Decoding the enigma of multiple primary lung cancers: from mechanism to bedside—a narrative review

Introduction

Lung cancer is one of the most common malignant tumours worldwide and the leading reason for cancer deaths (1). Incidence data show that lung cancer is at the top of incidence and mortality tables among all cancers (2). According to an International Agency for Research on Cancer (IARC) report, there were approximately 2.48 million new cases of lung cancer worldwide in 2022, which accounts for 12.4% of all newly diagnosed cancers. Same year, lung cancer caused 1.8 million deaths, accounting for 18.7% of cancer fatalities (3).

Multiple primary lung cancer (MPLC) is a clinically significant subtype characterized by two or more distinct primary lung tumors. These tumors may develop synchronously or metachronously and are confirmed as primary tumors through histological or molecular analysis (4,5). The exact mechanisms are unclear. A rising hypothesis for MPLC is “field cancerization”, wherein prolonged exposure to carcinogens induces widespread molecular alterations across the respiratory epithelium, creating a preconditioned “field” prone to the development of independent tumors. In addition to this process, MPLC arises through complex interactions between genetic predisposition, acquired somatic mutations, epigenetic alterations, and environmental exposures, such as tobacco smoke and air pollution (6).

Distinguishing MPLC from intrapulmonary metastases (IPM) remains a key diagnostic challenge in clinical practice. This difficulty stems from several factors. Firstly, conventional imaging alone is often insufficient for a definitive diagnosis. More importantly, clinical judgment can be influenced by ingrained assumptions, such as presuming synchronous nodules to be metastatic or classifying metachronous tumors within a certain period (e.g., 6 months to 2 years) as recurrences. Additionally, diagnostic ambiguity frequently persists even after histopathological assessment, especially when morphological features overlap or biomarkers yield equivocal results. It is precisely these limitations of conventional approaches that have driven the adoption of molecular diagnostics, particularly comprehensive genomic profiling, to establish a more objective and accurate diagnostic framework (7). The foundational approaches for treatment are surgical resection, radiotherapy, and systemic chemotherapy. However, recent advances in targeted therapies and immune checkpoint inhibitors (ICIs) have expanded therapeutic options, especially for advanced-stage disease (8).

In this review, we aim to provide a comprehensive summary of MPLC, exploring its pathogenesis from multiple key perspectives. Genetic factors significantly contribute to disease onset, and germline mutations and familial clustering patterns suggest potential genetic susceptibility. Tumor cells exhibit widespread somatic mutations, and the heterogeneity of driver gene expression across different lesions poses challenges for diagnosing and treating the disease at the molecular level. Epigenetic regulatory abnormalities are also critical, as disrupted DNA methylation patterns and dysregulated histone modifications collectively form the epigenetic foundation of tumorigenesis and progression. Furthermore, the tumor microenvironment (TME) undergoes continuous, dynamic changes. Immune cells and matrix components interact through complex signaling networks, jointly shaping the unique microenvironment of tumor evolution. Based on these mechanistic studies, the latest advances in clinical diagnosis and treatment are further integrated: at the diagnostic level, the evolution from traditional Martini-Melamed (M-M) criteria to modern precision diagnostic technologies such as radiomics and liquid biopsy is systematically reviewed; in the treatment domain, we comprehensively cover systemic strategies ranging from optimized surgical resection and stereotactic body radiation therapy (SBRT) for local treatment, to targeted therapy targeting driver genes such as EGFR/ALK and immunotherapy using programmed death receptor 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors. Finally, based on current research bottlenecks, we prospectively explored future development directions such as multi-omics integrated diagnostic models, artificial intelligence (AI)-assisted diagnosis and treatment, and personalized treatment based on tumor evolutionary clones, providing new ideas for translating MPLC research from basic science to clinical practice. We present this article in accordance with the Narrative Review reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-957/rc).

Methods

A literature search was conducted across PubMed/MEDLINE, Web of Science, and Google Scholar databases for articles published between January 2000 and September 2024. Search terms used included (“multiple primary lung cancer” OR “synchronous multiple primary lung cancer” OR “multiple primary lung neoplasm” OR “double primary lung cancer” OR “second primary lung cancer”) AND (“diagnosis” OR “classification” OR “molecular characteristics” OR “genomic profiling” OR “treatment” OR “management” OR “prognosis” OR “etiology” OR “risk factors”). The selection focused on English-language articles, including clinical trials, cohort studies, case-control studies, and review articles that addressed the epidemiology, diagnosis, molecular mechanisms, or treatment of MPLC. Articles were excluded if they were non-English, were case reports with fewer than 5 cases, or did not contain original data. The search strategy is summarized in Table 1.

