PD-L1 expression in primary lung tumor as a superior predictive biomarker to metastatic lymph nodes for first-line immunochemotherapy in advanced KRAS-mutant non-small cell lung cancer

Introduction

KRAS is among the most commonly mutated oncogenes in cancer and its mutations have been detected in ~30% of Caucasian and ~15% of Asian non-small cell lung cancer (NSCLC) patients (1). The most common KRAS mutation subtypes include G12C, G12D, and G12V, with G12C accounting for ~40% of all mutations (2,3). Notably, targeted drugs such as sotorasib and adagrasib have been approved as second-line interventions for KRAS G12C-mutant NSCLC. However, their clinical efficacy is suboptimal (4,5). Furthermore, despite the lack of KRAS mutation-specific stratifications in clinical trials evaluating immune checkpoint inhibitors (ICIs) for NSCLC, ICI monotherapy or immunochemotherapy remains the first-line therapeutic intervention for advanced KRAS-mutant NSCLC (6).

Patients with KRAS-mutant NSCLC often present with higher tumor immunogenicity and increased T-cell infiltration, suggesting that they may benefit from immunotherapy (7). Notably, programmed death-ligand 1 (PD-L1) expression is often employed as a biomarker for predicting ICI response (8). However, few studies have explored the precise correlation between PD-L1 expression and the efficacy of first-line immunotherapy in KRAS mutant NSCLC patients—a phenomenon that also remains controversial. For instance, while some studies linked higher PD-L1 expression with improved immunotherapy outcomes (9,10), others found no significant association between PD-L1 expression and therapeutic efficacy (11-14).

Several factors could contribute to the aforementioned inconsistencies in research findings regarding the correlation between PD-L1 expression and immunotherapy response in KRAS-mutant NSCLC. The presence of co-mutations is one of the potential explanations. Co-mutations in STK11 or KEAP1 were linked with poor responses to immunotherapy (15), whereas co-mutations involving TP53 were linked to more favorable outcomes (16). Different KRAS mutation subtypes could also exert distinct influences on immunotherapy efficacy (17,18). The spatial heterogeneity of PD-L1 expression across different tumor sites could be the other contributing factor.

Discrepancies in PD-L1 expression between primary lung and metastatic lesions were previously reported in NSCLC (19,20). Some studies reported that PD-L1 expression could be higher at metastatic sites—including lymph nodes, pleural effusions, soft tissues, and adrenal glands—than in primary lung lesions (20). Nonetheless, PD-L1 expression levels at various anatomical sites may not equally predict immunotherapy response (21).

Overall, the relationship between site-specific PD-L1 expression heterogeneity and the clinical efficacy of first-line immunochemotherapy in patients with advanced KRAS-mutant NSCLC is not completely understood. Consequently, we aimed to investigate the predictive value of PD-L1 expression heterogeneity—on the efficacy of first-line immunochemotherapy in primary lung lesions and metastatic lymph nodes using data from patients with advanced KRAS-mutant NSCLC. We present this article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-817/rc).

Methods

Patients

We conducted a retrospective review of the medical records of 4,213 patients diagnosed with KRAS-mutant NSCLC at the Shanghai Chest Hospital and Fujian Cancer Hospital between January 2018 and December 2022. The inclusion criteria were as follows: (I) patients with histologically confirmed stage IIIB–IV NSCLC according to the International Association for the Study of Lung Cancer’s (IASLC) tumor-node-metastasis (TNM) classification, 8th edition; (II) patients with confirmed KRAS mutations at initial diagnosis via an amplification refractory mutation system (ARMS) or next-generation sequencing (NGS); (III) patients whose tumor tissues were assessed for PD-L1 expression at diagnosis; (IV) patients whose pathological specimens obtained at diagnosis from either primary lung lesions or thoracic metastatic lymph nodes (stations 1–11, excluding stations 5, 6, 8, and 9) were available; and (V) patients who received first-line immunochemotherapy (ICI combined with chemotherapy).

Conversely, the exclusion criteria were: (I) patients who received non-immunotherapy-based chemotherapy as the first-line intervention; (II) patients who did not undergo PD-L1 testing; (III) patients who underwent PD-L1 testing for specimens from non-pulmonary primary lesions or non-metastatic lymph nodes; (IV) patients with squamous cell carcinoma (SCC) or adenosquamous carcinoma (ASC) histology; and (V) patients with incomplete clinical data.

Following the application of these criteria, 302 patients with advanced KRAS-mutant NSCLC who received first-line immunochemotherapy were included in the final analysis (Figure 1).

Figure 1 Flow chart of enrolled patients. PD-L1, programmed death-ligand 1.

This retrospective study was approved by the Shanghai Chest Hospital’s Ethics Committee (No. IS25076) and the Fujian Cancer Hospital’s Ethics Committee (No. K2025-187-01). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The patients’ right to informed consent was waived due to the retrospective nature of the study.

Clinical assessments

All patients were staged according to the IASLC’s TNM classification system, 8th edition. before the initiation of first-line immunochemotherapy. During treatment, patients underwent thoracic computed tomography (CT) and abdominal ultrasonography every 2–3 months to monitor therapeutic response. Brain magnetic resonance imaging (MRI) and bone emission computed tomography (ECT) were performed when necessary. Follow-up continued until disease progression, treatment discontinuation, or the last follow-up date, whichever occurred first.

