The pulmonary GGNs could be detected without any symptoms during regular health examinations. Although GGNs can grows in any part of the lung, they mostly reside in the bilateral upper lobes, especially in the right lung. According to Chen et al., the prevalence of lung cancer among people older than 45 years increases significantly with age. This report agrees with the findings of the present study; we found that patients aged 45 to 65 years had a higher risk of developing GGNs in LUAC, which was more common in female non-smokers. The GGNs have been identified as a signal of potential early LUAC. Unlike solid adenocarcinoma (SADC), GGNs had satisfactory prognoses of long-term OS in apparent characteristics of indolent growth and rare lymph node metastasis, including distant metastasis and inner changes of cancer cells and tumor microenvironment (TME) . Liu et al. found that the prognosis in patients with IAC is worse than in AIS/MIA; the five-year DFS in patients with AIS/MIA was 100% and 74.1% in IAC. Notably, GGNs smaller than 6 mm or stable for five years still had a risk of developing LUAC[21, 22]. Clinical management, taking measures, and continued follow-up mainly relied on morphological characteristics and behavior of GGNs. It is worth noting that an unfavorable location such as hilus pulmonis might influence disease progression and accelerate early intrapulmonary lymph node metastasis, as was observed in this study.
Early growth of the tumor is accompanied by neovascularization and vascular remodeling. Localized or extensive tumor cells typically infiltrate, invade the surrounding lung tissue, and fibrous tissue proliferates with shrinkage, stretching the adjacent visceral pleura, along with hyperplasia of alveolar epithelial and thickening of the alveolar septum[24–26]. Before tumor histopathological diagnosis is made, objective quantitative indicators of the maximum diameter and solid component of GGNs could effectively evaluate the degree of invasion in LUAC. The larger size of GGNs, the greater risk of malignancy. The solid component is representative of mGGNs, as its appearance showed a higher possibility of IAC. The transformation from pGGN into mGGN is more likely to a process of tumor-advanced evolution from AIS into IAC with a gradual increase in the proportion of solid components. The invasiveness improves until a complete solid nodule with strong invasiveness is achieved. Research has shown that, unlike SADC, GGNs of LUAC are characterized by slow growth, low-grade malignancy, and downregulation of signaling pathways related to cell proliferation and angiogenesis at the molecular level. Molecular alterations, biological characteristics, and clinical phenotypes of GGNs are also affected by intratumor heterogeneity (ITH), as oncogenic drivers of all tumor cells within the same primary tumor may be different[28–30].
Herein, EGFR was the most frequent driver for gene mutation, with exon 21 L858R mutation being the most prevalent, followed by exon 19 deletion. These findings concur with previous pandemic statistics[31, 32], considering cancer-promoting genes in tumor genesis and processing. Subtle alterations emerged in EGFR status as a tumor at an early stage of AIS was seen in the case of one AIS patient of pGGN with EGFR L858R- positive mutation. Besides, HER2, BRAF, KRAS, and MAP2K1 mutations were also seen in pre-invasive lesions, suggesting that multiple gene activation might induce early stage tumorigenesis. A study reported that mutations in the canonical cancer genes, EGFR, HER2, NRAS, and BRAF, participate in early genomic events before the acquisition of invasiveness. However, another study reported no significant difference in EGFR mutation alteration between early and advanced cancer stages, suggesting that EGFR mutation participates in tumor initiation and maintenance. A systematic review reported that EGFR mutation in GGNs had no apparent relationships in neither radiological progression nor tumor advancement. On the contrary, we found that the frequency of EGFR mutations in IAC was more than AIS/MIA. We, therefore, hypothesized that some special components were generated to promote tumor progress by positive feedback during the slow-growing phase of GGNs. Later we found that mGGNs were the predominant type in IAC patients with positive EGFR mutation, causing us to pay closer attention to solid components that could influence tumor invasiveness. The Solid component showed pathological changes, including increased fibrotic tissue, vastly infiltrated tumor cells, and angiogenesis, providing a comfortable TME for a variety of tumor-associated cells and promoting tumor growth as well as a malignant biological behavior. To some extent, early complex TME with high ITH attributed to different phenotypes and invasion of LUAC with EGFR mutation. Surprisingly, researchers have a strong interest in predicting EGFR mutation status in a non-invasive method with CT images, though it had not been sufficiently explored.