Pathogenesis of MPLC

MPLC develops through a dynamic interplay between environmental triggers and individual susceptibility. While tobacco smoke remains the predominant risk factor, emerging evidence highlights other important contributors, including chronic obstructive pulmonary disease (COPD) (9), and prolonged exposure to airborne pollutants—particularly fine particulate matter (PM2.5), which promotes carcinogenesis through oxidative damage mechanisms. Other established risk parameters include advancing age and previous radiation therapy exposure (10). Interestingly, epidemiological data consistently show higher incidence rates among women, suggesting potential sex-specific biological factors in MPLC pathogenesis. Notably, these risk factors frequently demonstrate synergistic effects. For instance, tobacco smokers with positive family history exhibit substantially higher MPLC incidence compared to either risk factor alone (11). The risks of MPLC are shown in Figure 1.

Figure 1 Risks of MPLC. The main risk factors for MPLC include tobacco smoking, COPD, air pollution exposure (especially to PM2.5), genetic susceptibility (germline mutations), as well as age and sex differences. Created in https://BioRender.com. COPD, chronic obstructive pulmonary disease; MPLC, multiple primary lung cancer; PM, particulate matter.

The origin of MPLC is a multifactorial, multistep, and complex process involving separate evolutionary pathways for different histologic types. Pathologically, MPLC originate mainly from two distinct epithelial cell lineages: the peripheral alveolar and fine bronchial epithelium predominantly develops into adenocarcinomas, whereas the pseudocomplex ciliated epithelium of the central airways predisposes to squamous carcinoma formation (12). These tumors may develop through two main modes: a completely independent clonal origin, in which the foci evolve from different precursor cells through independent oncogenic events; and a “field cancerization” effect, in which a wide range of epithelial areas accumulate similar genetic and epigenetic alterations in response to long-term carcinogen exposure, resulting in the formation of multiple foci of disease that develop independently of each other in a common context.

The pathogenesis of MPLC is fundamentally distinct from that of IPM, characterized by the development of multiple independent primary tumors. This “field cancerization” effect, driven by a convergence of germline susceptibility and sustained environmental insults, creates a preconditioned lung epithelium prone to synchronous or metachronous malignant transformation. Genomically, this is evidenced by the prevalence of discordant driver mutations and independent clonal origins (branching evolution) among different lesions, in contrast to the shared truncal mutations seen in metastatic disease. Epigenetic alterations, induced by chronic carcinogen exposure, can lead to widespread and coordinated silencing of tumor suppressor genes across broad respiratory fields, further enabling the parallel development of separate primary tumors. Concurrently, a permissive TME, potentially characterized by systemic immune dysregulation, fails to surveil and suppress the outgrowth of these multiple independent malignant clones. (Figure 2)

Figure 2 Pathogenesis of MPLC. The development of MPLC is driven by a complex interplay between genetic susceptibility and the gradual progression of carcinogenesis. Germline mutations (e.g., in EGFR or TP53) confer inherited predisposition, while subsequent acquisition of somatic driver mutations (e.g., KRAS, ALK) leads to the emergence of independent tumor clones. These clones progressively accumulate additional genetic and epigenetic alterations (including DNA methylation changes and histone modifications), which cooperate with a permissive tumor microenvironment featuring altered immune-stromal interactions to drive malignant progression. This multistep process explains both the molecular heterogeneity and clinical behavior of MPLC. Created in https://BioRender.com. circRNA, circular RNA; lncRNA, long-stranded non-coding RNA; miRNA, microRNA; MPLC, multiple primary lung cancer; TME, tumor microenvironment.

Germline mutations and genetic susceptibility

Many human cancers demonstrate heritable predisposition. However, MPLC represents a more complex disease entity involving multifactorial interactions between environmental exposures and genetic components. Consequently, the contribution of genetic factors to MPLC pathogenesis remains less clearly defined compared to other inherited cancer syndromes (13).

Familial history significantly increases the risk of MPLC compared to single lung tumors. Epidemiological evidence shows first-degree relatives of MPLC patients have a higher cancer risk than the general population, supporting genetic susceptibility (14). Multivariate analyses indicate genetic factors outweigh shared environmental exposures in familial clustering (15). Cohort studies confirm higher MPLC incidence in individuals with a family history, with a Swedish study of 55,238 lung cancer cases reporting a 4.78-fold higher standardized incidence rate for second primary lung cancers in those with familial history (16,17). EGFR germline mutations, notably p.T790M and p.R776H, are linked to MPLC predisposition and tyrosine kinase inhibitor resistance, with p.K757R prevalent in Chinese patients (18-20). Additionally, TP53 germline mutations, associated with Li-Fraumeni syndrome, may contribute to MPLC development due to their role in multiple early-onset malignancies (21,22).

