Clinical Characters in Pre-invasive and Invasive Adenocarcinoma with Pulmonary Ground-glass Nodules

Purpose: With the increasing prevalence of pulmonary ground-glass nodules (GGNs) among younger population, its clinicopathologic performance, lung cancer-associated genetic mutation, and immune landscape features between pre-invasive adenocarcinoma and invasive adenocarcinoma (IAC) need to be get well known. Methods: We retrospectively reviewed basic clinical information, analyzed radiological characteristics, and then evaluated the status of mutational hotspots and tumor mutational burden by sequencing genome in tissue. Programmed death ligand 1 (PD-L1) expression was detected by immunohistochemistry staining. Results: Nodules vastly increased the probability of IAC when the diameter of GGNs was more than 1.15 mm or the consolidation-to-tumor ratio was at least 8.5%, with the latter predictor having a better diagnostic specicity. Tumors positive for exon 19 deletion and exon 21 L858R in EGFR mutation had a higher prevalence in IAC. However, there was no difference in PD-L1 expression. As expected, tumor mutational burden in IAC was higher, despite a low background mutational burden as a whole. Conclusions: GGNs should be pay high attention when several aggressive behaviors showed in radiology and inner solid components increased gradually, providing more evidence apt to a diagnosis of IAC. We found that GGNs of IAC performed early genomic alternations events during the slow growth carcinogenesis stage of GGNs, including the most common proto-oncogene EGFR activation, which mainly concentrates on IAC. Indolent GGNs at an early stage usually have negative PD-L1 expression.


Introduction
The latest global cancer data shows that the incidence of lung cancer ranked second worldwide in 2020.
In China, the incidence and mortality rank highest, constituting 23.8% of the total cancer deaths [1] . The extensive application of low-dose thoracic computed tomography (CT) in the detection of ground-glass nodules (GGNs) has increased, raising widespread attention in recent years. The GGNs are round-like or irregular ground-glass opacities, with cloudy fuzzy density shadows. The GGNs were observed in lung windows on CT, in which the internal blood vessels and bronchi were uncovered completely and often closely related to lung adenocarcinoma (LUAC). The GGNs are divided into pure GGN (pGGN) and mixed GGN (mGGN) based on differences in density. It is still a challenge diagnosing GGNs using radiology.
Benign lesions are the leading cause of ground-glass changes, such as in ammatory infection, pulmonary brosis, granulomatous lesions, pulmonary haemorrhage, intrapulmonary lymph nodes, and carbon dust which could be contracted or absorbed gradually during long-term follow-up [2] . A small part of GGNs evolves into malignant tumors with slow growth in the early stage [3,4] . Thus, proper management of these indolent GGNs is needed [5,6] . Avoiding over-treatment and delayed surgery does not have a signi cant impact on prognosis [7,8] .
According to the recent WHO classi cation of thoracic tumors [9] , adenocarcinoma in situ (AIS) was reclassi ed as precursor glandular lesion, with its management different from minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma (IAC), it was certainly worth taking into consideration because of molecular alterations [10,11] . Wilshire et al. reported that disease-free survival (DFS) in AIS and MIA of lepidic adenocarcinomas in stage I could be 100% and 96% respectively and 80% in IAC after surgical resection. The overall survival in AIS and MIA was 100% more than in IAC, 90%. The GGNs of early LUAC possess better prognostic survival than solid nodules, especially pGGNs reaching nearly 100% DFS after resection [12][13][14][15] . In the early stages of LUAC, GGNs with unique biology still have their invasive behaviors from AIS/MIA to IAC. When nodules grew rapidly, solid components arose, or previous solid components increased during follow-up, the risk of developing IAC and invasiveness were relatively higher [16] .
Preoperative evaluation of the radiological features of pulmonary GGNs and prediction of the degree of tumor in ltration can greatly help clinicians make diagnosis and treatment plans for malignant nodules. Knowing the dynamic genomic alterations and immune checkpoint status in pre-invasive and invasive LUAC could provide better guidance for precision-targeted therapy and immunotherapy in the latest treatments.

Patient Selection
We retrospectively reviewed complete electronic medical records of patients who accepted surgical resection of pulmonary GGNs at the Tianjin Medical University Cancer Institute and Hospital from March 2017 to May 2021. All eligible lesions met the following criteria; GGNs detected by CT and resected later, nodules con rmed with a pathologic result of AIS, MIA, and IAC. And lastly, specimen detected by nextgeneration sequencing. Patients were divided into two groups (AIS/MIA group and IAC group) according to the nal pathological results of scope and degree of tumor cells in ltration.

