Objective: The present study is to investigate the expression of CD34, β-Tubulin-III and Collagen IV-Laminin in adenocarcinoma in situ (AIS), the AIS component of minimally invasive adenocarcinoma (MIA), and early invasive foci, in order to find a valuable immunohistochemical marker for discriminating AIS and its early invasive foci. Methods: A total of 51 AIS patients and 88 MIA patients were included in the present study. In addition, 40 atypical adenomatous hyperplasia (AAH) patients and 54 invasive adenocarcinoma (IA) patients were included as control. Immunohisto-chemical staining of β-Tubulin-III, CD34, CD31, F8 and Collagen IV-Laminin was performed by serial sectioning. β-Tubulin-III was used to show invasive adenocarcinoma foci, CD34 was used to indicate interstitial cells in AIS, CD31 and F8 were used to identify capillary endothelial cells in tumor tissues, and Collagen IV-Laminin was used to visualize the basement membrane component of AIS. Results: The basement membranes and interstitial cells of AAH, AIS and the AIS component of MIA had positive expression of CD34, while mucinous AIS and various invasive adenocarcinomas had no CD34-positive basement membranes or interstitial cells. Invasive cancers such as alveolar adenocarcinoma, papillary adenocarcinoma, micropapillary adenocarcinoma and solid adenocarcinoma had strong positive expression of β-Tubulin-III, while AAH, AIS and the AIS component of MIA, and invasive mucinous adenocarcinoma had negative expression of β-Tubulin-III. AAH, AIS and the AIS component of MIA were surrounded by basement membranes with positive expression of Collagen IV-Laminin, AIS and the AIS component of MIA had significantly thickened basement membranes, and none of invasive adenocarcinomas was surrounded by basement membranes. Conclusions: The present study demonstrates that immunohistochemical staining of CD34, β-Tubulin-III, and Collagen IV-Laminin discriminates AIS component of lung adenocarcinoma from early invasive foci, with the efficacy of β-Tubulin-III being the best. Staining of β-Tubulin-III precisely identifies the early invasive foci of MIA, and can be used as a marker for the identification of the early invasive foci of nonmucinous lung adenocarcinoma.
Treatment strategies for lung adenocarcinoma at different stages are different, and the pathological staging of lung adenocarcinoma has great significance in the clinical treatment and prognosis of the disease. Noguchi et al. show that adenocarcinoma in situ (AIS) may develop into invasive adenocarcinoma (IA), which is characterized as Noguchi type A (simple adherent growth), Noguchi type B (adherent growth with stromal hyperplasia and alveolar collapse), and Noguchi type C (adherent growth accompanied by local infiltration and stromal hyperplasia) [
Roh et al. report that interstitial cells of adherent adenocarcinoma have positive expression of CD34, while interstitial cells of invasive adenocarcinoma have negative expression of CD34 [
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 (
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 (EliVisionTM 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
Pathological types | N | Female (%) | Age (years) |
---|---|---|---|
AAH | 40 | 65 | 51.1 (30 - 71) |
AIS | 51 | 70.6 | 55.4 (26 - 76) |
Nonmucinous | 49 | 69.4 | 54.9 (26 - 76) |
Mucinous | 2 | 100 | 68.5 (66 - 71) |
MIA | 88 | 60.2% | 58.3 (26 - 84) |
Nonmucinous | 86 | 60.5 | 58.2 (26 - 84) |
Mucinous | 2 | 50.0 | 61 (60 - 62) |
IA | 54 | 44.4 | 56.85 (36 - 75) |
Adherent | 12 | 33.3 | 54.7 (47 - 69) |
Acinar | 10 | 40.0 | 58.4 (44 - 73) |
Papillary | 4 | 100 | 56 (56 - 56) |
Micropapillary | 8 | 25.0 | 64 (58-72) |
Solid adenocarcinoma | 10 | 40.0 | 58.8 (36-75) |
Mucinous adenocarcinoma | 10 | 60.0 | 50.6 (41-61) |
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 (
Groups | N | Tumor basement membrane and interstitial cells | Tumor cells |
---|---|---|---|
CD34 | β-Tubulin-III | ||
AAH | 40 | 1.67 ± 0.49 (75%) | 0 (0%) |
AIS | 51 | 3.73 ± 0.46 (100%) | 0 (0%) |
MIA | 88 | 3.82 ± 0.39 (100%) | 0.92 ± 0.58 (94.4%)* |
IA | 54 | 0.22 ± 0.49 (18.5%)** | 2.41 ± 1.15 (92.6%)** |
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 (
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 (
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) (
The early invasive boundaries of most tumors are defined as the breakthrough of the basement membranes. However, there are still some limitations when using basement membrane staining to observe cancer breakthrough, because local inflammation usually leads to blurry or disappeared staining of basement membranes in intraepithelial tumors. Some researchers use interstitial changes after breakthrough of basement membranes to judge the invasion of intraepithelial tumors. For example, the interstitial cells in bronchioloalveolar carcinoma [
In previous researches, β-Tubulin-III is mainly used in chemotherapy resistance studies of lung cancer or other cancers [
In the follow-ups of the 233 patients, recurrence and metastatic rate of AAH and AIS groups was 0%, that of MIA group was 1.14%, and that of IA group was 37.04%. Tumor-free cumulative survival rate of IA group was significantly lower than the other three groups. The result of follow-ups demonstrates that the three markers are reliable auxiliary indicators for the diagnosis of early invasive foci of lung adenocarcinoma. In conclusion, immunohistochemical staining of β-Tubulin-III, CD34 and Collagen IV-Laminin is effective in discriminating AIS component of lung adenocarcinoma from early invasive foci, with the efficacy of β-Tubulin-III being the best. Staining of β-Tubulin-III precisely identifies the early invasive foci of MIA, and can be used as a marker for the identification of the early invasive foci of nonmucinous lung adenocarcinoma.
In conclusion, when non-mucinous lung adenocarcinomas were in situ carcinomas, tumor cells were negative for β-Tubulin-III. When tumor invasion occurred, tumor cells became positive for β-Tubulin-III. β-Tubulin-III could be detected clearly in early invasive non-mucinous lung adenocarcinoma to avoid over- or inadequate diagnosis of microinvasive pulmonary adenocarcinoma.
Qinghai Yang,Huiling Chen,Dehua Zeng,Xuzhou Wang,Zhiyong Zheng, (2016) β-Tubulin-III Is an Immunohistochemical Marker for the Early Invasive Foci of Nonmucinous Lung Adenocarcinoma. Open Journal of Pathology,06,162-170. doi: 10.4236/ojpathology.2016.63019