First-degree relatives of lung cancer patients carrying pathogenic germline mutations in cancer predisposition genes—most notably EGFR (T790M, R776H), TP53, and BRCA2—face an elevated risk of developing lung cancer, including MPLC (23). Consequently, germline genetic testing in probands can enable at-risk relatives to quantify their inherited risk and adopt enhanced surveillance or preventive measures at an early stage. However, several limitations currently constrain the widespread clinical implementation of such screening. The prevalence of these germline mutations in lung cancer populations is generally low, and the associated penetrance and lifetime risk are not yet fully characterized, making individualized risk prediction challenging. From a practical standpoint, the clinical feasibility of population-wide germline testing in lung cancer remains limited by cost, accessibility to genetic counseling, and the need for clearer management guidelines for mutation carriers. Future studies should aim to establish genetic causality in familial lung cancer clusters, clarify the pathogenic mechanisms of these germline variants, and develop evidence-based protocols for integrating germline testing into standard lung cancer care.

Somatic mutations and clonal evolution

The clonal evolution theory explains that each primary tumor in MPLC arises from independent somatic mutational events in distinct mutant clones, leading to unique genetic profiles and molecular heterogeneity among lesions (24). This genetic diversity distinguishes MPLC primaries from metastatic lesions and reflects tumor cells’ independent accumulation of mutations during evolution (25).

In the study of MPLC, the discussion around discordant mutations and concordant mutations has become an increasingly important topic with significant clinical implications. Genomic analyses have shown that discordant mutations, which differ between distinct tumor foci within the same patient, are a prominent feature of MPLC. Even tumors within the same patient show considerable genetic divergence, indicating that these tumors evolve along different genetic paths rather than converging on a common set of mutations or molecular alterations. Takahashi et al. mentioned in their study that as tumor progression occurs, subsequent somatic mutations generate branching patterns that increase the genetic heterogeneity between distinct tumor foci (26). Similarly, Cheng et al. noted that the genetic divergence between different tumors from the same patient is comparable to that seen between tumors from different patients, suggesting the absence of evolutionary convergence at both genetic and functional levels (27). This challenges the hypothesis of convergent evolution, which posits that tumors might converge toward the same driver mutations or signaling pathways despite their genetic differences.

However, there is conflicting evidence. Ma et al. provided support for concordant mutations, finding that, despite the genetic heterogeneity of MPLC, distinct tumors within the same patient often converge on alterations in a limited set of critical signaling pathways. This type of convergent evolution suggests that, although the tumors may show considerable genetic diversity, certain oncogenic pathways may still exhibit convergence in response to similar selective pressures, offering a potential common therapeutic target across different tumor foci (28). This finding implies that while genetic diversity within tumors is high, some degree of convergence in molecular functions may still occur, providing a basis for targeting specific pathways in MPLC treatment. The difference between discordant and concordant mutations has a major impact on treatment decisions. If discordant mutations dominate, the genetic diversity across tumors within the same patient suggests that a one-size-fits-all treatment may not work. Each tumor focus could require a personalized approach based on its own genetic makeup. On the other hand, if concordant mutations are more prominent, targeting common pathways across tumors might simplify treatment by focusing on a narrower set of therapeutic targets.

In MPLC, some common somatic mutated genes such as EGFR, KRAS, BRAF, and TP53 are frequent in different primary tumors, and these mutations usually affect transcriptional regulation, MAPK signaling, cell adhesion, and cell survival. EGFR mutations are significantly more frequent in never or light smokers and women, while KRAS mutations are commonly found in lung cancers of smokers (29). Mutations in TP53 frequently occur in multiple primary tumors, suggesting its critical role in multi-tumorigenic processes. Notably, the diversity of TP53 mutations and their synergistic effects with other key mutations may provide a molecular basis for the multiple primaries of lung cancer. Overall, somatic mutation patterns in MPLC patients show diversity and complexity, suggesting that these tumors may originate from different pathogenic mechanisms.

