Prognostic implications of MDM2 expression in surgically resected epidermal growth factor receptor mutant lung cancer with pathological lymph node metastasis

Introduction

Epidermal growth factor receptor mutant (EGFRm) lung cancer accounts for about 30% to 50% of primary lung adenocarcinomas in Asia (1,2), with exon 21 L858R mutation (Ex21) and exon 19 deletion mutation (Ex19) accounting for approximately 90% of EGFRm lung cancer (3). The postoperative prognosis of pathological stage II–III EGFRm lung cancer patients was extremely poor prior to the ADAURA trial, with pathological stage II and IIIA having 5-year disease-free survival (DFS) rates of 46.6% and 17.4%, respectively (4). The ADAURA trial was a recent randomized phase III trial, which revealed that DFS was significantly prolonged with the use of adjuvant osimertinib versus placebo in patients with pathological stage II–IIIA EGFRm lung cancer [hazard ratio (HR), 0.23; 95% confidence interval (CI): 0.18–0.30; 4-year DFS, 70%] (5). The overall survival (OS) was also significantly prolonged with the use of osimertinib (HR, 0.49; 95% CI: 0.33–0.73; 5-year OS, 85%) (6).

However, osimertinib was associated with frequent side effects, such as diarrhea (47%), paronychia (27%), and dry skin (25%), as well as a 23% incidence of grade 3–5 adverse events in the ADAURA trial (5). Adjuvant osimertinib therapy has also been associated with higher healthcare costs with several studies demonstrating that osimertinib was less cost-effective than placebo (7,8). Therefore, adjuvant therapy with osimertinib should ideally be limited to patients with pN1–N2 EGFRm lung cancer who have a high likelihood of recurrence after curative surgery. However, no studies have identified factors that predict the recurrence of pN1–N2 EGFRm lung cancer, and new biomarkers are needed to prognosticate these patients.

MDM2 reportedly has prognostic implications in primary lung cancer (9-11). MDM2 is a p53-regulatory protein that binds to p53, causing its degradation through the proteasome pathway and inhibiting its role as a transcription factor (12,13). MDM2 protein also functions as a negative feedback regulator for p53; the increased MDM2 levels result in the binding and inactivation of p53 by directly blocking the p53 transactivation domain (14,15). MDM2 protein expression has been identified as a favorable prognostic factor for non-small cell lung cancer (NSCLC) (9,10), but some studies have shown that MDM2 overexpression is a poor prognostic factor in NSCLC (11,16). Nevertheless, there is still no consensus on the correlation between MDM2 protein expression and prognosis.

This study investigated the relationship between MDM2 protein expression and the prognosis of pN1–N2 EGFRm lung adenocarcinoma after resection. We present this article in accordance with the REMARK reporting (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-143/rc).

Methods

Patients

This study included 124 consecutive patients with pN1–N2 EGFRm lung adenocarcinoma (Ex19, n=61; Ex21, n=63) who underwent lobectomy or a greater degree of lung resection with lymph node dissection at Kanagawa Cancer Center between January 2010 and December 2020. Patients who received neoadjuvant therapy or postoperative EGFR-tyrosine kinase inhibitor therapy and those with minor EGFR mutations, were excluded. The study was approved by the institutional review board of Kanagawa Cancer Center (No. 2019 Eki-174) and individual consent for this analysis was waived due to the retrospective nature. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments

Pathological diagnosis and detection of Ex19 and Ex21

The pathological diagnosis was based on hematoxylin-eosin (HE) staining of formalin-fixed, paraffin-embedded tissue sections. Immunohistochemistry (IHC) staining for thyroid transcription factor 1 and Alcian blue staining were performed to confirm the diagnosis of adenocarcinoma. Synaptophysin, CD56, chromogranin A, p63, and p40 staining were also performed to differentiate between other histological types as needed. Vascular and pleural invasion were evaluated through Elastica Van Gieson staining, while lymphovascular invasion was evaluated using D2-40 as necessary. Several evaluations were performed on surgically resected specimens to identify the presence of EGFR exon 21 L858R mutation and EGFR Ex19 in lung adenocarcinomas, including the loop-hybrid mobility shift assay method (17), cycleave/fragment method (18), and the Cobas^®EGFR mutation test v2 (19).

