AAH, AIS, MIA, and IAC are currently diagnosed based on morphological analysis. However, this method does not accurately capture the biological characteristics of GGNs. In addition, although multiple studies have investigated mutational alterations in lung cancer (26), the gene mutational characteristics for the initiation and early progression of precancerous lesions remain poorly understood. Recent findings on gene mutation signatures in ground glass nodules have been summarized in Table 1 .

In the preinvasive phase, EGFR is the most common gene mutation in GGNs, followed by ERBB2, RBM10, and KRAS mutations (12–14, 27, 28, 30, 31, 33–35). The exon 21 L858R mutation is reportedly the most common EGFR mutation among GGNs, followed by exon 19 deletion mutations (30, 33). GGNs with mutations in RBM10 tend to present with a pathologically lepidic pattern. Lepidic pattern growth is characterized by tumor cell proliferation along intact alveolar walls. In addition, patients with lepidic pattern have a better prognosis than those with non-lepidic pattern tumors, suggesting that RBM10 mutations are associated with a better prognosis (13, 31). However, in some studies on lung adenocarcinoma, RBM10 acts as a tumor suppressor. When cancer is about to develop, RBM10 is mutated to reduce its inhibitory effect on c-Myc, which ultimately promotes tumor growth (36, 37). More studies may be needed to determine the role of RBM10 in the development of lung cancer. Affyl CoA synthase long chain (ACSL) plays a crucial role in the uncontrolled proliferation of cancer cells through abnormal lipid synthesis and extracellular lipid uptake (38). As the tissues change from normal to AAH and ADC, ACSL5 expression increases gradually, supporting the notion that ACSL5 contributes to tumor progression (32). In addition, FUT2 and SERINC2, which are involved in metabolism, are aberrantly expressed in precancerous lesions, aiding the identification of early-stage lung cancer (32).

EGFR, ERBB2, NRAS, and BRAF mutations contribute to the early clonal genomic events in lung ADC. In contrast, mutations in TP53 and genes associated with cell migration, gap junctions, and metastasis (such as FRG1, DOCK7, SH3BP1, and GJA1) may be late events that occur during subclonal diversification and tumor progression (12, 14). Similar proportions of EGFR mutations were observed in IAC and MIA/AIS, whereas TP53 was mutated in IAC at a much higher proportion than in MIA/AIS (12). TP53 mutation was the most abundant in the invasive phase, followed by EGFR and RB1 mutations when compared with the preinvasive and microinvasive phases. The frequency of loss of heterozygosity of human leukocyte antigen (HLA) also increased in the invasive phase (13). Conversely, the mutation frequencies of EGFR, RBM10, and TP53 in IAC cases presenting with GGNs were all significantly higher than those of AAH, AIS, and MIA (30).

The tumor mutational burden (TMB) increases gradually from AAH, AIS, and MIA to IAC (13, 30). TMB in lung adenocarcinoma (LUAD) presented as SN was significantly higher than that in LUAD with GGO components, and the proportion of GGO components was negatively correlated with TMB (35). Copy number variations (CNVs) reflect chromosomal instability, and gene gains and losses caused by CNVs can result in tumorigenesis. In a previous study, GGO displayed significantly lower leverage of arm-level CNVs and lower focal CNVs than LUAD, further indicating that GGO had fewer genomic alterations and simpler genomic profiles than LUAD (14). Li et al. discovered that malignant cells in GGNs have CNV patterns similar to those in SN, indicating that different radiological appearances are not correlated with genomic features (39). The CNV patterns of malignant cells within GGNs and solid component cells are similar. This indicates that genomic characteristics do not correspond to radiological characteristics (38). Hu et al. proposed that progression before tumor formation mainly follows the clonal clearance model, whereby partial clonal mutations discovered in early indeterminate lung nodules (IPNs) become clones in later IPNs, whereas inappropriate subclones are eliminated (29). Furthermore, somatic copy number alterations and allelic imbalance may occur during the transition from AAH to AIS and MIA to AIS, respectively (32) ( Figure 1 ).