Epigenetic modification

Lung cancer development involves the accumulation of genetic mutations and epigenetic changes, with epigenetic alterations like DNA methylation and histone modifications often playing a more prominent role than somatic mutations (30,31). Promoter hypermethylation of tumor suppressor genes (e.g., CDKN2A, RASSF1A) silences critical cell cycle and apoptotic pathways, driving malignant transformation of bronchial epithelium (32,33). Concurrently, widespread demethylation occurs at repetitive DNA elements, tissue-specific genes, and imprinted gene loci, resulting in global genomic hypomethylation (34). This hypomethylated state promotes genomic instability and accelerates the acquisition of oncogenic mutations. Comparative epigenomic analyses reveal distinct methylation patterns between MPLC and solitary lung cancers. Notably, different lesions from the same MPLC patient demonstrate remarkably similar methylation profiles, suggesting DNA methylation may participate in field cancerization—the process whereby large epithelial areas become predisposed to malignant transformation (35). Furthermore, aberrant DNA methylation patterns correlate strongly with dysregulation of immune-related genes, indicating a potential role in modulating anti-tumor immune responses within MPLC lesions.

The complex “histone code” comprises multiple post-translational modifications including acetylation, methylation, phosphorylation, ubiquitination, and glycosylation. Among these, histone acetylation dynamics, regulated by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs), play crucial roles in carcinogenesis. Aberrant HDAC overexpression is frequently observed in lung cancer, with various HDAC family members contributing to malignant transformation and metastatic progression (36). Histone methylation, controlled by histone methyltransferases (HMTs) and demethylases (HDMs), represents another critical epigenetic regulator. Lung cancer typically exhibits elevated global H3 and H4 methylation levels along with increased HMT expression, both correlating with poor clinical outcomes. The histone lysine methyltransferase family (KMTs), particularly EZH2, regulates fundamental cellular processes including DNA replication, damage response, cell cycle progression, and transcriptional regulation. EZH2 overexpression drives lung cancer progression through multiple oncogenic signaling pathways. Conversely, histone lysine demethylases (KDMs) such as LSD1 demonstrate oncogenic properties when overexpressed, promoting lung cancer cell proliferation and invasion.

Dysregulated non-coding RNAs (ncRNAs) play a crucial role in lung cancer development and participate in lung cancer development through a complex regulatory network. MicroRNAs (miRNAs) are a class of small ncRNAs, which have been found to have both tumor-suppressor and oncogenic roles in lung cancer. Abnormally low expression of the let-7 family leads to uncontrolled activation of RAS, MYC oncogenes such as RAS and MYC (37), while overexpression of miR-21 and miR-155 promotes malignant transformation of bronchial epithelial cells by suppressing oncogenes such as PTEN and p53 (38), and this disorder of miRNA expression profiles constitutes the molecular basis for the early onset of MPLCs. Long-stranded non-coding RNAs (lncRNAs) such as MALAT1 and HOTAIR regulate the localization and activity of chromatin remodeling complexes by acting as molecular scaffolds or decoys, inducing the process of EMT and the acquisition of metastatic potential (39), and their differential expression patterns in different foci provide molecular markers for the identification of independent primary foci and metastatic foci. Circular RNAs (circRNAs) such as CDR1as adsorb regulatory factors such as miR-7 through a sponge mechanism to deregulate signaling pathways such as EGFR and RAF, promoting adaptive evolution of each lesion of multi-primary lung cancer (40).

TME and field cancerization

The pathogenesis of MPLC involves genetic alterations and a dynamic TME, where tumor cells interact with immune cells, stromal components, and vascular networks to support growth and invasion (41). Despite TME heterogeneity across MPLC lesions, shared germline mutations and immune signatures, revealed by T-cell receptor sequencing, suggest biological connections that could guide novel therapeutic strategies (42).

The concept of field cancerization, first proposed by Slaughter and colleagues in 1953, describes the phenomenon where histologically normal-appearing tissues surrounding a primary tumor harbor molecular alterations resembling those found in the malignant lesion itself. This concept differs fundamentally from the TME, though both play important roles in multifocal tumor development. Field cancerization results from the evolutionary process of somatic cell alterations that create patches of epithelial cells exhibiting partial malignant phenotypes, while the TME represents the established ecosystem of stromal and immune components that interact with and support fully transformed tumor cells MPLC often develop through a process called field cancerization, where prolonged exposure to carcinogens induces molecular changes across large areas of lung tissue. This creates a genetically altered “field” where multiple tumors can emerge independently, explaining why these cancers may share certain molecular features while developing in different locations. Meanwhile, each developing lesion interacts with and shapes its own unique TME, contributing to the heterogeneity observed between different tumor foci despite their potential shared origin within a cancerized field (43).