Tumor response to treatment was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (version 1.1); classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). The primary endpoints of the study included progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR). Due to the limited maturity of overall survival (OS) data—as most patients began immunochemotherapy treatments within the past 4 years—OS was not included as an endpoint. The final follow-up date was December 30, 2024.

Gene testing and the PD-L1 expression

All patients underwent tissue biopsy prior to the initiation of treatment. KRAS mutation status was determined using ARMS or NGS. For NGS pre-processing, TrimmomaticPE was employed to remove adapter sequences, and low-quality sequences or those shorter than 50 bp were filtered out (parameter: ILLUMINACLIP:adapter.fa:2:30:10:1:true, TRAILING: 20, SLIDINGWINDOW: 30:25, MINLEN: 50). The Illumina TruSeq Amplicon Cancer Panel kit (Burning Rock Company, Guangzhou, China) was utilized for library preparation, and sequencing was conducted using the MiSeq instrument. The variant allele frequency (VAF) distribution of KRAS p.G12C mutations identified by NGS is presented in Table S1. PD-L1 expression in tumor cells was evaluated by two experienced pathologists using the Dako PD-L1 immunohistochemistry (IHC) 22C3 pharmDx assay (Shanghai Chest Hospital) and the Dako PD-L1 IHC 28-8 pharmDx assay (Fujian Cancer Hospital), with grading based on the tumor proportion score (TPS). Scores were categorized as follows: PD-L1 <1% (negative), PD-L1 =1–49% (moderate-to-low expression), and PD-L1 ≥50% (high expression).

Statistical analysis

Statistical analyses were conducted using SPSS software, version 24.0 (IBM, Armonk, NY, USA). Age, treated as a continuous variable, was expressed as a median, while categorical variables were presented as counts (N) and percentages (%). PFS was defined as the interval from the initiation of first-line immunochemotherapy to disease progression, regimen change, or the last follow-up, whichever occurred first. ORR was defined as the proportion of patients achieving CR or PR. DCR was defined as the proportion of evaluable patients who achieved CR, PR, or SD. Comparisons of continuous variables between groups were performed using non-parametric tests, while comparisons of categorical variables utilized Pearson’s Chi-squared test or Fisher’s exact test, as appropriate. Kaplan-Meier curves were generated to estimate PFS, and univariate and multivariate Cox proportional hazard regression analyses were carried out to identify correlating factors. All tests were two-sided, with results considered statistically significant at P<0.05.

Results

Baseline characteristics

This study involved 302 patients diagnosed with advanced NSCLC harboring KRAS mutations who received first-line immunochemotherapy. The majority of participants were male (84.4%, 255/302) with a median age of 65.7 years. Smoking status was reported in 57.9% of patients (175/302). At diagnosis, the distribution of disease stages among patients was as follows: 13.6% (41/302) in stage IIIB, 3.0% (9/302) in stage IIIC, and 83.4% (252/302) in stage IV. Additionally, brain, bone, and liver metastases were observed in 18.9% (57/302), 38.7% (117/302), and 9.3% (28/302) of the patients, respectively (Table 1, Figure 2). Table S2 summarizes the clinical landscape of all KRAS-mutant NSCLC patients by PD-L1 status, and Table S3 presents the corresponding data for the KRAS p.G12C subset.

Figure 2 Clinical and genomic features associated with first-line immunochemotherapy efficacy in all patients. Brain/bone/liver: indicates metastasis status “yes”. BOR, best overall response; PD, progressive disease; PD-L1, programmed death-ligand 1; PR, partial response; PFS, progression-free survival; SD, stable disease.

Predictive factors for first-line immunochemotherapy efficacy in advanced KRAS-mutant NSCLC

The median follow-up duration was 29.5 months, during which 226 patients (74.8%) experienced disease progression. The median PFS duration for the entire cohort was 9.07 months [95% confidence interval (CI): 7.80–10.34], with ORR and DCR values of 35.4% and 88.4%, respectively.

Patients were stratified into three subgroups based on PD-L1 expression: PD-L1 <1% (n=103, 34.1%), PD-L1 =1–49% (n=102, 33.8%), and PD-L1 ≥50% (n=97, 32.1%). The ORR values for these groups were 20.4%, 34.3%, and 52.6% (P<0.001), while the DCR values were 86.4%, 86.3%, and 92.8% (P=0.26), respectively. Although the groups exhibited significant differences in ORR, the differences in DCR were not statistically significant.

Kaplan-Meier analysis indicated that the median PFS durations were 7.27 months (95% CI: 5.51–9.03), 8.30 months (95% CI: 6.67–9.93), and 15.00 months (95% CI: 11.00–19.00) for the PD-L1 <1%, PD-L1 =1–49%, and PD-L1 ≥50% groups, respectively, with statistically significant differences observed (P<0.001) (Figure 3, see 3A). This finding suggests a positive correlation between PD-L1 expression and clinical benefit from first-line immunochemotherapy in patients with advanced KRAS-mutant NSCLC.