Approximately 20% of patients with GGNs (3% of the screened population) have synchronous multiple ground-glass nodules (SM-GGNs) (11). Clinical decisions regarding the treatment of SM-GGNs are complicated by the difficulty in distinguishing synchronous multiple primary lung cancers from intrapulmonary metastases. According to some studies, SM-GGNs may have a common origin, and intrapulmonary metastasis may occur even in the early stages of lung cancer (11, 31, 40). However, some studies have found that SM-GGNs mostly originate independently (28, 33). There is no genetic linkage between independently cloned SM-GGNs, and they do not have the same driver mutations, nor typical susceptibility mutations. Therefore, they occur randomly (28). SM-GGNs that were not surgically resected did not exhibit changes in size or characteristics during long-term follow-up (41). It is recommended to interpret patients with partially solid GGN lesions as surgical candidates even if they have multiple lesions (42). A good prognosis after surgical resection of SM-GGNs has been reported, supporting the strategy of considering multiple GGNs in patients as primary tumors, and resection may be an appropriate option for the subgroup (33). Most previous studies have shortcomings, such as small sample sizes, relatively low purity of tumors in GGN samples, and geographic limitations of the study population. Thus, in the future, more prospective clinical trials are needed to better understand GGNs in greater depth.

T cells are key mediators of tumor destruction, and their specificity for tumor-expressed antigens is of paramount importance. CD3+ in T lymphocyte subsets is responsible for antigen presentation on the surface of mature T cells and assists in the recognition of T cell antigen receptors. CD4+ T cells are known as helper T cells (Th cells) and act to modulate immune responses by producing cytokines to enhance or suppress inflammation (49). CD4+ T cells can be differentiated into Th1 (antiviral/antitumor), Th2 (allergic), Th17 (autoimmune and pathogen responses), and Tregs (immunosuppressive) (50–53). CD8+ T cells are known as cytotoxic T cells (TC cells) and act to kill pathogen infected or malignant cells by secreting inflammatory cytokines along with cell lytic molecules such as perforin and granzyme (54). Studies have shown that most T cells, macrophages, mast cells, and type I alveolar epithelial cells are derived from normal tissues adjacent to tumors. Most plasma, dendritic, endothelial, and B cells are usually derived from tumor tissues, suggesting that tumors can stimulate their infiltration (15).

In the immune microenvironment, the infiltration of regulatory T (Treg) cells and CD8+ cytotoxic T cells are predominant, whereas the differential Treg genes are enriched in the G2M checkpoint pathways and E2F target pathways (15, 48). In addition, the proportions of effector CD4+ T cells, cytotoxic CD8+ T cells, DCs, and natural killer cells are higher in subsolid nodules (SSNs) than in advanced adenocarcinoma. In contrast, the proportions of regulatory and depleted CD4+ T and depleted CD8+ T are lower in advanced lung adenocarcinoma. These findings suggest that immune surveillance functions well in SSNs (46). CD4+ T cells, Tregs, and DC subtypes in GGN-ADC are comparable to those in solid nodular lung adenocarcinoma (SN-ADC) but significantly higher than those in normal lung tissues. However, the cytotoxicity scores of CD8+ effector memory T cells, gzmk+CD8+ effector T cells, and the natural killer (NK) cell subtypes are reportedly significantly lower in SN-ADC than in GGN-ADC, indicating similar immunity in both radiological components but attenuated cytotoxic function in the more aggressive components (39). This may be because CD8+ T cells derived from GGN-ADC enable cells to function by activating metabolic pathways, oxidative phosphorylation, and enhanced antigen presentation and processing pathways (44). The immunosuppressive microenvironment plays a major role in the occurrence and development of tumors, and T cell-mediated immunosuppression begins in preinvasive nodules and continues to increase in intensity during invasive tumor progression. Increased Treg density and T cell exclusion from cancer nests are the major immunosuppressive mechanisms of pure non-solid (PNS) nodules (17).

T cell types and numbers vary across pathological subtypes. A gradual decrease in CD8+ T lymphocytes, B lymphocytes, NK cells, and granulocytes occurs from normal lungs to AAH, AIS, MIA, and IAC, whereas the CD4+ T lymphocytes and CD4/CD8 ratio increase significantly under such conditions, suggesting that adaptive immune responses and immune escape begin in the preneoplastic stage (15, 32, 45, 47). In addition, exhausted CD8+ T cells and Tregs increase continuously as the pathology progresses, indicating further negative immune regulation as the tumor progresses (47). Meanwhile, as tumor heterogeneity increases with the transition from AIS to IAC, CD8+ effector T cells, NK cells, and myeloid cells act as promoters, and B cells act as suppressors of tumor heterogeneity (15).