Regional oncogenesis involves interactions between driver mutations and the microenvironment. Normal cell lineages can acquire pro-tumorigenic mutations or epimutations that are cumulatively selected in the microenvironment, and the region may or may not exhibit morphological changes. The cancerous region may be induced by “unfortunate mutations”, after mutagenic damage, mutant clones of multiple genes may be created, and if the mutagenic damage is sustained, perhaps because it is caused by ongoing environmental factors such as tobacco carcinogens, diet, or infections, then new clones will be created. Eventually, all these clones will be selected to adapt to the current microenvironment and develop into tumors (44).

The immunoediting process can selectively favor tumor clones with reduced immunogenicity, leading to the outgrowth of immune-evasive populations (45). In MPLC, this selection pressure, operating independently across distinct primary tumors, may contribute to their typically lower tumor mutational burden (TMB) and PD-L1 expression compared to IPM. This phenomenon can be attributed to their independent clonal origins; unlike IPM, which arise from a common, often highly mutated and immunogenic ancestor clone that has undergone extensive immune selection during metastasis, each MPLC tumor originates de novo. These independent primaries may thus begin their evolution with a lower neoantigen load and develop within a potentially less inflamed microenvironment, resulting in less pressure to upregulate PD-L1 as an adaptive resistance mechanism (46).

Classification and diagnosis of MPLC

Classification and diagnostic criteria

The classification of MPLC into synchronous and metachronous types has been guided by evolving diagnostic criteria, with the foundational M-M standard and subsequent American College of Chest Physicians (ACCP) guidelines providing key clinical benchmarks (46,47) (Table 2). While these criteria established critical frameworks based on histology, anatomy, and time interval, several complex diagnostic scenarios require further elaboration. First, the diagnosis of metachronous tumors within the same lobe is challenging and necessitates confirmation of independent origin through divergent histologic patterns or discordant molecular profiles. Second, tumors with identical histology arising in different lobes in the absence of nodal involvement may still be classified as MPLC if comprehensive molecular profiling demonstrates distinct clonal origins. Third, significant variation exists in the recommended disease-free interval for defining metachronous MPLC across different guidelines, ranging from 6 months [International Association for the Study of Lung Cancer (IASLC)] to 2–4 years (Colice criteria), reflecting ongoing clinical debate on optimally distinguishing second primaries from late metastases (48). The integration of molecular profiling into later diagnostic standards, such as the ACCP guidelines, has been instrumental in addressing these ambiguities and enhancing diagnostic precision for clinically ambiguous cases (49,50).

The diagnostic differentiation between MPLC and IPM continues to rely on histopathological evaluation as the gold standard, though this approach faces limitations when tumors share identical histological features. This diagnostic challenge has prompted the development of integrated approaches that combine conventional pathology with molecular profiling. Mansuet-Lupo et al.’s work demonstrated an integration of World Health Organization (WHO) 2015 histological classification and multi-gene panel sequencing significantly improves diagnostic sensitivity, and predominantly in differential diagnosis of multiple pulmonary adenocarcinomas (51). MPLC exhibits significantly greater tumor heterogeneity compared to IPM, which manifests across multiple molecular and microenvironmental dimensions (Figure 3). Therefore, the rapid development of genomic technology and molecular biology technology has greatly expanded the diagnostic possibilities of MPLC. Characteristic mutation patterns, comparative analysis of copy number abnormalities, epigenetic profiling, and comprehensive analysis of the TME have made it possible to more accurately distinguish between independent primary and metastatic lesions and reveal tumor progression pathways.

Figure 3 Differences between MPLC and IPM. MPLC demonstrate significantly greater intertumoral heterogeneity compared to IPM, manifesting across multiple molecular and microenvironmental dimensions. At the genomic level, MPLC lesions typically harbor distinct driver mutations, independent copy number variation patterns, and divergent clonal evolutionary trajectories, reflecting their independent cellular origins. In contrast, IPM tumors maintain remarkably similar genetic profiles due to their derivation from a common progenitor clone through metastatic dissemination. Created in https://BioRender.com. IPM, intrapulmonary metastases; MPLC, multiple primary lung cancer.