Figure 3 Kaplan-Meier estimates of PFS. (A) PFS of all patients. The median PFS was statistically significantly different among the three groups. (B) PFS in the primary lung lesions group. The median PFS was statistically significantly different among the three groups. (C) PFS in the lymph nodes group. No statistically significant differences in median PFS were observed across the three groups. (D) PFS in the TP53 co-mutation group. No statistically significant differences in median PFS were observed between the two groups. (E) PFS in the STK11 co-mutation group. The median PFS was statistically significantly different between the two groups. (F) PFS in the ATM co-mutation group. No statistically significant differences in median PFS were observed between the two groups. *, statistical significance at P<0.05. PD-L1, programmed death-ligand 1; PFS, progression-free survival.

Univariate Cox regression analysis identified bone metastases [hazard ratio (HR) =1.341; 95% CI: 1.028–1.749; P=0.03] as a factor associated with shorter PFS. Furthermore, compared to the PD-L1 <1% group, both the PD-L1 =1–49% (HR =0.704; 95% CI: 0.515–0.964; P=0.03) and PD-L1 ≥50% (HR =0.523; 95% CI: 0.378–0.725; P<0.001) groups were significantly correlated with longer PFS (Figure 4).

Figure 4 Univariate Cox regression analysis of PFS. *, statistical significance at P<0.05. CI, confidence interval; HR, hazard ratio; PD-L1, programmed death-ligand 1; PFS, progression-free survival.

Multivariate analysis further confirmed that bone metastasis (HR =1.378; 95% CI: 1.056–1.800; P=0.02), PD-L1 =1–49% (HR =0.685; 95% CI: 0.500–0.939; P=0.02), and PD-L1 ≥50% (HR =0.513; 95% CI: 0.370–0.711; P<0.001) were independent prognostic predictors of PFS, with PD-L1 <1% serving as the reference category.

PD-L1 expression in primary lung lesions is correlated with improved PFS following first-line immunochemotherapy in advanced KRAS-mutant NSCLC

Patients were initially stratified into two subgroups based on the anatomical site of pathological sampling at diagnosis: primary lung lesions (n=211) and metastatic lymph nodes (n=91) (Table 2).

Within the primary lung lesions group (n=211), PD-L1 expression was further categorized into three subgroups: PD-L1 <1% (n=83), PD-L1 =1–49% (n=69), and PD-L1 ≥50% (n=59), which differed significantly in ORR, with values of 20.5%, 30.4%, and 52.5%, respectively (P<0.001). Conversely, the corresponding DCR values were 86.7%, 89.9%, and 91.5%, respectively, although without statistical significance (P=0.65) (Table 3; Figure 5).

Figure 5 The response rate of different groups. (A) ORR for all patients, patients in the primary lung lesions group, and those in the lymph nodes group. The ORR was statistically significant within both the all patients and the primary lung lesions group. (B) DCR for all patients, patients in the primary lung lesions group, and those in the lymph nodes group. No statistically significant difference was observed among the three groups. DCR, disease control rate; LN, lymph node; ORR, objective response rate; PD-L1, programmed death-ligand 1.

Following first-line immunochemotherapy, the median PFS durations were 7.23 months (95% CI: 5.14–9.33), 8.30 months (95% CI: 6.49–11.12), and 15.03 months (95% CI: 11.47–18.59) for the PD-L1 <1%, PD-L1 =1–49%, and PD-L1 ≥50% groups, respectively, attaining statistical significance (P=0.001). This observation indicates a trend toward prolonged PFS with increasing PD-L1 expression (Figure 3, see 3B). Pairwise comparisons further revealed significant PFS differences between the PD-L1 <1% and PD-L1 ≥50% groups (P<0.001).

Univariate Cox regression analysis indicated that within the primary lung lesions group, patients with PD-L1 ≥50% experienced significantly longer PFS compared to those with PD-L1 <1% (HR =0.484; 95% CI: 0.324–0.722; P<0.001) (Table 4).

Collectively, these findings suggest that higher PD-L1 expression in primary lung lesions is positively correlated with improved clinical outcomes from first-line immunochemotherapy in advanced KRAS-mutant NSCLC patients.

PD-L1 expression in metastatic lymph nodes is not associated with PFS benefit from first-line immunochemotherapy in advanced KRAS-mutant NSCLC

In the metastatic lymph nodes group, 91 patients were further stratified based on PD-L1 expression into three subgroups: PD-L1 <1% (n=20), PD-L1 =1–49% (n=33), and PD-L1 ≥50% (n=38). The ORR values for these subgroups were 20.0%, 42.4%, and 52.6%, respectively (P=0.057). The corresponding DCR values were 85.0%, 78.8%, and 94.7%, respectively (P=0.13). Notably, there were no statistically significant differences in ORR and DCR values among the three groups (Table 5; Figure 5).

Following first-line immunochemotherapy, the median PFS durations were 8.20 months (95% CI: 4.92–11.48), 9.50 months (95% CI: 4.01–15.00), and 11.80 months (95% CI: 7.72–15.88) for the PD-L1 <1%, PD-L1 =1–49%, and PD-L1 ≥50% groups, respectively, with no statistical significance (P=0.17) (Figure 3, see 3C). Univariate Cox regression analysis further indicated no significant correlation between PD-L1 expression in metastatic lymph nodes and PFS (Table 4). To assess the robustness of this finding, we conducted a post-hoc power analysis for the comparison between the PD-L1 ≥50% and <1% groups (HR =0.558). The estimated power was approximately 46%, suggesting that the non-significant result may have been influenced by limited statistical power.

