duration have yet to be elucidated.
Next-generation sequencing (NGS) in clinical oncology can be implemented to address various co-occurring genetic alterations in patients with documented driver oncogenes. Previous studies have suggested a potential association between TP53 mutations, RB1 mutations, and MDM2 amplification and poor survival outcomes in EGFR mutant NSCLC patients, which was detected by targeted panel sequencing (6,7). However, a comprehensive co-occurring genetic landscape profile in EGFR mutant patients treated with first-line osimertinib remains limited. Hence, this study aimed to investigate the co-occurring genetic alteration profiles assessed by NGS for patients treated using first-line osimertinib and the significance of the duration of response to osimertinib. We present this article in accordance with the REMARK reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-880/rc).

Methods

Study population and specimens
A total of 48 patients with advanced NSCLC were tested for clinically relevant genes by targeted NGS using tumor tissue samples acquired before administering Osimertinib between 2018 and 2022. Patient information, including sex, age, baseline EGFR mutation types, histological diagnoses, treatment histories, and treatment and survival outcomes, was retrieved from electronic medical records. Time to treatment discontinuation (TTD) and OS were examined. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. This study was conducted retrospectively and was approved by the Institutional Review Board of Asan Medical Center (IRB No. 2020-1204), with informed consent waived due to the retrospective nature.

Targeted NGS
DNA was extracted from previously collected formalin-fixed, paraffin-embedded (FFPE) tissue specimens. To determine the frequency of co-occurring genes in NSCLC patients with EGFR mutations and the predictive effect of co-mutations on the efficacy of osimertinib, genetic sequencing and mutation calling were performed using an in-house panel of the AMC, Seoul, Korea (OncoPanel AMC, versions 3 and 4), as previously described (8,9). The OncoPanel AMC version 3 (OP AMC v3) and version 4 (OP AMC v4) were conducted using the MiSeq platform (Illumina, San Diego, CA, USA) and captured 383 and 322 cancer-related genes, respectively (OP AMC v3: 199 genes for entire exons, 8 genes for partial introns, and 184 genes for hotspots; OP AMC v4: 225 genes for entire exons, 6 genes for partial introns, and 99 for hotspots).
The sequence mapping steps for OP AMC v3 and v4 were performed in accordance with a previously described method (10,11). Somatic variant calling for single-nucleotide variants and short indels was conducted using VarDict (12). Germline variants in candidates relating to somatic variants (found in ≥1% of samples) were filtered out using a common germline variants database (dbSNP build 141, gnomAD; common germline variants from 1,100 healthy Korean participants) (13). The normal panel was also used for variant calling. Tumor mutational burden (TMB) was calculated as the number of nonsynonymous alterations per megabase (Mb) in the examined genome.

Immunohistochemistry (IHC)
For the programmed death-ligand 1 (PD-L1) assay, FFPE tissue sections (4 µm thick) were dried at 60 ℃ for 30 minutes. The PharmDx assay (Dako, Carpinteria, CA, USA) involved staining using an anti-PD-L1 22C3 mouse monoclonal primary antibody. For the mesenchymal-epithelial transition factor (MET) assay, the 4 µm thick FFPE tissue sections were deparaffinized and rehydrated, and antigen retrieval was performed in citrate buffer (pH 6.1) at 95 ℃ for 40 minutes. The CONFIRM anti-total c-MET SP44 antibody (Ventana Medical Systems) was used for MET IHC staining. IHC data were categorized according to the following staining scores: 0, negative (no staining, or <50% of the tumor cells with any intensity); 1, weak (≥50% of the tumor cells with weak or higher staining, but <50% with moderate or higher intensity); 2, moderate (≥50% of the tumor cells with moderate or higher staining, but <50% with strong intensity); 3, strong (≥50% of the tumor cells staining with strong intensity) (14).

cBioPortal (RRID:SCR_014555) database analysis
Information regarding TP53 and EGFR alterations, the survival time, and live/dead status at the 5-year follow-up in NSCLC patients was downloaded from the cBioPortal (RRID:SCR_014555) using the cgdsr package (version 1.3.0) (15,16). The Memorial Sloan Kettering Cancer Center - Integrated Mutation Profiling of Actionable Cancer Targets (MSKCC-IMPACT) 2021 (17), Oncology Singapore (OncoSG) (18), The Cancer Genome Atlas (TCGA) (19), and National Cancer Institute (NCI) (20) datasets were selected. Among a total of 1,196 NSCLC samples, we identified 579 samples derived from 379 patients harboring EGFR mutations. For survival analysis, we excluded patients with mutations affecting multiple TP53 exons or those lacking either OS duration or survival status data. Consequently, a total of 359 EGFR-mutated patients were analyzed.