Differential diagnosis

Imaging

Chest computed tomography (CT) and positron emission tomography (PET)-CT are the primary imaging modalities used to differentiate IPM from MPLC pre-operatively. MPLC lesions are spiculated mixed-density nodules, while IPM lesions are solid nodules with metabolic heterogeneity. Clinicians typically use assessment of lesion morphology and tumor doubling time to aid in differential diagnosis (52). PET-CT further aids in this distinction by evaluating differences in standardized uptake values (SUV) between lesions. Studies have demonstrated that the ΔSUV (difference in SUV between tumors) is significantly higher in synchronous MPLC (sMPLC) than in metastatic disease, providing a potential diagnostic marker (53,54). This difference can be attributed to the distinct biological nature of these entities: sMPLC comprises independent primary tumors that may arise from different cellular origins and exhibit divergent metabolic phenotypes and glycolytic activities. In contrast, IPM lesions, originating from a common ancestral clone, typically display more uniform metabolic characteristics due to their shared genetic background and driver mutations, resulting in lower inter-lesion metabolic heterogeneity. However, PET-CT alone remains insufficient for definitive differentiation. To improve diagnostic accuracy, Suh et al. developed an integrated algorithm combining SUV analysis with CT imaging features, demonstrating promising positive predictive value when validated against histopathological results. While further large-scale studies are needed to strengthen its reliability, this approach offers a non-invasive and clinically practical diagnostic tool (55). The integration of AI and deep learning represents a transformative advancement in this field. Current deep learning models have achieved diagnostic performance comparable to or exceeding that of radiologists in analyzing CT images for lung cancer classification (56). As AI continues to evolve, it is poised to revolutionize the diagnosis of multiple lung cancers by enhancing precision, reducing subjectivity, and enabling earlier and more accurate differentiation between MPLC and IPM (57). Future developments in AI-driven imaging analysis may further refine non-invasive diagnostic strategies, ultimately improving patient stratification and personalized treatment planning.

Pathological histology

Histopathological evaluation remains the cornerstone for distinguishing MPLC from IPM in clinical practice. Clear differentiation can be made when tumors exhibit distinct histological types. Additionally, multiple lesions demonstrating carcinoma in situ, minimally invasive adenocarcinoma, or lepidic-predominant adenocarcinoma patterns, especially when accompanied by precancerous lesions or in situ components, are usually classified as distinct primary tumors (51,58). Nevertheless, histopathological diagnosis has limitations. A lepidic growth pattern does not invariably indicate a precancerous lesion, as it may simply represent a tumor’s growth morphology; thus, IPM cannot be entirely excluded. Moreover, most invasive mucinous adenocarcinomas are monoclonal in origin and more likely to represent metastatic spread. For invasive non-mucinous adenocarcinomas, histology alone is often insufficient, necessitating integration with molecular or genomic profiling to accurately differentiate MPLC from IPM. In international lung cancer pathology studies, comprehensive histologic assessment (CHA) demonstrates good interobserver consistency. However, a subset of cases remains ambiguous even after detailed evaluation. Refining diagnostic accuracy may require further correlation with growth patterns, such as assessing lepidic spread in conjunction with molecular features. While histopathology provides a foundational diagnostic framework, its limitations underscore the need for multimodal approaches incorporating genomic, radiological, and clinical data to optimize MPLC and IPM differentiation.

Molecular genetics

With the advancement and development of modern molecular biotechnology, methods for molecular genetics and molecular biology analysis have been increasingly applied to the differential diagnosis of MPLCs. Researchers focus on molecular characteristics to explain whether there is a clonal relationship between multiple lesions, primarily distinguishing between them based on the heterogeneity within MPLC and the relative uniformity of IPM (59). Currently, mutation detection, chromosomal variation detection, and DNA methylation sequencing technologies have been reported as useful for differentiating MPLC from IPM.

Initially, only a few representative genes frequently found in lung cancer, such as p53, EGFR, and KRAS, were tested to assist in diagnosing. However, due to shared genetic backgrounds and environmental exposures, there is a high likelihood that multiple primary tumors may coincidentally share a hotspot driver gene mutation. Therefore, testing for a small number of oncogenic driver mutations is insufficient to distinguish between the two (60,61). Large-scale lung cancer driver gene mutation testing is necessary to improve the accuracy of distinguishing between MPLC and IPM. Next-generation sequencing (NGS) can achieve broader detection coverage and clearly differentiate clonal relationships between different lesions, but the limitations of NGS mainly lie in implementation costs, the need for personnel skilled in bioinformatics and data analysis, and the longer time required compared to pathological examination.