These findings collectively imply that PD-L1 expression in metastatic lymph nodes may not serve as a reliable predictor of the efficacy of first-line immunochemotherapy in advanced KRAS-mutant NSCLC patients.

Association between co-mutations, PD-L1 expression, and treatment with first-line immunochemotherapy

Of the 302 patients with KRAS-mutant NSCLC, 257 underwent NGS testing at initial diagnosis. Among these, 118 (45.9%), 38 (14.8%), and 28 (10.9%) exhibited TP53, STK11, and ATM co-mutations, respectively, while other co-mutations accounted for <10%.

In the cohort with TP53 co-mutations, 26.3%, 35.6%, and 38.1% were categorized into the PD-L1 <1%, PD-L1 =1–49%, and PD-L1 ≥50% groups, respectively. Conversely, 65.8%, 26.3%, and 7.9% of patients with STK11 co-mutations were found in the PD-L1 <1%, PD-L1 =1–49%, and PD-L1 ≥50% groups, respectively. Finally, 28.6%, 46.4%, and 25.0% of patients with ATM co-mutations fell into the PD-L1 <1%, PD-L1 =1–49%, and PD-L1 ≥50% groups, respectively. Notably, TP53 (P=0.08) and ATM (P=0.35) co-mutations did not significantly influence PD-L1 expression. In contrast, STK11 co-mutations were significantly associated with lower PD-L1 expression (P<0.001) (Table 6).

Among the patients who received first-line immunochemotherapy, those with TP53 (9.80 vs. 8.97 months, P=0.56) or ATM (11.07 vs. 9.07 months, P=0.31) co-mutations demonstrated longer median PFS durations compared to those without these co-mutations, although this difference was not statistically significant. Conversely, patients with STK11 co-mutations exhibited a trend toward shorter median PFS durations compared to those without STK11 co-mutations (5.27 vs. 9.93 months, P=0.01), with a statistically significant difference observed (Figure 3, see 3D-3F).

Univariate Cox regression analysis indicated that patients with TP53 (HR =0.919; 95% CI: 0.693–1.220; P=0.56) and ATM (HR =0.772; 95% CI: 0.469–1.270; P=0.31) co-mutations who received first-line immunochemotherapy demonstrated a trend toward prolonged PFS compared to patients without these co-mutations, although this difference did not reach statistical significance. Conversely, patients with STK11 co-mutations exhibited a trend toward shorter PFS relative to those without STK11 co-mutations (HR =1.615; 95% CI: 1.110–2.351; P=0.01), achieving statistical significance (Figure 6, see 6A).

Figure 6 Univariate Cox regression analysis of co-mutations, G12C and non-G12C. (A) Univariate Cox regression analysis of the relationship between co-mutations and PFS. (B) Univariate Cox regression analysis of the relationship between PD-L1 expression in different lesions and G12C PFS. (C) Univariate Cox regression analysis of the relationship between PD-L1 expression in different lesions and non-G12C PFS. *, statistical significance at P<0.05. CI, confidence interval; HR, hazard ratio; PD-L1, programmed death-ligand 1; PFS, progression-free survival.

Impact of KRAS mutation subtypes on the efficacy of first-line immunochemotherapy

In this study, the G12C mutation was identified as the most prevalent KRAS mutation (34.1%), followed by G12V (18.5%), G12D (17.2%), and G12A (10.6%). The remaining mutation subtypes each accounted for <5% (Figure 7, see 7A).

Figure 7 KRAS mutation subtypes. (A) Proportional distribution diagram of KRAS mutation subtypes in all patients. (B) PFS in the G12C and non-G12 groups. No statistically significant differences in median PFS were observed between the two groups. PFS, progression-free survival.

Patients were subsequently classified into two groups: those with the KRAS G12C mutation (n=103) and those with non-G12C mutations (n=199). Although patients with KRAS G12C mutation NSCLC exhibited a longer median PFS following first-line immunochemotherapy compared to those with non-G12C mutations (10.50 vs. 8.83 months, P=0.24), this difference was not statistically significant (Table 7; Figure 7, see 7B).

Additionally, we compared PD-L1 expression levels between the KRAS G12C and non-G12C groups, revealing significantly higher expression in the G12C group (P=0.02). The KRAS G12C mutation group also showed a significantly greater proportion of patients with high PD-L1 expression (42.7% vs. 26.6%, P=0.005) compared to the non-G12C mutation group. However, no significant differences were observed in the proportions of patients with negative (P=0.19) or moderate-to-low (P=0.14) PD-L1 expression between the two groups (Table 7). Univariate Cox regression analysis in the KRAS G12C subgroup (Figure 6, see 6B) demonstrated that PD-L1 expression, whether evaluated in primary lung lesions or metastatic lymph nodes, did not significantly stratify PFS outcomes. In contrast, within the KRAS non-G12C subgroup (Figure 6, see 6C), higher PD-L1 expression in primary lung lesions was significantly correlated with longer PFS (HR =0.402; 95% CI: 0.239–0.678; P=0.001), while PD-L1 expression in metastatic lymph nodes showed no correlation with PFS. These findings suggest that PD-L1 heterogeneity is more pronounced in the non-G12C subgroup.

