Non-small cell lung cancer (NSCLC) is the most common type of lung cancer worldwide, comprising approximately 85% of all cases (Miao et al., 2024). Patients diagnosed at advanced stages typically face a poor prognosis (Chen et al., 2014). In recent years, immunotherapy, particularly immune checkpoint inhibitors targeting programmed death protein-1 (PD-1) and its ligand (PD-L1), has significantly improved outcomes for a subset of NSCLC patients (Kanabar et al., 2022). As an emerging therapeutic approach, neoadjuvant immunochemotherapy (nICT) involves the administration of chemotherapy and immunotherapeutic agents prior to surgical resection, which not only contributes to tumor downstaging but may also activate systemic anti-tumor immune responses, thereby enhancing long-term survival (Zheng et al., 2023). Nonetheless, substantial variability exists among individuals regarding their response to nICT, and reliable methodologies for predicting therapeutic outcomes remain scarce (Fei et al., 2023). To address this challenge, we aimed to identify appropriate predictive biomarkers to better assess treatment responses, optimize therapeutic strategies, and improve clinical outcomes for NSCLC patients undergoing nICT.

In recent years, the 14-gene molecular assay, a quantitative PCR-based method for molecular detection, has been widely utilized in prognostic studies for post-surgical NSCLC patients (Jiang et al., 2023). This assay assesses the expression levels of 11 cancer-related target genes (BAG1, BRCA1, CDC6, CDK2AP1, ERBB3, FUT3, IL11, LCK, RND3, SH3BGR, Wnt3A) and 3 reference genes (ESD, TBP, YAP1) within tissue samples to generate a continuous risk score. Using this score, patients are classified into low-risk, intermediate-risk, or high-risk categories. The reliability and clinical relevance of this molecular signature for risk stratification and prognostic prediction have been verified through multiple independent international validation cohorts (Jiang et al., 2023; Huang et al., 2024). Nonetheless, its utility in evaluating the efficacy of nICT in patients as well as its association with post neoadjuvant therapy changes in the immune microenvironment, including TIL levels and TLS positivity rates, remains to be clarified.

This study aimed to retrospectively analyze 14-gene molecular profiling in NSCLC patients who underwent surgical treatment following nICT. It also sought to evaluate the correlation between 14-gene risk stratification and pathological response rates after nICT, along with its association with the immune microenvironment, to explore potential biomarkers for predicting nICT efficacy in NSCLC and providing more precise guidance for individualized treatment of NSCLC patients.

In this study, we conducted a 14-gene molecular assay on NSCLC patients undergoing nICT, alongside an evaluation of tumor microenvironment factors such as TIL and TLS, and demonstrated that the 14-gene molecular assay holds significant predictive value for pathological response and DFS. Specifically, patients classified into the high-risk group based on the 14-gene signature exhibited substantially lower pCR rates, markedly elevated %RVT, and significantly shortened DFS compared to those in the low-risk group. Furthermore, high-risk patients showed notably reduced proportions of TLS positivity and lower levels of TIL infiltration, indicating that the 14-gene molecular assay not only correlates with pathological response but also reflects the immune status of the tumor microenvironment. These findings provide important evidence for optimizing neoadjuvant treatment strategies in NSCLC patients.

As the 14-gene molecular risk stratification progresses from low to high risk, pathological response steadily decreases, characterized by significant reductions in the pCR rate and pronounced elevations in RVT. Cox regression analysis further confirms the independent prognostic value of the 14-gene molecular assay in predicting DFS, with high-risk patients exhibiting substantially lower DFS compared to those in the low- and intermediate-risk groups. Previous studies have established a strong association between the 14-gene molecular stratification and the pathological features of lung cancer, malignant biological behavior, and immune regulation, underscoring its ability to reflect tumor biology and patient prognosis (Ding et al., 2025). Among the 14 genes, BRCA1 is associated with DNA damage repair, and its low expression may enhance tumor cell sensitivity to treatment (Yoshida and Miki, 2004); conversely, high Wnt3A expression may promote tumor cell proliferation and differentiation, correlating with poorer pathological response and prognosis (Lin and Liu, 2021). Furthermore, elevated CDC6 expression may promote tumor cell proliferation by regulating the cell cycle (Borlado and Méndez, 2008), while high LCK expression may enhance T cell activity, thereby improving immunotherapy efficacy (Moogk et al., 2016). These findings further highlight the promising application of the 14-gene molecular assay for evaluating nICT efficacy in NSCLC patients.