Chromosomal variations are reliable markers for distinguishing MPLC from pulmonary metastases. Research indicates MPLC tumors have significantly less inter-lesion copy number variation (CNV) than metastatic lesions (62). Array comparative genomic hybridization (aCGH) effectively identifies these genomic differences, enabling accurate differentiation between primary and metastatic tumors (63). Chromosome rearrangement breakpoints in MPLC are also clear markers for clonal analysis. Researchers used paired sequencing technology to detect somatic breakpoint connections to determine the lineage of tumors, finding no common genomic rearrangements in MLPC tumors. In IPM patients, common rearrangements were found in all tumor pairs, which seems to be used to differentiate between MPLC and IPM (64). Loss of heterozygosity (LOH), where germline heterozygosity at polymorphic loci shifts to somatic homozygosity, is a marker of allele loss. Tumors from the same patient showing identical patterns of allele loss are considered to have a common clonal origin, while different patterns of allele loss are thought to represent tumors with independent clonal origins (65). Using LOH assays can successfully distinguish synchronous and asynchronous lung tumors with the same histology from IPM.

DNA methylation is a ubiquitous epigenetic silencing mechanism that exhibits higher levels of universality, tissue specificity, and relative stability compared to somatic mutations. Studies have found that identifying methylation profiles can lead to MPLCs and intra-pulmonary metastases (66). The differences in TME markers routinely tested in tumors also provide potential insights into recognizing clonal-related aspects of MPLC. Compared to MPLC lesions, PD-L1 expression shows higher consistency between IPM lesions, although the differences are not significant enough to distinguish them, they still hold some research potential (67).

Treatment strategies

The management of MPLC presents unique challenges distinct from those of single primary lung cancers, primarily revolving around the need to achieve oncologic control of multiple lesions while preserving sufficient pulmonary function. For sMPLC, the surgical strategy must balance the extent of resection for each lesion against the cumulative impact on respiratory capacity. Evidence suggests that parenchyma-sparing sublobar resections can provide comparable outcomes to lobectomy for appropriately selected, smaller tumors, which is a critical consideration when planning treatment for multiple ipsilateral lesions (68-70). For bilateral disease, the decision between staged versus simultaneous resections requires careful assessment of the patient’s functional reserve and perioperative risk.

In medically inoperable patients, the application of localized therapies like stereotactic ablative radiotherapy (SABR) or thermal ablation must be carefully planned across multiple tumor sites. The challenge lies in delivering effective tumoricidal doses to all lesions while minimizing the composite toxicity to the surrounding lung parenchyma, a consideration that is magnified in MPLC compared to solitary tumors (71-73).

Targeted therapy has been an effective treatment of end-stage non-small cell lung cancer (NSCLC) and has shown therapeutic efficacy on patients with MPLC who have residual disease intraoperatively. A distinctive feature of MPLC is that different lesions often harbor unique driver gene mutations, with a highly heterogeneous mutation profile, necessitating the adoption of personalized treatment strategies. Clinical data indicate that KRAS and EGFR are the most common driver gene mutations in MPLC patients, providing a theoretical basis for targeted therapy (74,75). Ye et al.’s study confirmed that after surgical resection of the invasive primary lesion, maintenance therapy with gefitinib can achieve complete remission of unresected ground-glass nodules (GGNs) (>10 mm), providing practical evidence for the combined approach of “surgical resection of drug-resistant lesions + targeted drug therapy for sensitive lesions” (76).

ICIs, especially therapeutic antibodies to PD-1/PD-L1, are currently the main choice for first-line treatment of patients with advanced NSCLC without driver mutations (77). However, there are few studies on PD-L1 expression levels in MPLC patients. A study by Wu et al. demonstrated interesting findings in sMPLC patients with predominantly GGN lesions (78). The characteristic “tailing effect” of PD-1 antibodies resulted in nearly complete radiological remission of residual GGNs in these cases, suggesting immunotherapy may represent a viable treatment option for select MPLC patients. However, the inherent biological complexity of MPLC presents unique challenges for immunotherapy. The significant genomic heterogeneity between different tumor lesions and variations in their immune microenvironments may lead to differential responses to immune checkpoint blockade. The current understanding of immunotherapy in MPLC reveals both promising opportunities and substantial challenges. While case reports have documented remarkable responses in some patients, the molecular heterogeneity characteristic of MPLC complicates treatment response prediction and patient selection. The observed “tailing effect” following PD-1 inhibition suggests sustained immune activity that continues after treatment cessation, potentially offering prolonged clinical benefit. However, the variability in immune microenvironment composition and PD-L1 expression across different tumor foci within the same patient underscores the need for comprehensive immune profiling of multiple lesions. Future research efforts should focus on systematic evaluation of ICI efficacy in molecularly defined MPLC subsets, development of predictive biomarkers for pan-lesional response, and investigation of rational combination strategies with targeted therapies or local ablative treatments.