Discussion

This study investigated the spatial heterogeneity of PD-L1 expression in advanced NSCLC with KRAS mutations, as well as its predictive value for the efficacy of first-line immunochemotherapy. In primary lung lesions, PD-L1 expression was positively correlated with PFS benefit from first-line immunochemotherapy. On the other hand, PD-L1 expression levels in metastatic lymph nodes demonstrated no predictive significance—a phenomenon that was more pronounced in patients with KRAS non-G12C mutations.

Among genetic alterations in NSCLC, KRAS mutations represent a notably distinct category. Despite the availability of targeted therapies for these mutations, they have demonstrated a suboptimal overall efficacy and are presently approved only for second-line treatment (4,5). Notably, NSCLC patients with negative driver mutations received interventions such as ICI monotherapy or chemo-immunotherapy regimen as first-line approaches similar to patients with KRAS-mutant. Additionally, compared to KRAS wild-type NSCLC patients, KRAS-mutant NSCLC patients have a higher PD-L1 expression (22). Furthermore, patients with KRAS mutations are more likely to derive greater benefit from immunotherapy (23-26).

The mechanisms underlying PD-L1 upregulation in NSCLC patients with different genetic mutations are multifaceted. For instance, in EGFR-mutant and ALK-translocated NSCLC, PD-L1 is upregulated via PI3K/AKT signaling pathway activation (27,28). Meanwhile, oncogenic RAS signaling upregulates PD-L1 expression via a mechanism involving AU-rich element-binding protein tristetraprolin (TTP)-mediated changes in mRNA stability (29). Besides pathway-mediated transcriptional regulation, the tumor immune microenvironment (TIME) could also influence PD-L1 expression (30,31). Notably, TIME differences between primary lung lesions and metastatic lymph nodes are well-documented (32-34), potentially explaining the differential regulatory mechanisms of PD-L1 expression. Compared to primary lung lesions, metastatic lymph nodes are enriched with tumor-infiltrating lymphocytes (TILs) and regulatory T cells (Tregs) (35), which could secrete substantial amounts of IFN-γ and TGF-β, potentially enhancing PD-L1 expression in tumor cells (36,37). Nonetheless, such microenvironment-driven PD-L1 upregulation mechanisms could only reflect localized immunosuppression rather than an intrinsic tumor cell escape avenue. Conversely, PD-L1 expression in primary lung lesions correlates more with KRAS-driven autoregulatory mechanisms. Discrepancies in the KRAS mutational status between the primary and metastatic lesions were also reported, with primary lesions exhibiting a higher mutation frequency (38,39). Therefore, compared to those in metastatic lesions, PD-L1 expressions in primary lesions could more accurately reflect KRAS-driven tumor biology, highlighting PD-L1’s direct and autonomous regulatory role via the KRAS signaling pathway. This phenomenon can potentially explain the differential predictive value of PD-L1 expression in primary lung lesions against metastatic lymph nodes for immunotherapy efficacy. Overall, PD-L1 expression levels in primary lung lesions in KRAS-mutant NSCLC patients could more reliably predict first-line immunotherapy efficacy.

Through stratified analysis of KRAS mutation subtypes, we further established that PD-L1 expression in metastatic lymph nodes was not significantly correlated with PFS after first-line immunochemotherapy, regardless of whether the mutation was G12C or non-G12C. Conversely, in the non-G12C subgroup, higher PD-L1 expression in primary lung lesions correlated positively with prolonged PFS, a trend that was not observed in the G12C subgroup. This discrepancy could be attributed to the different regulatory mechanisms of PD-L1 expression across various KRAS mutation subtypes in NSCLC. For instance, KRAS G12V activation was reported to promote reactive oxygen species (ROS) generation and induce fibroblast growth factor receptor 1 (FGFR1) expression, ultimately leading to significant PD-L1 upregulation (40). On the other hand, KRAS G12D mutations suppressed PD-L1 expression via the P70S6K/PI3K/AKT axis (41). Additionally, compared to KRAS G12D-mutant NSCLC, KRAS G12C-mutant NSCLC exhibited higher levels of ERK1/2 phosphorylation (p-ERK1/2) (42), with p-ERK signaling driving PD-L1 upregulation in lung adenocarcinoma (22). These findings suggest that distinct KRAS subtypes could activate different signaling cascades, regulating PD-L1 expression and ultimately influencing its predictive value for immunotherapy. Potential differences in PD-L1’s predictive efficacy for immunotherapy may exist between the KRAS G12C and non-G12C mutations in primary lung lesions.

In KRAS non-G12C mutant NSCLC, we found that PD-L1 expression in primary lung lesions could predict immunotherapy efficacy, a correlation that was absent in G12C-mutant cases. These findings suggest that both the spatial heterogeneity of PD-L1 and the characterization of KRAS mutation subtypes should be considered when assessing PD-L1 expression as a predictive marker for immunotherapy. However, these analyses were based on relatively small sample sizes, and the statistical power of the subgroups was limited. Therefore, the results should be interpreted with caution and validated in larger cohorts.