Statistical analysis
The Fisher’s exact test, or Wilcoxon rank-sum test, was performed as appropriate. TTD was defined as the date of osimertinib treatment started and the date of treatment discontinuation or death from any cause. OS was defined as the time from the start of osimertinib treatment to death from any cause. TTD and OS were evaluated using Kaplan-Meier methods and compared using the log-rank test. For analyses involving biomarkers with missing data (PD-L1 and MET IHC), patients were excluded only from the specific analyses requiring those markers but were included in all other analyses. Missing data were handled using complete-case analysis for each specific comparison. Multivariable Cox regression analyses were not performed due to the limited sample size and low number of events, which would risk model overfitting and unreliable estimates. Stratified analyses were conducted to examine the combined effects of TP53 mutations and PD-L1 expression. A P value <0.05 was considered statistically significant, and R (version 4.1.0) was used for all statistical analyses.

Results

Patient characteristics
We retrospectively identified 48 patients with EGFR-mutated NSCLC whose pretreatment tumors had been assessed using targeted gene panel NGS and who had undergone first-line osimertinib treatment for recurrent or initially metastatic lung adenocarcinoma between 2018 and 2022 (Table 1). Among these 48 patients, 22 (45.8%) were male, 34 (70.8%) were non-smokers, and osimertinib treatment was initiated at a median age of 62 years. At the time of data cutoff (31 March 2022), 14 (29.2%) patients had died; the median follow-up was 19.7 months. The median duration of osimertinib treatment was 11.1 months (range, 0.9–38.7 months). Detailed clinicopathological characteristics are shown in Table 1.

Landscape of genomic alterations
Regarding the founder EGFR mutation, 23 patients (47.9%) possessed the L858R point mutation, 22 (45.8%) patients harbored an exon 19 deletion, and three patients carried atypical mutations, such as L861Q, G719A/T790M, and L694_T698delinsQ/K754E (Figure 1 and Figure S1). T790M mutations that occurred de novo were detected in four patients: two patients with the L858R mutation, one patient with the exon 19 deletion, and one patient with the G719A mutation. A comparison was performed between the patients with the exon 19 deletion and those with the L858R mutation and identified a numerical trend that favored the exon 19 deletion compared with the L858R mutation in the TTD and OS {TTD: HR =2.31 [95% confidence interval (CI): 0.69–7.70]; P=0.17; OS: HR =2.39 (95% CI: 0.72–7.94); P=0.16} (Figure S2A,S2B).
In total, 47 patients (47/48, 97.9%) were found to have co-occurring genetic alterations (Figure 2). The median TMB was 12.5 mutations/Mb (range, 3.1–25.0 mutations/Mb). The most common co-mutated genes were TP53 (62.5%, 30/48), ATM (16.7%, 8/48), FLT1 (16.7%, 8/48), SLX4 (12.5%, 6/48), CTNNB1 (12.5%, 6/48), and BARD1 (12.5%, 6/48). A total of 31 TP53 alterations in 30 patients were evaluated in parallel with data analysis from TCGA database (21,22). In total, 26 mutations were identified in TP53 that affected the DNA-binding domain, and two mutations affected the proline-rich domain. Additionally, one truncating mutation (Q317*) and two splicing mutations (c.560-1G>T and c.920-1G>T) were observed (Figure 3). Baseline brain metastasis was present in 17 patients (35.4%), and the frequency of TP53 mutations (76.5% vs. 54.8%, P=0.21) and TP53 exon 5 mutations (35.3% vs. 9.7%, P=0.051) did not differ significantly between patients with and without brain metastasis. Among the four patients who had a post-treatment biopsy, one had the C797S mutation in the post-osimertinib specimens. The evident on-target or off-target resistance profiles were not identified in the remaining three patients.
Of the 48 patients, 6 patients had insufficient tissue samples for the PD-L1 assay, 28 (28/41, 68.3%) showed PD-L1 ≥1%, and 14 (14/41, 34.1%) had PD-L1 negative tumors. Furthermore, 5 of the 28 patients with PD-L1 ≥1% were strongly positive for PD-L1 (≥50%). In the MET IHC analysis, 22 patients had insufficient tissue for MET IHC, eight patients (8/25, 32.0%) were MET IHC 3+, and 18 patients (18/25, 72%) were MET IHC 0–2+ (Table 1). Analysis of NGS data revealed MET gene amplification in 3 patients (6.3%) at baseline. There was poor concordance between MET amplification detected by NGS and MET protein overexpression detected by IHC (concordance rate: 12.5%, 1 out of 8 MET IHC 3+ cases showed amplification).