Histopathologic Classi cation
After surgical resection, tissue specimens were used to make a histological examination of immunohistochemistry staining (IHC). Each lesion was classi ed as AIS, MIA, and IAC according to the WHO classi cation of lung adenocarcinoma. The growth pattern and range of invasion of cancer cells were observed under a light microscope. Senior pathologists made the nal identi cation of pathologic subtypes.

Acquisition of Chest CT Images
Thoracic CT scans were performed in a 64-slice helical multidetector scanner (Optima CT680 Expert, USA) with multiple-plane reconstruction. Quantitative indexes were measured precisely using a pitch of 1.25mm in lung window settings (window level, 35 Houns eld units [HU]; window width, 320 HU). Mixed components in nodules were also observed to exclude calci cation in the mediastinal window (window level, -500 HU; window width, 1200 HU). Meanwhile, the largest diameter was measured and analyzed when the lesion shapes were irregular or multiple lesions were present. Consolidation-to-tumor ratio (CTR) was de ned as the ratio of the maximum size of the solid component against full lesion size in the axial plane (for pGGN, CTR=0; for mGGN, 0 CTR 1; for pure solid nodule, CTR=1). These images were evaluated for malignant performance combined with relative clinical information and history of past illness by two experienced radiologists.

Biomarker Detection
Sections of tumor tissue specimens were sent for next-generation sequencing in the Illumina sequencing platform. Comprehensive mutation information of the tumor tissues such as DNA point mutations, fusion, deletion, insertion, or germline mutation of lung cancer-relative hotspots were acquired using the GRCh37/hg19 genome as control. Subsequently, ALK-EML4, the most frequent fusion form, were evaluated by IHC with Ventana anti-ALK rabbit monoclonal primary antibody (D5F3, Roche). Not all patients admitted the detection of PD-L1 expression and tumor mutational burden (TMB) quanti cation in this study. In total, we collected 185 cases to assess the level of immune checkpoint PD-L1 expressed in tumor cells by Ventana PD-L1 Assay (SP263, Roche). According to the tumor proportion score (TPS), the results were marked as a negative or positive expression. The results could also be divided into low expression (1%-49%) and high expression (≥50%). In addition, TMB was de ned as the number of somatic mutations per megabase of the analyzed genomic sequence and was detected in 228 patients by high throughput sequencing.

Statistical Analysis
Categorical variables were shown as numbers or percentages and compared by the Pearson chi-square test or Fisher exact test. Continuous variables were compared by directly applying the Mann-Whitney U Test, and values are described as mean ± standard deviation. Logistic regression analysis was performed to determine the relationship between potential risk predictors closely advancing into IAC. The receiver operating characteristic curve (ROC) was made, and the area under the curve (AUC) was calculated to identify a proper diagnostic index with its best cut-off of distinguishing nodules between the pre-invasive lesion and IAC. The statistical analyses were done using SPSS software (version 26.0, IBM Corp, Armonk, NY) and GraphPad Prism software (version 8.0, GraphPad Software, La Jolla, CA). Tables and gures were generated using Microsoft O ce 2019 (Microsoft, Redmond, WDC, USA). Inspection levels α = 0.05 were considered statistically signi cant when the p-value was less than 0.05.
These meaningful univariate features were analyzed using multivariate indexes in logistic regression.
There were two EML4-ALK fusion mutations from six ALK mutations in AIS/MIA and IAC, respectively.
The three ROS1-mutated patients with one positive fusion mutation belonged to IAC. Only one specimen had BRAF V600E mutation in 20 BRAF-mutated patients (Fig.4e). Thirty patients had HER2 mutation, out of which 22 were in AIS/MIA. About 22 specimens had KRAS mutation, of which 9 were AIS/MIA and 13 IAC, with two having both EGFR L858R and TP53 inactivation. About 44 patients had the tumorsuppressor gene (TSG) TP53 inactivation, followed by other gene mutations. Among 35 patients with TP53 mutation carried by EGFR mutation, four patients were AIS/MIA, and 31 were IAC (Fig.4f). Eight IAC positive specimens had one exon 14 skipping mutation and one gene ampli cation. Three of the six specimens had RET mutation with EGFR L858R mutation, and the other fused with CCDC6 KIF5B, and NTRK3. Ampli cation of MDM2 was observed in 11 specimens, of which seven were IAC. Gene fusion of NTRK1/2/3 could also be seen in 10 specimens, of which 7 were IAC. In addition, other gene mutations of PIK3CA, MAP2K1, and CDKN2A are detailed in Figure 4b.