Although in our study both PD-L1 expression and KRAS mutation status were evaluated using tissue specimens, liquid biopsy is considered a feasible alternative when tumor samples are limited (43). KRAS alterations can be reliably detected through circulating tumor DNA (ctDNA) assays (44), while PD-L1 expression can be explored using circulating tumor cells (CTCs) or exosome-based approaches (45,46). Nevertheless, liquid biopsy assays have several key challenges, including the need for extremely high sensitivity to detect the small fraction of ctDNA within total cell-free DNA (cfDNA), the risk of false negatives at low tumor burden, and the lack of standardized protocols across platforms (47,48). Therefore, tissue biopsy remains the gold standard, with liquid biopsy serving as a complementary tool that may become increasingly valuable in future clinical practice.

Despite its valuable insights, there are several notable limitations in this study. First, its retrospective nature led to inherent biases that might have affected the accuracy of the results. Second, the marked disparity in sample sizes between the primary lung lesion group and the metastatic lymph node group may have compromised the statistical robustness of our analyses. Specifically, a post-hoc power analysis for the metastatic lymph node cohort (TPS ≥50% vs. <1%) yielded an estimated power of approximately 46%, indicating that the lack of statistical significance should be interpreted cautiously, as it likely reflects limited sample size and inadequate statistical power rather than a genuine absence of predictive association. Third, two different PD-L1 IHC assays (22C3 and 28-8) were employed in different institutions. Although previous studies have reported high concordance between these assays (49), and the distribution of patients tested with each was comparable in our cohorts, inter-assay variability cannot be entirely excluded. Future prospective studies using a standardized assay platform are advocated to enhance the reliability of such analyses. Finally, the low number of mortality events at the time of analysis precluded a meaningful OS evaluation. To further validate the clinical relevance of PD-L1 expression at different tumour sites in predicting response to first-line immunochemotherapy in advanced KRAS-mutant NSCLC patients, additional large-scale, prospective studies will be required in the future.

Conclusions

Our findings revealed that PD-L1 expression in primary lung lesions in advanced KRAS-mutant NSCLC patients may serve as a predictive biomarker for the clinical efficacy of first-line immunochemotherapy. Conversely, PD-L1 expression in metastatic lymph nodes demonstrated no predictive value—a phenomenon that was more pronounced in patients with KRAS non-G12C mutations.

Acknowledgments

The authors thank all team members and participants from the Shanghai Chest Hospital and the Fujian Cancer Hospital.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-817/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-817/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-817/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-817/coif). The 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. This retrospective study was approved by the Shanghai Chest Hospital’s Ethics Committee (No. IS25076) and the Fujian Cancer Hospital’s Ethics Committee (No. K2025-187-01). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The patients’ right to informed consent was waived due to the retrospective nature of the study.

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/.