Predictive and prognostic value of TP53 co-mutation and other genomic alterations
Analysis of pre-osimertinib alterations associated with TTD and OS revealed a trend towards worse outcomes in patients with TP53 alterations (Figure 4A,4B). Given that different types of TP53 genetic mutations tend to have different effects on the functionality of the protein (7,21-25), we classified TP53 mutations in other sites. The various TP53 mutations detected in our study and their distribution are shown in Figure 3 and Table S1. We speculated that mutations on different TP53 exons might show distinct prognostic or predictive effects (21,23,26,27); each mutation was analyzed separately according to the exon. TP53 exon 5 mutations (18.8%, 9/48) showed an association with a shorter TTD when compared with TP53 wild-type (WT) controls (HR =7.67; 95% CI: 1.77–33.32; P=0.007; Figure 4C). Other TP53 exon mutations (including exons 4, 6, 7, 8, and 9; n=21, 43.8%) did not show significant associations with TTD compared to TP53 WT (HR =1.87; 95% CI: 0.47–7.48; P=0.38; Figure 4C). Similarly, in the prognostic analysis, we found that TP53 exon 5 mutations were significantly associated with poor survival (HR =9.29; 95% CI: 2.06–41.86; P=0.004; Figure 4D), while other TP53 exon mutations showed no significant association with OS (HR =1.63; 95% CI: 0.41–6.60; P=0.49; Figure 4D).
We next sought to understand how TP53 alterations promote resistance by evaluating whether TP53 alterations are associated with specific mutagenesis patterns or increased genomic instability, as these processes might act as potential mechanisms in osimertinib resistance. The TTD for patients with TP53 exon 5 mutations and PD-L1 expression remained on osimertinib treatment for a significantly shorter time than for those without TP53 exon 5 mutations and PD-L1 expression (HR =9.27; 95% CI: 1.42–60.34; P=0.02; Figure 4E). Furthermore, patients with TP53 exon 5 mutations and elevated PD-L1 expression showed a significantly reduced OS compared to those lacking these mutations and PD-L1 expression (HR =14.54; 95% CI: 2.03–104.32; P=0.008; Figure 4F). Predictive and prognostic values of various alterations were also analyzed, including MET overexpression, TMB status and PD-L1. As demonstrated in Figure S3, MET overexpression, TMB and PD-L1 expression alone did not exhibit significant predictive and prognostic values.

Validation of clinical value of TP53 exon 5 co-mutations in patients with EGFR mutations
We analyzed a publicly available database to validate the prognostic value of TP53 exon 5 mutations identified from the current analysis in independent cohorts. NSCLC sequencing and clinical metadata from four cohorts were obtained from cBioPortal (15,16), MSKCC-IMPACT 2021 (17), OncoSG (18), TCGA (19), and NCI (20). Among 359 NSCLC patients with a mutation in EGFR, TP53 mutations were discovered in 49.6% (178/359) of the patients, of which 41.8% (150/359) possessed mutations in the DNA-binding domain (exons 5–8). Thus, based on the data collected from these four cohorts, mutations in TP53 presented poor prognostic value (HR =2.20; 95% CI: 1.54–3.13; P<0.001; Figure 5A). Then, we subdivided TP53 mutations into exons. The common TP53 mutation site was in exon 5, accounting for about 13.1% (47/359) of the observed mutations. Consistent with our findings, the patients with the TP53 exon 5 mutations had the worst 5-year survival compared with those possessing the WT TP53 (HR =3.18; 95% CI: 1.97–5.12; P<0.001; Figure 5B).

