A total of 51 AIS patients and 88 MIA patients admitted in our hospital between January 2007 and September 2014 were included in the present study. In addition, 40 AAH patients and 54 IA patients were included as control (Table 1). All patients received high-definition CT chest radiography. AAH was mainly treated by local excision, and AIS, MIA, and IA were mainly treated by lobectomy. In addition, lymphadenectomy and postoperative chemotherapy were also performed on IA patients. Samples excised during surgeries were fixed by neutral formalin, paraffin-embedded, and subjected to hematoxylin and eosin staining and immunohistochemical staining (TTF-1, Napsin A, CK7, P63, CK5/6, CD56, CgA, and Syn). Diagnosis of lung adenocarcinoma was performed following the new classification by 2011 IASLC/ATS/ERS. Follow-ups of patients were conducted via outpatient, letter or telephone. Follow-ups were terminated when recurrence or metastasis occurs. All procedures were approved by the Ethics Committee of Dongfang Hospital, Fujian Medical University. Written informed consents were obtained from all patients or their families.

For the staining of β-Tubulin-III, CD34, CD31, F8 and Collagen IV-Laminin, paraffin-embedded specimens were cut into 4 μm sections. Sections for CD34 (QBEnd/10; Thermo Fisher Scientific, Waltham, MA, USA), β- Tubulin-III (TUJ1; Covance, Princeton, NJ, USA), CD31 (Thermo Fisher Scientific, Waltham, MA, USA) and F8 (Zymed, Thermo Fisher Scientific, Waltham, MA, USA) staining were submerged into citrate (0.01 M, pH 6.0), and autoclaved for antigen repair. For Collagen IV (PHM-12; Thermo Fisher Scientific, Waltham, MA, USA)-Laminin (LAM-89; Sigma-Aldrich, St. Louis, MO, USA) staining, the samples were digested with 0.05% protease XXIV (P8038; Sigma-Aldrich, St. Louis, MO, USA) for 40 min. Other procedures were performed according to the manufacturer’s manuals (EliVision^TM plus; Maixin Biotech. Co., Ltd., Fuzhou, China). CD31 and F8 were used to visualize the capillary endothelial cells in lung adenocarcinoma tissues, being in comparison to

Note: AAH, atypical adenomatous hyperplasia; AIS, adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma; IA, invasive adenocarcinoma.

CD34-positive interstitial cells. Collagen IV-Laminin was used to identify basement membrane components of lung adenocarcinoma that were in contrast to CD34-positive basement membrane components in lung adenocarcinoma.

To evaluate the immunohistochemical scores, positive staining of β-Tubulin-III was indicated by cytoplasm staining, and positive staining of CD34 was indicated by staining of basement membrane and interstitial cells. Negative expression scored 0 point, positive expression <25% scored 1 point, positive expression between 25% and 50% scored 2 points, positive expression between 50% and 75% scored 3 points, and positive expression between 75% and 100% scored 4 points. Average positive expression score = total positive expression score/ number of cases.

All results were analyzed using SPSS 19.0 statistical software (IBM, Armonk, NY, USA). Data of each group were expressed as means ± standard deviations. Comparison between two groups was performed using Mann- Whitney U test. Comparison among multiple groups was performed using Kruskal-Wallis H test. Lung cancer recurrence or metastatic rate were subjected to univariate survival analysis by Kaplan-Meier method, and tested using log-rank method. All tests were bilateral. Differences are statistically significant if P < 0.05.