Preparation of the tissue microarray (TMA) and IHC analyses of MDM2 and p53 protein expression

The most representative tumor area (i.e., containing tumor cells and excluding necrotic areas) was marked on the HE-stained slides. Tumor samples were collected using 2-mm cores and embedded in paraffin acceptor blocks to create TMA blocks, which were cut in 4-µm sections. These were subjected to IHC staining according to standard protocols using anti-MDM2 mouse monoclonal antibody (1:100, 33-7100, Invitrogen, Rockford, IL, USA). Antigen retrieval was performed by autoclaving at 110 ℃ for 15 minutes in 10 mM sodium citrate buffer (pH 6.0). Color development was performed using a fully automated HISTOSTAINER (Nichirei Biosciences Inc., Tokyo, Japan), Histofine Simple Stain MAX-PO kit, and 3,3-diaminobenzidine (Nichirei Biosciences Inc.) according to the manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin and coverslipped. Sections from a multitissue block served as positive and negative controls.

After more than 1,000 tumor cells were confirmed on the HE-stained TMA, MDM2 protein expression was determined. MDM2 staining ≥1% of the tumor nuclei was defined as positive expression (MDM2⁺), whereas staining <1% of the tumor nuclei was defined as negative expression (MDM2⁻) (Figure 1). For p53 staining using the mouse anti-p53 antibody (mouse monoclonal DO-7) (1:100, #M7001, Dako, Agilent, Santa Clara, CA, USA), positive expression (p53⁺) was defined as staining ≥1% of the tumor nuclei, whereas staining <1% was defined as negative expression (p53⁻).

Figure 1 MDM2 protein expression on immunohistochemistry (original magnification ×400). MDM2 expression was considered positive if ≥1% of the nuclei of the tumor cells in the TMA were stained. TMA, tissue microarray.

Adjuvant chemotherapy and follow-up for the patients after surgery

The use of adjuvant chemotherapy was defined as completing at least three courses of a platinum-based regimen. The platinum-based agents included cisplatin, carboplatin, and nedaplatin, whereas the combination drugs included vinorelbine, paclitaxel, irinotecan, and pemetrexed. The use of adjuvant chemotherapy and the specific regimen was on the clinician’s discretion, based on the patients’ background and overall condition.

Chest and abdominal computed tomography (CT) were performed postoperatively every 6–12 months for 5 years. Additionally, carcinoembryonic antigen (CEA) was evaluated postoperatively every 3–6 months for the first 3 years, then every 6–12 months thereafter up to 5 years. Positron emission tomography-CT or magnetic resonance imaging of the head was performed in cases wherein recurrence was suspected.

Primary/secondary outcomes of this study and word definitions

The primary outcome of the study was to compare the postoperative relapse-free survival (RFS) between patients with MDM2⁺ and MDM2⁻ with pN1–N2 EGFRm lung adenocarcinoma. The secondary outcome was to compare OS and recurrence patterns between the two groups. The prognosis and recurrence patterns between patients with MDM2⁺ and MDM2⁻ were also compared by EGFRm subtype (Ex19 and Ex21).

The recurrence site was defined as the recurrent metastatic organ found during surveillance at the time of initial recurrence. Intrathoracic recurrence includes recurrence at the hilar and mediastinal lymph nodes, lungs, and pleura. Distant metastatic recurrence includes recurrence at the central nervous system (CNS), bone, and abdominal organs (e.g., liver and adrenal gland). RFS was defined as the time from surgery to recurrence or death from any cause; patients without recurrence were censored at the last observation period. OS was defined as the time from surgery to all-cause death; patients were censored at the date of last follow-up.

Statistical analysis

Continuous variables between the two groups were compared using the Mann-Whitney U test. Categorical variables between the two groups were compared using Fisher’s exact test. The RFS and OS curves of patients with pN1–N2 EGFRm lung adenocarcinoma were determined using the Kaplan-Meier method. The RFS and OS between the MDM2⁺ and MDM2⁻ groups were compared using the log-rank test. Univariable and multivariable analyses for RFS and OS were performed using the Cox proportional hazards regression model with the following variables: age, sex (male vs. female), smoking history (present vs. absent), CEA level (≤10 vs. >10 ng/mL), pathological tumor size (cm), lymphatic vessel invasion (present vs. absent), blood vessel invasion (present vs. absent), pathological lymph node metastasis (pN1 vs. pN2), adjuvant chemotherapy (present vs. absent), p53 protein expression (negative vs. positive) and MDM2 protein expression (negative vs. positive). Multivariable analysis was performed with the significant variables (P<0.10) identified on univariable analysis. Gray’s test was used to analyze the cumulative incidence of recurrence. Associations between the hazard of recurrence and the variables above were estimated using the Fine and Gray competing-risk model. Statistical significance was set at P<0.05. EZR on R commander version 1.61 was used for all statistical analyses (20).