Discussion
We explored the use of targeted NGS in NSCLC patients with EGFR mutations who had received first-line osimertinib treatment in a clinical setting. TP53 co-mutations were the most commonly associated finding with inferior TTD and OS in contrast to TP53 WT in EGFR-mutant NSCLC patients; in particular, mutations in exon 5 of the TP53 gene, which encodes a critical region of the DNA-binding domain. In addition, patients with a TP53 exon 5 mutation and PD-L1 22C3 ≥50% showed the worst TTD and OS compared with TP53 WT and low PD-L1 expression. Considering that first-line osimertinib is the standard treatment in patients with EGFR mutants and NGS is an essential test in stage IV NSCLC patients, our results suggest that TP53 exon 5 mutations alone or in combination with PD-L1 expression might be a stratifying risk factor for predicting the response and survival durations.
TP53 alterations have been suggested to contribute to poor outcomes in EGFR TKI-treated patients (6,23-25,28-31). Although an underlying prognostic effect cannot be excluded, we observed the poor predictive effects of TP53 exon 5 alterations, whereby TP53 exon 5 alterations were associated with decreased TTD. Concurrent TP53 exon 5 mutations may alter clinical outcomes; however, the mechanism through which TP53 mutations are associated with worse outcomes remains largely unknown. We observed that TP53 alterations were associated with increased TMB (mean: 14.01 vs. 10.15; Wilcoxon: P=0.006; Figure S4A), suggesting increased genomic instability. Exon-level analysis demonstrated that TP53 exon 5 mutations were associated with a greater increase in TMB compared with mutations in other TP53 exons (Figure S4B). Overall, tumors with concomitant mutations in EGFR and TP53 may bypass EGFR as a target and activate alternative pathways. Thus, there is a need for combination therapeutic strategies to prolong the duration of initial benefit from EGFR-targeted therapy. Further studies are needed to determine whether comprehensive co-occurring profiling adds clinically relevant information to patients treated with osimertinib.
The varied prognostic effects of TP53 mutations in NSCLC patients have also been reported previously (7), and those conflicting findings were probably due to the heterogeneity of TP53. Moreover, findings remain ambiguous as to whether co-mutations in exon levels within the TP53 gene, and the observed consequences of these mutations are associated with the clinical outcomes. In contrast to previous reports of poor clinical outcomes for mutations in exon 8 of TP53 (21,32-34), no associations between TP53 exon 8 alterations and outcome were observed (Figure S5A,S5B). Instead, consistent with Liu et al. (27), we observed that TP53 mutations in exon 5 were associated with a worse outcome than those in TP53 WT. To validate these findings, we further explored the association of exon-level events with OS using data from the cBioPortal database (n=359). In this larger independent cohort, alterations in DNA-binding domain exons (exons 5–8, analyzed collectively) showed reduced OS compared to TP53 WT (Figure 5A). Among the modifications in exon 5–8 (Figure 5A,5B), Importantly, among DNA-binding domain mutations, alterations in exon 5, presented the worse outcomes (HR =3.18; 95% CI: 1.97–5.12; P<0.001, Figure 5B), suggesting a possible relationship between TP53 exon 5 alterations and outcome (Figures 4C,4D,5A,5B). Particularly, we observed that a combination of TP53 exon 5 alterations and PD-L1 expression was associated with decreased TTD and OS (Figure 4E,4F). Given the potential impacts of specific TP53 alterations on the osimertinib therapeutic response, future studies are needed to determine the effect of exon-level mutations on the outcomes of patients. Also, functional studies investigating the molecular mechanisms by which TP53 exon 5 mutations confer resistance to osimertinib would provide valuable insights for developing combination therapeutic strategies targeting these specific alterations. Although pretreatment PD-L1 expression and MET overexpression per se did not provide any predictive value in patients with EGFR mutants treated using osimertinib, their potential as predictive biomarkers warrants renewed focus in the era of first-line osimertinib therapy. Previous literature has also reported that PD-L1 expression did not affect the osimertinib treatment outcome (35). However, this study did not consider the impact of PD-L1 expression in the context of coexisting genetic alterations, nor did it stratify patients based on strong PD-L1 expression (≥50%). Other studies have demonstrated that high PD-L1 expression is associated with poor outcomes in patients treated with EGFR-TKIs (36-38), including osimertinib (39). Furthermore, a strong association between high PD-L1 expression and TP53 mutations