References

1. Oya Y, Imaizumi K, Mitsudomi T. The next-generation KRAS inhibitors…What comes after sotorasib and adagrasib? Lung Cancer 2024;194:107886. [Crossref] [PubMed]
2. Ricciuti B, Alessi JV, Elkrief A, et al. Dissecting the clinicopathologic, genomic, and immunophenotypic correlates of KRAS(G12D)-mutated non-small-cell lung cancer. Ann Oncol 2022;33:1029-40. [Crossref] [PubMed]
3. Lim TKH, Skoulidis F, Kerr KM, et al. KRAS G12C in advanced NSCLC: Prevalence, co-mutations, and testing. Lung Cancer 2023;184:107293. [Crossref] [PubMed]
4. de Langen AJ, Johnson ML, Mazieres J, et al. Sotorasib versus docetaxel for previously treated non-small-cell lung cancer with KRAS(G12C) mutation: a randomised, open-label, phase 3 trial. Lancet 2023;401:733-46. [Crossref] [PubMed]
5. Jänne PA, Riely GJ, Gadgeel SM, et al. Adagrasib in Non-Small-Cell Lung Cancer Harboring a KRAS(G12C) Mutation. N Engl J Med 2022;387:120-31. [Crossref] [PubMed]
6. Reck M, Carbone DP, Garassino M, et al. Targeting KRAS in non-small-cell lung cancer: recent progress and new approaches. Ann Oncol 2021;32:1101-10. [Crossref] [PubMed]
7. Liu C, Zheng S, Jin R, et al. The superior efficacy of anti-PD-1/PD-L1 immunotherapy in KRAS-mutant non-small cell lung cancer that correlates with an inflammatory phenotype and increased immunogenicity. Cancer Lett 2020;470:95-105. [Crossref] [PubMed]
8. Mok TSK, Wu YL, Kudaba I, et al. Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial. Lancet 2019;393:1819-30. [Crossref] [PubMed]
9. Falk AT, Yazbeck N, Guibert N, et al. Effect of mutant variants of the KRAS gene on PD-L1 expression and on the immune microenvironment and association with clinical outcome in lung adenocarcinoma patients. Lung Cancer 2018;121:70-5. [Crossref] [PubMed]
10. Sebastian M, Eberhardt WEE, Hoffknecht P, et al. KRAS G12C-mutated advanced non-small cell lung cancer: A real-world cohort from the German prospective, observational, nation-wide CRISP Registry (AIO-TRK-0315). Lung Cancer 2021;154:51-61. [Crossref] [PubMed]
11. Jeanson A, Tomasini P, Souquet-Bressand M, et al. Efficacy of Immune Checkpoint Inhibitors in KRAS-Mutant Non-Small Cell Lung Cancer (NSCLC). J Thorac Oncol 2019;14:1095-101. [Crossref] [PubMed]
12. Tsiouda T, Domvri K, Boutsikou E, et al. Prognostic Value of KRAS Mutations in Relation to PDL1 Expression and Immunotherapy Treatment in Adenocarcinoma and Squamous Cell Carcinoma Patients: A Greek Cohort Study. J Pers Med 2024;14:457. [Crossref] [PubMed]
13. Qiang H, Wang Y, Zhang Y, et al. Efficacy of first-line chemotherapy combined with immunotherapy or anti-angiogenic therapy in advanced KRAS-mutant non-small cell lung cancer. Transl Oncol 2025;53:102317. [Crossref] [PubMed]
14. Fancelli S, Caliman E, Mazzoni F, et al. KRAS G12 isoforms exert influence over up-front treatments: A retrospective, multicenter, Italian analysis of the impact of first-line immune checkpoint inhibitors in an NSCLC real-life population. Front Oncol 2022;12:968064. [Crossref] [PubMed]
15. Ricciuti B, Arbour KC, Lin JJ, et al. Diminished Efficacy of Programmed Death-(Ligand)1 Inhibition in STK11- and KEAP1-Mutant Lung Adenocarcinoma Is Affected by KRAS Mutation Status. J Thorac Oncol 2022;17:399-410. [Crossref] [PubMed]
16. Budczies J, Romanovsky E, Kirchner M, et al. KRAS and TP53 co-mutation predicts benefit of immune checkpoint blockade in lung adenocarcinoma. Br J Cancer 2024;131:524-33. [Crossref] [PubMed]
17. Zhao R, Shu Y, Xu W, et al. The efficacy of immunotherapy in non-small cell lung cancer with KRAS mutation: a systematic review and meta-analysis. Cancer Cell Int 2024;24:361. [Crossref] [PubMed]
18. Shahnam A, Davis A, Brown LJ, et al. Real-World outcomes of Non-Small cell lung cancer patients harbouring KRAS G12C and KRAS G12D mutations. Lung Cancer 2025;201:108421. [Crossref] [PubMed]
19. Pinato DJ, Shiner RJ, White SD, et al. Intra-tumoral heterogeneity in the expression of programmed-death (PD) ligands in isogeneic primary and metastatic lung cancer: Implications for immunotherapy. Oncoimmunology 2016;5:e1213934. [Crossref] [PubMed]
20. Moutafi MK, Tao W, Huang R, et al. Comparison of programmed death-ligand 1 protein expression between primary and metastatic lesions in patients with lung cancer. J Immunother Cancer 2021;9:e002230. [Crossref] [PubMed]
21. Hong L, Negrao MV, Dibaj SS, et al. Programmed Death-Ligand 1 Heterogeneity and Its Impact on Benefit From Immune Checkpoint Inhibitors in NSCLC. J Thorac Oncol 2020;15:1449-59. [Crossref] [PubMed]
22. Chen N, Fang W, Lin Z, et al. KRAS mutation-induced upregulation of PD-L1 mediates immune escape in human lung adenocarcinoma. Cancer Immunol Immunother 2017;66:1175-87. [Crossref] [PubMed]
23. Landre T, Justeau G, Assié JB, et al. Anti-PD-(L)1 for KRAS-mutant advanced non-small-cell lung cancers: a meta-analysis of randomized-controlled trials. Cancer Immunol Immunother 2022;71:719-26. [Crossref] [PubMed]
24. Uehara Y, Watanabe K, Hakozaki T, et al. Efficacy of first-line immune checkpoint inhibitors in patients with advanced NSCLC with KRAS, MET, FGFR, RET, BRAF, and HER2 alterations. Thorac Cancer 2022;13:1703-11. [Crossref] [PubMed]