Results

Out of 124 patients with EGFRm lung adenocarcinoma, 56 patients (45.2%) were MDM2⁺ (Table 1). The patients with MDM2⁺ and MDM2⁻ had comparable clinicopathological backgrounds; however, the p53⁺ was more frequent in patients with MDM2⁺ than those with MDM2⁻ (83.9% vs. 67.6%, P=0.04, Table 1). The median follow-up time was 65.0 months. The RFS was significantly lower in patients with MDM2⁻ than those with MDM2⁺ (5-year RFS: 14.8% vs. 35.5%; median: 21.1 vs. 33.2 months, P=0.04; Figure 2A). The OS tended to be worse in patients with MDM2⁻, but this was not statistically significant (5-year OS: 61.0% vs. 79.8%; median: 78.1 vs. 98.5 months, P=0.17; Figure 2B).

Figure 2 Comparison of RFS (A) and OS (B) between the MDM2⁻ and MDM2⁺ groups among patients with stage pN1–N2 EGFRm lung adenocarcinoma. EGFRm, epidermal growth factor receptor mutant; OS, overall survival; RFS, relapse-free survival.

Multivariable analysis revealed the following independent prognostic factors for RFS: MDM2⁺ (HR, 0.64; 95% CI: 0.42–0.98; P=0.04), smoking history (HR, 1.74; 95% CI: 1.10–2.74; P=0.006), pathological tumor size (HR, 1.24; 95% CI: 1.06–1.44; P=0.006), and pN2 (HR, 2.19; 95% CI: 1.46–3.28; P<0.001) (Table 2). The independent prognostic factors for OS were pathological tumor size (HR, 1.21; 95% CI: 1.02–1.44; P=0.03) and pN2 (HR, 1.66; 95% CI: 1.01–2.71; P=0.044) (Table S1). In patients with Ex19 lung cancer, RFS and OS were comparable regardless of MDM2 expression (Figure 3A,3B). Among patients with Ex21 lung cancer, patients with MDM2⁺ had significantly better RFS (5-year RFS: 36.4% vs. 9.2%, P=0.005; Figure 3C) and OS (5-year OS: 81.8% vs. 39.3%, P=0.002; Figure 3D). Among patients with p53⁻, RFS was comparable regardless of MDM2 expression (Figure S1A). Among patients with p53⁺, patients with MDM2⁺ had significantly better RFS (5-year RFS: 40.3% vs. 14.8%, P=0.03; Figure S1B).

Figure 3 Comparison of relapse-free survival and overall survival among patients with Ex19 lung cancer (A,B) and among those with Ex21 lung cancer (C,D). OS, overall survival; RFS, relapse-free survival.

Among patients with Ex21 lung cancer, patients with MDM2⁺ were significantly older than those with MDM2⁻ (72 vs. 66 years, P=0.04), but the other clinicopathologic characteristics were comparable (Table S2). Multivariable analysis among patients with Ex21 lung cancer revealed the following independent prognostic factors for RFS: MDM2⁺ (HR, 0.43; 95% CI: 0.23–0.80; P=0.008), smoking history (HR, 2.03; 95% CI: 1.08–3.82; P=0.03), and pN2 (HR, 3.19; 95% CI: 1.62–6.29; P<0.001) (Table 3). The only independent prognostic factor for OS was MDM2⁺ (HR, 0.32; 95% CI: 0.15–0.67; P=0.003) (Table 3).

Among those with EGFRm lung adenocarcinoma, the cumulative incidence of overall recurrence tended to be higher in patients with MDM2⁻ (Figure 4A). Among patients with Ex19 lung cancer, the cumulative incidence of all types of recurrence was similar regardless of MDM2 expression (Figure S2A), but for patients with Ex21 lung cancer, this was significantly higher in patients with MDM2⁻ (Figure 4B).

Figure 4 Comparison of the cumulative incidence of overall recurrence between the MDM2⁻ and MDM2⁺ groups among patients with lung cancer patients with EGFRm (A) and those specifically with the Ex21 subtype (B). Comparison of the cumulative incidence of distant recurrence (C) and central nervous system recurrence (D) between the MDM2⁻ and MDM2⁺ groups among patients with Ex21 lung cancer. CNS, central nerve system; EGFRm, epidermal growth factor receptor mutant.

The cumulative incidence of distant recurrence was higher in patients with MDM2⁻ among those with Ex21 lung cancer (Figure 4C). The cumulative incidence of CNS recurrence (Figure 4D) and abdominal organ recurrence (Figure S2B) was higher in patients with MDM2⁻. The rates of intrathoracic recurrence, bone recurrence, and pleural recurrence were similar regardless of MDM2 expression (Figure S2C-S2E).