Besides, the status of ALK rearrangement was also crucial among LUAC. Patients with stage IA of ALK-positive adenocarcinoma had a worse prognosis and higher chances of developing regional lymph node metastasis. Whereas ALK-positive mutation is common in GGNs, his study recorded only two in six patients with ALK fusion mutations, and no ALK fusion co-existed with any other mutation. However, this result could be attributed to the few samples examined in the present study. Similar findings were recorded in other genetic markers. TP53 as a co-existing gene mutation in LUAC with GGNs played a crucial role in maintaining normal cell growth and inhibiting tumor proliferation, of which inactivation could promote carcinogenesis and cancer metastasis. A previous study reported that TP53 mutation could disrupt the regular cell cycle control, becoming an early tumor-initiating event in EGFR-mutant LUAC. Herein, we found 16.2% EGFR-mutant patients with TP53 mutation, which is less than a previous study that reported 30–60% mutation. Xu et al. found that TP53 mutation could significantly affect the sensitivity of tumor cells to EGFR-TKI and long-term prognosis, resulting in primary resistance. Besides, TP53 was thought to have an association with germline mutations in LUAC. Since the solid component of GGNs was less than that of nodules and had lower invasiveness, we speculate that TP53 mutation in GGNs was unfavorable and might reduce the efficacy of targeted therapy of EGFR.
The expression of PD-L1 in tumor tissues was up-regulated to escape immune response due to acquiring resistance to molecular targeted therapy. As such, immunotherapy compensates for wild-type driver genes and disease progression, achieving significant clinical benefits[43–46]. In the present study, the expression level of PD-L1 positively correlated with the therapeutic effect; the higher the TPS, the greater the clinical benefit[46, 47]. Following previous studies, the PD-L1 expression was pretty lower in GGNs, predicting that GGNs had their unique immune mechanism and patients may have a less sensitive response to PD-L1 inhibitors than SADC[35, 48]. D.P. Carbone et al. reported that patients with PD-L1 ≥5% and a higher TMB level respond better to immunotherapy. Another study proposed that EGFR-mutated patients with primary resistance might benefit from immunotherapy if both PD-L1 and CD8 + are positive. Notably, high PD-L1 expression was found when TP53 mutation was without co-occurring gene alterations. The study found that pathological subtypes in GGNs do not influence the expression of PD-L1 in tumor cells. In contrast, PD-L1-negative patients also benefited significantly, making the benefit group no longer limited to PD-L1-positive patients.
The present study revealed that changes in TMB appear in the early stage of lesions. Notably, IAC patients had a higher TMB, suggesting that an activation pathway accelerated gene mutation in IAC even if GGNs had a low mutational burden. TMB played a predictive effect on immunotherapy since somatic mutations could produce new antigens and then be recognized by T cells to kill tumor cells[53–55].
In fact, some shortcomings of this study, such as manual measurement of the nodule diameter, and subjective judgment of CT signs, might cause deviations in the research findings to a certain extent, and reflecting the clinical-pathological characteristics of GGNs in an unprecise way. Limiting to a small sample size, the result could not represent overall data well. On the other hand, tumors had ITH not only in multifocal lesions, but in different region of the same nodule, small tissue specimens could not provide complete information about genetic alterations in GGNs. Last but not the least, we did not conduct the whole exome sequencing in those tumor tissue specimens, and the thorough molecular mechanism behind slow growth of GGNs was not refer to. All of these limitations mentioned above will be take into consideration in further exploration.