25. Sun L, Hsu M, Cohen RB, et al. Association Between KRAS Variant Status and Outcomes With First-line Immune Checkpoint Inhibitor-Based Therapy in Patients With Advanced Non-Small-Cell Lung Cancer. JAMA Oncol 2021;7:937-9. [Crossref] [PubMed]
26. Pathak GP, Shah R, Castonguay M, et al. Temporal Effect on PD-L1 Detection and Novel Insights Into Its Clinical Implications in Non-Small Cell Lung Cancer. Cancer Med 2024;13:e70262. [Crossref] [PubMed]
27. Guo R, Li Y, Wang Z, et al. Hypoxia-inducible factor-1α and nuclear factor-κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non-small-cell lung cancer cells. Cancer Sci 2019;110:1665-75. [Crossref] [PubMed]
28. Ma L, Lv J, Dong Y, et al. PD-L1 Expression and Its Regulation in Lung Adenocarcinoma with ALK Translocation. Interdiscip Sci 2019;11:266-72. [Crossref] [PubMed]
29. Coelho MA, de Carné Trécesson S, Rana S, et al. Oncogenic RAS Signaling Promotes Tumor Immunoresistance by Stabilizing PD-L1 mRNA. Immunity 2017;47:1083-1099.e6. [Crossref] [PubMed]
30. Huynh TG, Morales-Oyarvide V, Campo MJ, et al. Programmed Cell Death Ligand 1 Expression in Resected Lung Adenocarcinomas: Association with Immune Microenvironment. J Thorac Oncol 2016;11:1869-78. [Crossref] [PubMed]
31. Zhang L, Chen Y, Wang H, et al. Massive PD-L1 and CD8 double positive TILs characterize an immunosuppressive microenvironment with high mutational burden in lung cancer. J Immunother Cancer 2021;9:e002356. [Crossref] [PubMed]
32. Haragan A, Field JK, Davies MPA, et al. Heterogeneity of PD-L1 expression in non-small cell lung cancer: Implications for specimen sampling in predicting treatment response. Lung Cancer 2019;134:79-84. [Crossref] [PubMed]
33. Reticker-Flynn NE, Zhang W, Belk JA, et al. Lymph node colonization induces tumor-immune tolerance to promote distant metastasis. Cell 2022;185:1924-1942.e23. [Crossref] [PubMed]
34. Basto PA, Reticker-Flynn NE. Interrogating the roles of lymph node metastasis in systemic immune surveillance. Clin Exp Metastasis 2024;41:351-9. [Crossref] [PubMed]
35. Schneider T, Kimpfler S, Warth A, et al. Foxp3(+) regulatory T cells and natural killer cells distinctly infiltrate primary tumors and draining lymph nodes in pulmonary adenocarcinoma. J Thorac Oncol 2011;6:432-8. [Crossref] [PubMed]
36. Alspach E, Lussier DM, Schreiber RD. Interferon γ and Its Important Roles in Promoting and Inhibiting Spontaneous and Therapeutic Cancer Immunity. Cold Spring Harb Perspect Biol 2019;11:a028480. [Crossref] [PubMed]
37. Du F, Qi X, Zhang A, et al. MRTF-A-NF-κB/p65 axis-mediated PDL1 transcription and expression contributes to immune evasion of non-small-cell lung cancer via TGF-β. Exp Mol Med 2021;53:1366-78. [Crossref] [PubMed]
38. Schmid K, Oehl N, Wrba F, et al. EGFR/KRAS/BRAF mutations in primary lung adenocarcinomas and corresponding locoregional lymph node metastases. Clin Cancer Res 2009;15:4554-60. [Crossref] [PubMed]
39. Sun L, Zhang Q, Luan H, et al. Comparison of KRAS and EGFR gene status between primary non-small cell lung cancer and local lymph node metastases: implications for clinical practice. J Exp Clin Cancer Res 2011;30:30. [Crossref] [PubMed]
40. Glorieux C, Xia X, He YQ, et al. Regulation of PD-L1 expression in K-ras-driven cancers through ROS-mediated FGFR1 signaling. Redox Biol 2021;38:101780. [Crossref] [PubMed]
41. Liu C, Zheng S, Wang Z, et al. KRAS-G12D mutation drives immune suppression and the primary resistance of anti-PD-1/PD-L1 immunotherapy in non-small cell lung cancer. Cancer Commun (Lond) 2022;42:828-47. [Crossref] [PubMed]
42. Li S, Liu S, Deng J, et al. Assessing Therapeutic Efficacy of MEK Inhibition in a KRAS(G12C)-Driven Mouse Model of Lung Cancer. Clin Cancer Res 2018;24:4854-64. [Crossref] [PubMed]
43. Heitzer E, Haque IS, Roberts CES, et al. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet 2019;20:71-88. [Crossref] [PubMed]
44. Heitzer E, van den Broek D, Denis MG, et al. Recommendations for a practical implementation of circulating tumor DNA mutation testing in metastatic non-small-cell lung cancer. ESMO Open 2022;7:100399. [Crossref] [PubMed]
45. Gong Y, Yao Y, Cheng W, et al. Highly Sensitive Detection of the Immune Checkpoint PD-L1-Positive Circulating Tumor Cells Based on Steric Hindrance. Anal Chem 2022;94:11767-72. [Crossref] [PubMed]
46. He Y, Zeng X, Xiong Y, et al. Portable Aptasensor Based on Parallel Rolling Circle Amplification for Tumor-Derived Exosomes Liquid Biopsy. Adv Sci (Weinh) 2024;11:e2403371. [Crossref] [PubMed]
47. Godsey JH, Silvestro A, Barrett JC, et al. Generic Protocols for the Analytical Validation of Next-Generation Sequencing-Based ctDNA Assays: A Joint Consensus Recommendation of the BloodPAC's Analytical Variables Working Group. Clin Chem 2020;66:1156-66. [Crossref] [PubMed]
48. Wan JCM, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer 2017;17:223-38. [Crossref] [PubMed]
49. Ratcliffe MJ, Sharpe A, Midha A, et al. Agreement between Programmed Cell Death Ligand-1 Diagnostic Assays across Multiple Protein Expression Cutoffs in Non-Small Cell Lung Cancer. Clin Cancer Res 2017;23:3585-91. [Crossref] [PubMed]