T cells are at the core of antitumor immunity in various tumors, and the T cell receptor immune repertoire reflects the strength and diversity of T cell responses; however, chromosomal instability and HLA loss inhibit T cell responses (47, 55). Dejima et al. (47) observed that T-cell clonal strength and clonality were the highest in normal lung tissues and decreased gradually in AAH, AIS, MIA, and invasive ADC. Conversely, T-cell density and diversity increased gradually. They also reported that T-cell receptor oligoclonality and the proportion of the top 10 clonal expansions in PSN-containing GGO components were indistinguishable from those in benign nodules but lower than those in SN, suggesting that T-cell clonal expansion was lower in GGNs (35).In addition, it was found that the infiltration of activated CD4+ T cells, activated CD8+ T cells, and regulatory T cells was negatively correlated with the proportion of GGO components (35).

B cells play a key role in acquired immunity. They can perform various antibody-independent functions, including engulfing and processing antigens for T cell presentation, secreting soluble mediators (such as interleukin [IL]-10), providing costimulatory signals, and participating in lymphoid tissue development (56). B cells play diverse roles in the tumor immune microenvironment. They can aggregate with T cells and dendritic cells to form tertiary lymphoid structures as an adaptive immune response to persistent tumor antigens (57). Immune escape from tumors is facilitated by regulatory B cells, which have potent immunosuppressive properties (58). GGN-ADC has significantly stronger immune responses of B cells, and antigen processing and presentation are enhanced significantly; NK cell-mediated cytotoxicity and allogeneic rejection pathways are also enhanced significantly in GGN-ADC compared to SN-ADC (44). Studies have shown that B cells in SSN exhibit inflammatory gene expression patterns, whereas B cells in advanced lung ADC are actively transcribed and have a stronger secretory phenotype (46). B cells increase gradually from normal lung tissues to ADC (32). This indicates that as the disease progresses, the number and function of B cells change further.

As the primary effector cells of the natural immune system, NK cells play an important role in controlling tumor development (59). NK cells maintain homeostasis through the expression of regulatory receptors related to their killing function. NK cells have two cell subtypes, NK-C1 and NK-C2. Compared to NK-C2, NK-C1 represents the most cytotoxic cluster. Normal lung tissues and SSN have comparable percentages of NK cells; however, NK-C1 cells in SSN have a markedly higher cytotoxic proportion than in LUAD with lymph node metastasis (46). Multiple studies have revealed that NK cell activation induces a stronger immune response in GGN-ADC than in SN-ADC, and the number of NK cells decreases gradually with pathological progression, which may promote a more aggressive PNS nodule nature and immune escape (32, 39, 44).

Macrophages are critical components of the innate immune system. Their functions include phagocytosis, antigen presentation, defense, repair, and metabolism. In GGNs, macrophages tend to polarize toward M1, suggesting a stronger proinflammatory function than in SN (39). Macrophages derived from GGO-LUAD also secrete more proinflammatory cytokines (IL-1 and TNF-α) and chemokines (CCL4), whereas proinflammatory cytokines can recruit cytotoxic T cells to attack cancer cells (44). The macrophage subtype in precancerous lesions is dominated by alveolar macrophages, whereas that in invasive ADC is dominated by tumor-associated macrophages (TAMs), which promote tumor angiogenesis, migration, and invasion and form an immunosuppressive TME (45). In a previous study, TAM exhibited significantly higher infiltration in IAC than in AIS/MIA, whereas TAM infiltration was significantly lower in GGO-LUAD than in SN-LUAD (16). The myeloid subset of AIS is dominated by mast cells, whereas MIA and IAC are dominated by macrophages.

CAFs, a heterogeneous population, are the most prominent stromal components outside tumor cells; they can secrete various cytokines to regulate the occurrence and development of tumor cells (60). Fibroblast activation protein-α (FAP) is one of the specific markers of CAFs and is generally not expressed in normal tissue cells but is highly expressed in more than 90% of malignant CAFs derived from epithelial cells; it promotes tumor cell proliferation, metastasis, and immunosuppression (61). A previous study demonstrated that GGN-ADC fibroblasts expressed lower levels of collagen than cells derived from SN-ADC, whereas the cell metabolism of the derived fibroblasts was inhibited (44). However, GGN-ADC also had a similar distribution of fibroblast subtypes as SN-ADC (39). Reproducible detection of myofibroblasts (ACTA2 and RGS5) in AAH and AIS is achievable, indicating that they may represent the TME in early-stage lung ADC (45). Xing et al. discovered that fibroblasts from SSN and normal lung tissues were enriched in immunoregulatory features; however, fibroblasts from advanced lung ADC exhibited tumor-supportive features, such as oxidative phosphorylation, angiogenesis, epithelial–mesenchymal transition, and active transcriptional pathways (46). Furthermore, tumor-related fibroblasts negatively regulate protein kinase activity and proliferation of endothelial cells (48). The preinvasive state is characterized by genetic variations that affect the extracellular matrix and fibroblasts (17).