Based on the aforementioned diagnostic and treatment strategies for MPLC, clinical management should be multidisciplinary and comprehensive. Patients identified through risk factor assessment and low-dose computed tomography (LDCT) screening should undergo further precise diagnosis via imaging, pathology, and molecular analysis. Subsequent treatment regimens should integrate molecular subtype data and TME characteristics, prioritizing targeted/immunotherapy combinations effective against different lesions. Post-treatment follow-up is equally critical, with patients required to undergo regular follow-up to enable early detection of new primary lesions or treatment-resistant mutations, and timely revision of intervention strategies. This “diagnosis-treatment-monitoring” closed-loop management system fully reflects the personalized management requirements of MPLC as a special subtype of lung cancer. The entire process is illustrated in Figure 4.

Figure 4 Strategies for screening, diagnosis, and treatment of MPLC. First, high-risk individuals are identified through risk factor assessment or LDCT screening. Subsequently, diagnosis is made through imaging and pathological tissue section analysis, and molecular testing can be performed to further refine the type of cancer. After diagnosis, molecular profiling helps to develop a precise treatment plan. Finally, regular follow-up after treatment can identify disease progression and facilitate timely intervention. Created in https://BioRender.com. IPM, intrapulmonary metastases; LDCT, low-dose computed tomography; MPLC, multiple primary lung cancer; SABR, stereotactic ablative radiotherapy.

Conclusions

MPLC is a complex and diverse type of lung cancer with unique characteristics in terms of pathogenesis, diagnosis, and treatment. Studies in recent years have gradually revealed the important roles played by dynamic interactions among genetic factors, somatic mutations, epigenetic regulation, and the TME in MPLC development. The discovery of germline mutations has provided important clues to the genetic susceptibility of MPLC, and in particular, the association of germline variants in genes such as EGFR and TP53 with familial MPLC has been preliminarily validated. However, how these mutations specifically regulate the development of MPLC and whether there are other undiscovered genetic risk factors still require more in-depth studies. The theory of clonal evolution of somatic mutations provides a framework to explain the heterogeneity of MPLC, but the conclusions of different studies on clonal heterogeneity and convergent evolution are controversial and need further explanation. The role of the TME in MPLC has also been gradually emphasized, and the discovery of regional cancerization and immunoediting has provided a new perspective to understand the coexistence of multiple foci, but how the microenvironment drives the independent development or co-evolution of different foci still needs more experimental and clinical data support. Similarly, although epigenetic regulation has been shown to be involved in the development of MPLC, there is currently a lack of systematic research on whether these epigenetic changes are lesion-specific and how they interact with genetic variations to promote the formation of multifocal tumors.

Future studies should focus on the in-depth exploration of molecular mechanisms, combining multi-omics technologies, such as genomics, transcriptomics, and proteomics, to comprehensively resolve the molecular features of MPLC. By integrating data from different genomics, the pathogenesis of MPLC can be more comprehensively revealed and new therapeutic targets can be identified, especially the interactions between germline and somatic mutations, the process of clonal evolution, and the dynamic changes in the TME. The development of early diagnostic techniques is also an important direction for future research. The combination of liquid biopsy and imaging techniques, especially the use of somatic mutation profiles and circulating tumor DNA (ctDNA) as biomarkers, will be the key to early detection and intervention of MPLC. Meanwhile, as targeted therapy and immunotherapy continue to evolve, the optimization of individualized treatment strategies is equally critical. Exploring how to combine these emerging therapies with traditional chemotherapy and radiotherapy to enhance the outcome of MPLC patients will be the focus of future research. In addition, for screening and preventive measures for high-risk groups, especially individuals with family history or other high-risk factors, future research should explore more personalized screening and preventive strategies for early detection and intervention of MPLC. Overall, the study of MPLC not only helps us to gain a deeper understanding of the pathogenesis of lung cancer but also provides a new direction for precision therapy. Combined with the advances in multi-omics technology and modern treatments, it will be able to bring better prognosis and quality of life to MPLC patients in the future.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-957/rc

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-957/prf

Funding: This work was supported by the National Natural Science Foundation of China (Nos. 32370628, 92159302, and 32170592), the Science and Technology Project of Sichuan (Nos. 2022ZDZX0018, 2023NSFSC004, and 2024NSFSC0402), State Key Laboratory Special Fund (No. 2060204), Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (No. 2023-12M-2-001), Key R&D Support Plan of Chengdu Science and Technology Bureau (No. 2023-YF09-00039-SN) and 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (No. ZYGD22009).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-957/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.

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