To measure the expression of CD34 and F8, immunohistochemistry was performed. Strong positive expression of CD34 was observed in the basement membrane of AIS, while moderate positive expression of CD34 was observed in interstitial cells of AIS (Figure 1(a)). In addition, strong positive expression of CD34 was observed in the basement membrane and interstitial cells of adherent adenocarcinoma of MIA and IA (Figure 1(b)). All interstitial cells of acinar adenocarcinoma in MIA and IA showed negative expression of CD34, but acinar adenocarcinoma with non-invasive growth was surrounded by basement membranes with positive expression of CD34 (Figure 1(c)), and acinar adenocarcinoma with invasive growth was not surrounded by CD34-positive basement membranes (Figure 1(d)). In CD34 staining images, it is difficult to discriminate tumor interstitial cells with positive expression of CD34 from vascular endothelia. And the vascular endothelial cells showed positive expression of CD31 and F8. By comparing CD31 and F8 staining images, basement membranes with positive expression of CD34 were differentiated from interstitial cells (Figure 1(b) and Figure 2(a), Figure 2(b)). The basement membranes and interstitial cells of AAH had negative or weak positive expression of CD34 (Figure 1(e)). Other invasive cancers such as papillary adenocarcinoma, micropapillary adenocarcinoma and solid adenocarcinoma were not surrounded by basement membranes, but their interstitial cells had negative CD34 expression. In addition, the basement membranes and interstitial cells of noncancerous alveolar tissues and mucinous adenocarcinoma had no CD34 expression (Figure 1(f)). The vascular endothelial cells in all groups showed positive expression of CD34. Statistical analysis showed that the average CD34-positive expression score of IA patients was significantly lower than those of AAH, AIS and MIA patients (P < 0.05) (Table 2). These results suggest that the basement membranes and interstitial cells of AAH, AIS and the AIS component of MIA have positive expression of CD34, while mucinous AIS and various invasive adenocarcinomas have no CD34-positive basement membranes or interstitial cells.

Note: AAH, atypical adenomatous hyperplasia; AIS, adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma; IA, invasive adenocarcinoma. ^*P < 0.05 compared with AAH or AIS; ^**P < 0.05 compared with AAH, AIS or MIA.

To detect the expression of β-Tubulin-III, immunohistochemistry was employed. Tumor cells of both AAH and AIS patients showed negative expression of β-Tubulin-III. In addition, adherent adenocarcinoma of MIA and IA group showed negative expression of β-Tubulin-III. Alveolar adenocarcinoma in MIA and IA groups that was surrounded by basement membranes had positive expression of β-Tubulin-III (Figure 1(c) and Figure 3(a)). Invasive alveolar adenocarcinoma that was not surrounded by basement membranes showed strong positive expression of β-Tubulin-III (Figure 1(d) and Figure 3(b)). Other invasive cancers such as papillary adenocarci- noma, micropapillary adenocarcinoma and solid adenocarcinoma showed strong positive expression of β-Tub- ulin-III (Figures 3(c)-(f)). Noncancerous alveolar tissues and mucinous adenocarcinoma showed negative expression of β-Tubulin-III. Statistical analysis showed that the average β-Tubulin-III-positive expression score of IA patients was significantly higher than those of AAH, AIS and MIA patients (P < 0.05) (Table 2). These results indicate that invasive cancers such as alveolar adenocarcinoma, papillary adenocarcinoma, micropapillary adenocarcinoma and solid adenocarcinoma have strong positive expression of β-Tubulin-III, while AAH, AIS and the AIS component of MIA, and invasive mucinous adenocarcinoma have negative expression of β-Tubulin- III.

For the staining of Collagen IV-Laminin, immunohistochemistry was used. Both AAH and AIS had complete basement membranes, while AIS had significantly thickened basement membranes. Adherent adenocarcinoma of MIA and IA showed significantly thickened basement membrane surrounding. Non-invasive alveolar carcinoma of MIA and IA was surrounded by basement membranes, while invasive alveolar carcinoma was not surrounded by basement membranes. Other invasive cancers such as papillary adenocarcinoma, micropapillary adenocarcinoma, solid adenocarcinoma and invasive mucinous adenocarcinoma were not surrounded by basement membranes. After comparing with CD34 staining images, the basement membranes of tumor tissues had expression of CD34 (Figure 1(c) and Figure 4). These results suggest that AAH, AIS and the AIS component of MIA are surrounded by basement membranes with positive expression of Collagen IV-Laminin, AIS and the AIS component of MIA have significantly thickened basement membranes, and none of invasive adenocarcinomas is surrounded by basement membranes.

To determine the tumor-free cumulative survival rate, 233 patients were followed up in 3 - 85 months after the surgery and the data were analyzed using Kaplan-Meier method. The recurrence and metastasis rate for AAH group was 0/40 (0%); that for AIS group was 0/51 (0%); that for MIA group was 1/88 (1.14%); and that for IA group was 20/54 (37.04%). Statistical analysis showed that the survival rate of IA group was significantly lower than those of AAH, AIS and MIA groups (P < 0.05) (Figure 5). The result indicates that invasive adenocarcinomas result in the lowest cumulative survival rate among all groups.