Multivariable analysis using competing-risk regression revealed that pN2 (HR, 3.29; 95% CI: 1.34–8.09; P=0.01) and MDM2 expression (HR, 0.42; 95% CI: 0.20–0.87; P=0.02) were associated with overall distant recurrence in patients with Ex21 lung cancer. Additionally, pathological tumor size (HR, 1.32; 95% CI: 1.13–1.54; P<0.001) and MDM2 expression (HR, 0.23, 95% CI: 0.07–0.80; P=0.02) were associated with CNS recurrence in patients with Ex21 lung cancer (Table 4).

Discussion

MDM2⁺ on IHC was identified as an independent favorable prognostic factor for RFS in patients with pN1–N2 EGFRm lung adenocarcinoma after curative surgery. Among patients with Ex21 lung cancer, MDM2 protein expression was a favorable prognostic factor for both RFS and OS, and it was also associated with a low cumulative incidence of distant recurrence, especially CNS recurrence, after curative surgery.

MDM2 regulates p53 through a variety of mechanisms. MDM2 is a regulatory protein comprising 491 amino acids, consisting of an N-terminal p53-binding domain, a nuclear localization signal (NLS), a nuclear export signal (NES), an acidic structural domain, a zinc finger structural domain, and a C-terminal RING-finger domain (21). The RING-finger domain of MDM2 functions as an E3 ubiquitin ligase for p53 in the nucleus, which causes the ubiquitination of p53, while its degradation is caused by the proteasome mechanism for cell survival (15,22-24). The NLS and NES of MDM2 transport p53 between the nucleus and cytoplasm (21,25,26). By preventing the interaction of p53 with coactivators, MDM2 inhibits the transcriptional activity of p53 (22,27,28). Meanwhile, the zinc finger structural domain of MDM2 inhibits p53 degradation (21).

The autoregulation of p53 is facilitated by modulations of MDM2 activity. Specifically, p53 activates MDM2 oncoprotein expression by binding to its promoter, and the increased level of MDM2 causes p53 to bind MDM2. Consequently, the p53 transactivation domain is directly blocked, and p53 is targeted for E3 ligase activity by the proteasome, ultimately resulting in p53 inactivation (13-15,29). This autoregulation of p53 via MDM2 helps regulate intracellular p53 levels and protects cells from the detrimental effects of high p53 concentrations (30). Additionally, p53 also negatively regulates EGFR function via miR-193a, while EGFR also negatively regulates miR-193a expression via specific EGFR-binding motifs. This double-negative feedback loop mechanism reportedly affects NSCLC tumorigenesis (31), and thus MDM2 may indirectly affect EGFR function.

MDM2 protein expression is positive in 33–51.5% of patients with primary lung cancer, and MDM2 is known to play an important role in tumorigenesis (10,32,33). In the present study, 45.2% of patients with EGFRm lung cancer had MDM2⁺, which is similar to previous reports. However, the prognostic significance of MDM2 expression in lung cancer after curative surgery is not well-understood. A few previous reports claim that MDM2 expression is associated with better prognosis in NSCLC (9,10). Ko et al. reported that patients expressing MDM2 mRNA had a longer median survival versus those that did not (50.9 vs. 21.7 months) (10). Higashiyama et al. reported that MDM2 protein expression was associated with better prognosis in stages I–IIIB of NSCLC (including 57.2% of stage I NSCLC) without p53 protein accumulation (<10%) (9). In contrast, our results revealed that p53 expression was more prevalent in MDM2⁺ lung cancers, and MDM2⁺ was associated with a better prognosis in p53⁺ lung cancers (9). Other studies suggest that the prognostic value of MDM2 expression extends to esophageal (32) and ovarian cancers (33) as well. In contrast, some reports found that MDM2 expression was not associated with prognosis in NSCLC (34,35). Another study reported that MDM2 expression was associated with worse survival in hepatocellular carcinoma, epithelial ovarian cancer, and breast cancer after surgical resection (36-38).