There are also limitations to the previous research on this topic. First, most of the samples studied were not large enough, and most studies were conducted in Asian non-smoking patients, requiring international collaborative studies in other ethnicities and smokers. Second, the biological complexity of GGNs may vary owing to differences in pathology and single-cell sequencing sampling locations, as well as intra-tumor heterogeneity. In the future, studies of larger cohorts of GGNs are needed to dissect the evolutionary trajectories of GGNs and their underlying molecular mechanisms using tools designed to deal with increasingly complex biological features.

Video-assisted surgery is increasingly applied to the treatment of GGNs instead of conventional thoracotomy with fewer ports and smaller incisions. Different surgical approaches need to be developed depending on the size and location of the GGNs. Although lobectomy is the standard treatment for early-stage lung cancer because the tumor biology of GGNs may differ from a previous diagnosis of lung cancer, lung segmental resection and lung wedge resection are surgical options. In patients with mixed GGNs, wedge resection should be considered with caution because of the high recurrence rate (67). If the lesion is deeper but still within one of the lung segments, then segmental resection may be considered. If the lesion is located between two or more segments or in a bronchial root segment, then lobectomy is indicated (68). In addition, intraoperative frozen pathology is suggestive of the choice of surgical approach for early-stage lung cancer with GGNs. Multiple studies have drawn similar conclusions and reported no cases of hilar and mediastinal lymph node metastases in GGO-predominant lung tumors, and mediastinal lymph node sampling/selective lymph node dissection is acceptable in patients with c-stage I non-small-cell lung cancer (NSCLC) with GGO-predominant tumors (69, 70).

When multiple GGNs are present in different locations, an appropriate surgical approach based on the principles of surgical oncology that preserves lung function as much as possible is needed (71, 72). Several studies have demonstrated a satisfactory prognosis for patients undergoing resection of multiple GGNs (71–74). Previous studies on the surgical treatment of GGNs have been mostly retrospective, and further large-sample, prospective trials are needed to determine the effect of surgical modality on the prognosis of GGNs as well as interactions between multiple GGNs to determine the optimal surgical treatment strategy.

SBRT is an alternative therapy for patients with inoperable early-stage NSCLC who are unable or unwilling to undergo surgical resection. Multiple studies have reported no significant difference in survival between patients with stage I lung cancer with GGNs who underwent surgery or SBRT (75, 76). As a precise minimally invasive technique, image-guided thermal ablation has been increasingly used to treat early−stage lung cancer (77–79). Studies have found that either radiofrequency ablation, cryoablation, or microwave ablation (MWA) are safe, minimally invasive, and have good survival rates for the treatment of lung cancer with GGNs (80–82). Huang et al. (83) retrospectively evaluated the MWA efficacy in the treatment of pulmonary multiple concurrent GGNs. The results showed that CT-guided percutaneous MWA for the treatment of multiple simultaneous pulmonary GGNs was feasible, safe, and effective. Radiofrequency ablation may be a suitable option for patients with GGNs who refuse surgery.

Systemic therapy for lung cancer with GGNs has also been reported in some retrospective studies. It was found that chemotherapy was not efficacious against lung adenocarcinoma with GGNs; therefore, chemotherapy should not be a treatment option (84, 85). In multiple primary lung adenocarcinomas, most lesions show multiple GGNs in imaging, many of which have EGFR mutations (86). Cheng et al. (87) conducted a study to determine whether postoperative oral EGFR-TKIs were effective in patients with unresected GGNs with synchronous multiple primary lung cancer (sMPLC) and EGFR mutations. The results showed that EGFR-TKIs showed limited efficacy against these unresected GGO lesions. This may be related to the inability to access the genetic status of each GGN and the heterogeneity among GGNs. Xu et al. (88) showed that the use of immune checkpoint inhibitors was efficacious and safe for patients with sMPLC, which exhibited promise as a neoadjuvant or adjuvant immunotherapy for such patients. Additional multicenter prospective randomized controlled trials are needed to determine whether immunotherapy is effective for treating GGNs.