has been reported (40,41). In this study, while PD-L1 expression alone did not significantly impact treatment outcomes, the coexistence of a TP53 mutation and high PD-L1 expression was associated with worse TTD and OS. These findings suggest that patients harboring both TP53 mutations and high PD-L1 expression may represent a subgroup with suboptimal PFS, underscoring the need for alternative therapeutic strategies in the context of EGFR-sensitizing mutations. Moreover, secondary EGFR mutations, such as C797X and G724S—“on-target resistance”—were commonly acquired resistance mechanisms of second-line osimertinib treatment, which was administered only to patients with an acquired EGFR T790M mutation. In contrast, “off-target resistance” accounts for up to 30–50% of cases in which first-line osimertinib was administered. Among off-target resistance, MET amplification is the most common mechanism (42). With the TATTON trial and the upcoming interim analysis of the SAFFRON trial, combining osimertinib and MET targeting agents can be considered a treatment option for patients who experienced disease progression following the first-line osimertinib treatment (43). In the TATTON trial, the combination treatment of savolitinib and osimertinib demonstrated meaningful antitumor activity in patients with EGFR mutants, MET-amplified, or overexpressed NSCLCs that had progressed after prior EGFR-TKI therapy, showing objective response rates up to 67% and a median PFS of up to 11.1 months.
Our study highlights the additional prognostic value of NGS beyond solely performing PCR analysis for EGFR, even when EGFR activating mutations are identified through PCR. A secondary analysis from the MARIPOSA trial demonstrated that TP53 mutations were associated with worse PFS. However, amivantamab treatment in combination with lazertinib significantly improved PFS compared to osimertinb (HR =0.72; 95% CI: 0.58–0.90; P=0.004). These findings suggest that NGS results provide further benefits in guiding treatment decisions (44). Furthermore, upfront platinum combination therapy could be a viable consideration for patients with EGFR and TP53 co-mutations, as explored by FLAURA2. Recently, FLAURA2 showed that the addition of a platinum-pemetrexed alongside osimertinib treatment promoted significantly increased PFS than osimertinib alone (HR for disease progression or death =0.62; 95% CI: 0.49–0.79; P<0.001) (45). Notably, following the FLAURA2 results, the FDA approved osimertinib treatment in combination with platinum-pemetrexed; however, the clinical question of who would benefit from adding intravenous chemotherapy every three weeks will be raised. Furthermore, given that adjuvant osimertinib treatment for patients with an EGFR-sensitizing mutation has become the standard of care based on the ADAURA trial, post-hoc analyses or real-world evidence studies are necessary to evaluate the predictive value of TP53 mutations in this setting.
These findings should be interpreted in the context of the study’s exploratory nature and potential limitations. First, we retrospectively extracted patient-level data. The clinical status of enrolled patients was heterogeneous, and the results of this study may have limited generalizability to all patients. Second, the small sample size precluded drawing firm conclusions. Given that PCR analysis is widely used in routine molecular diagnostics in NSCLC and that additional NGS is rarely implemented when activating mutations are confirmed by EGFR PCR, a pragmatic number of patients was included in this study Also, patients were selected from a consecutive cohort of EGFR-mutant NSCLC patients who received NGS testing at our center, thereby minimizing potential selection bias. Additionally, considering the turnaround time for NGS results is typically 4 to 6 weeks, prospectively collecting a comparable number of NGS data in patients with EGFR mutations is likely to remain challenging. Third, the relatively small sample size and limited number of events precluded us from performing multivariable analyses, which made it challenging to fully adjust for potential confounders. However, to mitigate this limitation, we provided stratified analyses examining the combined impact of TP53 mutations and PD-L1 expression, and validated our findings in independent, publicly available cohorts with larger sample sizes.

Conclusions
In conclusion, TP53 exon 5 alterations were associated with poor clinical outcomes of first-line osimertinib treatment of NSCLC. Our analyses further suggest that the patients possessing combined TP53 exon 5 alterations and PD-L1 expression may result in highly unfavorable outcomes and require intensified combination strategies. Additional studies with larger sample sizes are essential to validate these findings.

Supplementary
The article’s supplementary files as