Ex21 and Ex19 cancer cells have structural differences, specifically in EGFR-tyrosine kinase (39), and significant differences have been reported across various EGFR mutations (40). Ex21 lung cancer cells have a high cell proliferation capacity owing to highly phosphorylated proteins like Shc (41). Furthermore, EGFR compound mutations are more frequent in these cells, especially compared to Ex19 lung cancer cells (19.5% vs. 4.7%) (42). These molecular genetic differences may contribute to the heterogeneity of EGFRm lung cancers, potentially affecting the prognostic implications of MDM2 protein expression in Ex21 lung cancers; however, the mechanism behind this remains unclear. One possible reason is that MDM2 protein expression in Ex21 tumor cells may result from the activation of the MDM2 promoter by a normal autoregulatory feedback loop caused by mutant p53, resulting in the suppression of mutant p53 activity to inhibit tumor cell growth (13,43). Moreover, specific splice variants such as MDM2-A that enhance the inhibitory effect on tumor cell proliferation may result in the favorable prognosis of patients with MDM2⁺ with Ex21 lung cancer (13,44). Nevertheless, further biomolecular studies are needed to elucidate the mechanisms behind the association of MDM2 protein expression with prognosis and metastasis in Ex21 lung cancer.

In the present study, patients with MDM2⁻ were significantly younger than those with MDM2⁺ and MDM2⁻ status predicted poor prognosis in Ex21 lung cancer (5-year RFS: 9.2%, 5-year OS: 39.3%) and a high incidence rate of CNS recurrence (5-year recurrence rate: 34.5%). The early onset of lung cancer is likely attributed to the high rates of carcinogenesis and potential proliferation of MDM2⁻ tumors. Moreover, these findings also highlight the importance of postoperative adjuvant therapy with osimertinib and postoperative CNS surveillance for patients with MDM2⁻ Ex21. However, patients with MDM2⁺ Ex21 had a remarkably favorable 5-year OS (81.8%), which was comparable to that of p-stage IA3 (8^th TNM) based on the International Association for the Study of Lung Cancer (IASLC) database (80%) (45) and p-stage II–III EGFRm lung cancer treated with osimertinib in the ADAURA trial (85%) (6). Moreover, patients with MDM2⁺ Ex21 may have less prognostic benefit from adjuvant osimertinib therapy; however, further clinical studies are needed to determine whether they should receive adjuvant osimertinib.

Only a few studies have described the predictors of CNS recurrence after lung cancer surgery. CEA, a non squamous histology, and metastatic lymph node metastasis (which occurs in ≥30%) were previously identified as risk factors for CNS recurrence in NSCLC after curative resection (46,47). Another study found that patients with EGFRm lung cancer (48), especially the Ex21 subtype, had a higher risk of CNS metastasis (49). However, no studies have explored the factors associated with brain metastasis after curative surgery for EGFRm lung cancer. In the present study, we found that MDM2⁻ predicts CNS recurrence after surgery for Ex21 lung cancer; however, further large-scale clinical analyses are needed to demonstrate the clinical utility of MDM2 protein expression.

This study has several limitations. First, this was a single-center retrospective study with a small sample size. The small sample size makes it difficult to draw definitive conclusions regarding the prognostic significance of MDM2 in Ex21 lung cancer. A prospective study with a larger sample size is needed to confirm our conclusions. Second, because MDM2 protein expression was analyzed using TMAs, we were unable to account for intratumoral heterogeneity in this study. Third, this study did not analyze TP53 mutations. Reportedly, these have potential prognostic value in NSCLC (50). However, it remains unclear whether these EGFRm subtypes are also associated with MDM2 expression. Fourth, the prognostic impact of MDM2 protein expression during the ADAURA era remains unclear. Further studies are needed to elucidate the prognostic impact of MDM2 protein expression in patients receiving adjuvant osimertinib.

Conclusions

MDM2⁺ was associated with favorable RFS and OS and a low cumulative incidence of distant recurrence, including CNS recurrence, among patients with pN1–N2 EGFRm lung adenocarcinoma, especially the Ex21 subtype, after curative surgery. Our findings suggest the potential of MDM2 as a novel biomarker that can be used to determine the need for adjuvant osimertinib therapy and for predicting CNS recurrence in Ex21 lung cancer.

Acknowledgments

We thank Mr. Nakamura Yoshiyasu and Ms. Yoshihara Mitsuyo (Kanagawa Cancer Center Research Institute) for their excellent technical assistance.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-143/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-143/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-143/prf

Funding: This study was funded by a grant from Kanagawa Cancer Foundation and JSPS KAKENHI (Grant No. JP21K1651 to T.I.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-143/coif). T.I. reports grant from Kanagawa Cancer Foundation and JSPS KAKENHI (Grant No. JP21K1651). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the institutional review board of Kanagawa Cancer Center (No. 2019 Eki-174) and individual consent for this analysis was waived due to the retrospective nature.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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