The 14-gene molecular assay was strongly correlated with immune cell infiltration within the tumor microenvironment. As the 14-gene risk stratification increased, patients showed markedly decreased levels of TIL infiltration and TLS positivity rates. The low-risk group exhibited significantly higher proportions of robust TIL infiltration (3+) compared to the high-risk group, along with notably elevated TLS positivity rates. These observations suggest that the 14-gene molecular test effectively reflects the immune status of the tumor microenvironment, underscoring its potential utility in assessing the efficacy of immunotherapy. Prior research has established the pivotal roles of TIL and TLS in shaping the tumor immune microenvironment, with high levels of TIL infiltration generally linked to stronger anti-tumor immune responses and improved prognoses (Noël et al., 2021; Ruffin et al., 2021). Similarly, the presence and maturity of TLS have been shown to enhance both local and systemic anti-tumor immune responses by facilitating the activation of B cells and T cells (Fridman et al., 2023; Teillaud et al., 2024). Genes within the 14-gene signature may play critical roles in immune microenvironment regulation. For instance, elevated IL11 expression may suppress anti-tumor immune activity by fostering the development of an immunosuppressive microenvironment (Tang et al., 2024), while increased ERBB3 expression may impact immunotherapy outcomes by modulating the interplay between tumor cells and immune cells (Yang et al., 2022). Additionally, LCK, a crucial component of T cell receptor signaling pathways, may influence T cell activation and anti-tumor immune response (Wu et al., 2021). Furthermore, BRCA1, beyond its established role in DNA repair, may affect immune surveillance mechanisms and the tumor’s ability to evade immune recognition through its involvement in maintaining genomic stability (Yoshida and Miki, 2004). Further investigation into the mechanisms by which specific genes within the 14-gene signature interact with the immune microenvironment could unveil novel therapeutic targets, enabling the optimization of immunotherapy strategies. For these high-risk patients, potential treatment modifications could include intensified neoadjuvant regimens with additional cycles, closer monitoring during treatment, and more frequent surveillance schedules during follow-up.

Despite highlighting the significant value of the 14-gene molecular assay in evaluating the efficacy of nICT in NSCLC patients, this study has notable limitations. First, as a single-center retrospective study with a relatively small sample size, the potential for selection bias cannot be overlooked. Our current study represents an important initial step by demonstrating the signature’s predictive capability in a real-world clinical cohort, but external validation in independent cohorts across diverse patient populations and different institutions is essential for clinical implementation. Only through rigorous external validation can we ensure the signature’s robustness and reliability across different clinical settings before considering its integration into routine clinical practice. Second, the 14-gene molecular assay was conducted exclusively on post-surgical tumor tissue samples, with no analysis of pre-neoadjuvant treatment biopsy specimens. This limitation hinders the dynamic assessment of changes in gene expression before and after treatment and their potential impact on therapeutic efficacy. Third, given that the original 14-gene assay was primarily validated in early-stage NSCLC patients, its direct application to the neoadjuvant treatment context may benefit from model recalibration to optimize predictive performance. Nevertheless, we believe the 14-gene signature’s predictive value remains relatively stable across different treatment regimens because it captures intrinsic tumor biological characteristics that are more deterministic of treatment response than treatment variations themselves. Furthermore, the study did not comprehensively investigate the mechanisms of interaction between specific genes within the 14-gene signature and the immune microenvironment. Future research should prioritize larger-scale, multi-center prospective studies, integrating advanced technologies such as single-cell sequencing and spatial transcriptomics. These efforts would allow for a more in-depth exploration of the relationship between the 14-gene signature and the tumor immune microenvironment, ultimately providing more precise and tailored guidance for the individualized treatment of NSCLC patients.

In conclusion, this study highlights the significant value of the 14-gene molecular assay in assessing the efficacy of nICT for NSCLC. Additionally, the 14-gene risk stratification demonstrates a clear association with TLS and TIL within the immune microenvironment after nICT. The 14-gene molecular signature holds promise as a valuable tool for personalized medicine, enabling pre-treatment identification of high-risk patients, guiding individualized treatment decisions and follow-up strategies, and providing clinicians with molecular-level prognostic information that complements conventional TNM staging. Future research should focus on conducting large-scale multicenter prospective validation studies, exploring the molecular mechanisms underlying 14-gene interactions with the tumor immune microenvironment, integrating multidimensional biomarkers to construct more precise prognostic models, and establishing standardized testing procedures and clinical application guidelines to facilitate clinical translation. These findings collectively indicate that the 14-gene molecular signature represents a promising advancement toward optimizing efficacy evaluations of neoadjuvant therapy in NSCLC, potentially facilitating the development and implementation of patient-specific therapeutic algorithms and precision treatment stratification approaches in clinical practice.