Immune-relative landscape Feature Analysis
We evaluated the expression of PD-L1 in 185 patients (67 AIS/MIA and 118 IAC) and (109 pGGNs and 76 mGGNs). Sixty-seven expressed PD-L1, while 118 were negative. Twenty-two (32.8%) of the PD-L1 positives were AIS/MIA and 45 (38.1%) IAC (p=0.471) (Fig.5a). Neither GGNs in the negative PD-L1 expression nor in two level positive expression, there were no difference of between AIS/MIA and IAC (Fig.5b). Among the distribution of three expression levels of PD-L1 accompanied with diverse driver gene mutations, negative PD-L1 expression with EGFR-mutated GGNs were the predominant part, followed with low PD-L1 expression with EGFR-mutated GGNs, and those small part of high PD-L1 expression GGNs mainly had EGFR mutation (Fig.5c).

Discussion
The pulmonary GGNs could be detected without any symptoms during regular health examinations [17] .
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 signi cantly with age [18] . This report agrees with the ndings 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 identi ed 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) [19] . Liu et al. found that the prognosis in patients with IAC is worse than in AIS/MIA; the ve-year DFS in patients with AIS/MIA was 100% and 74.1% in IAC [20] . Notably, GGNs smaller than 6 mm or stable for ve 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 [23] . It is worth noting that an unfavorable location such as hilus pulmonis might in uence 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 in ltrate, invade the surrounding lung tissue, and brous tissue proliferates with shrinkage, stretching the adjacent visceral pleura, along with hyperplasia of alveolar epithelial and thickening of the alveolar septum [24][25][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 [27] . 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 [19] . 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][29][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 ndings concur with previous pandemic statistics [31,32] , considering cancer-promoting genes in tumor genesis and processing [33] . 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 [10] . However, another study reported no signi cant difference in EGFR mutation alteration between early and advanced cancer stages [34] , 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 [35] . 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 in uence tumor invasiveness. The Solid component showed pathological changes, including increased brotic tissue, vastly in ltrated 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 [36] . Surprisingly, researchers have a strong interest in predicting EGFR mutation status in a non-invasive method with CT images, though it had not been su ciently explored [37] .
Besides, the status of ALK rearrangement was also crucial among LUAC. Patients with stage IA of ALKpositive adenocarcinoma had a worse prognosis and higher chances of developing regional lymph node metastasis [38] . 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 ndings 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 [28] . Herein, we found 16.2% EGFR-mutant patients with TP53 mutation, which is less than a previous study that reported 30-60% mutation [39] . Xu et al. found that TP53 mutation could signi cantly affect the sensitivity of tumor cells to EGFR-TKI and long-term prognosis, resulting in primary resistance [40] . Besides, TP53 was thought to have an association with germline mutations in LUAC [41] . 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 e cacy 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 [42] . As such, immunotherapy compensates for wild-type driver genes and disease progression, achieving signi cant clinical bene ts [43][44][45][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 bene t [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 [49] . Another study proposed that EGFR-mutated patients with primary resistance might bene t from immunotherapy if both PD-L1 and CD8 + are positive [50] . Notably, high PD-L1 expression was found when TP53 mutation was without co-occurring gene alterations [51] . The study found that pathological subtypes in GGNs do not in uence the expression of PD-L1 in tumor cells. In contrast, PD-L1-negative patients also bene ted signi cantly, making the bene t group no longer limited to PD-L1-positive patients [52] .
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][54][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 ndings to a certain extent, and re ecting 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.

Conclusion
In summary, the sluggish growth of GGNs usually have a safe follow-up period. Surgical intervention should be timely when the nodules grow, new solid components create, or typical malignant signs of lobulation, spicule sign, and pleural traction are showed on CT, which are considered as re ections of IAC possibly. Besides size of nodules, the index CTR is a more favorable predictor to distinguish IAC from GGNs. There must be some special single pathways activated to modify the growth pattern of GGNs, different from the mechanism in SADC. Early events of genetic alterations like EGFR, KRAS, TP53 mutations emerge to regulate the tumorigenesis and advance. Consistent with most LUAC, protooncogene EGFR activation is the most frequent mutation in GGNs of IAC. In addition, we reveal that most GGNs have negative expression of PD-L1, indicating immunological escape does not play a predominant role in tumorigenesis at the early stage. Though low mutational burden in GGNs, we do not sure whether those patients are sensitive to immunotherapy or not. In general, patients with pulmonary GGNs of LUAC often have a preferable prognosis and survival and EGFR-mutated patients may bene t a lot from molecular targeted adjuvant therapy.  Tables   Table 1 Basic information of 328 patients with ground glass nodules     The receiver operating characteristic curve (ROC) and the area under the curve (AUC) of nodule diameter and consolidation tumor ratio are showed to better diagnose pre-invasive and invasive adenocarcinoma with best sensitivity and speci city. The maximum diameter is a comprehensive index to apply in clinical activity, while CTR has more accurate speci city.