Bronchioloalveolar invasion in non-small cell lung cancer is associated with expression of transforming growth factor-β1

Adenocarcinoma is the most common histological type among lung cancers. As with malignancies in other organs such as the breast and cervix, where carcinomas are defined as non-invasive, micro-invasive or invasive, the extent of the invasive component in lung adenocarcinomas is associated with clinical outcomes [1]. To address advances in oncology, molecular biology, pathology, radiology, and surgery, an international multidisciplinary classification was sponsored by the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society, and the European Respiratory Society [2]. Adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) with fibrous stromal invasion are newly introduced subtypes, while the older term of bronchioloalveolar carcinoma does no longer exist according to new IASLC classification of lung adenocarcinoma. AIS is a localized small (≤3 cm) adenocarcinoma, the growth of which is restricted to neoplastic cells along preexisting alveolar structures without stromal, vascular or pleural invasion. Papillary or micropapillary patterns and intra-alveolar tumor cells are absent. MIA is a small, solitary adenocarcinoma growing in a predominantly lepidic pattern and showing ≤5 mm invasion in its greatest dimension at any one focus. If these tumors are completely resected, there will be 100% or near 100% disease-specific survival.

Transforming growth factor (TGF)-β reportedly promotes cancer metastasis by affecting the tumor microenvironment in a manner that facilitates tumor cell invasion [3, 4]. Several tumors, including those arising in the lung [5‐7], express high levels of TGF-β, which correlate with tumor progression and clinical prognosis. Humans express three highly homologous isoforms of TGF-β (TGF-βs 1, 2, and 3), which share 70% sequence identity in their biologically active C-terminal regions, and all of which bind to the same receptor complex and activate the same downstream signaling pathways. TGF-β1 is the most abundant and most extensively studied isoform [8].

We previously showed that tumor-derived TGF-β1 causes a reduction in the number of dendritic cells within the sentinel lymph node in lung cancer [9]. Our findings also suggest that TGF-β1 29T>C genetic polymorphism is associated with lymph node metastasis in patients with adenocarcinoma of the lung [10]. In addition, we showed that overexpression of TGF-β1 by tumor cells promotes metastasis into tumor-draining lymph nodes in mice, most likely by inhibiting dendritic cell migration from tumors towards tumor-draining nodes [11]. Collectively, these results suggest that TGF-β1 is a key mediator fostering tumor invasion and metastasis. We therefore analyzed the relationship between TGF-β1 expression and clinicopathological microinvasion in patients with small lung adenocarcinoma.

There were no differences between the AIS and MIA groups with respect to age, gender, nodal involvement or pathological stage; however, tumor size was greater in the MIA group than the AIS group (Table 1). Table 2 shows the incidence of TGF-β1 expression detected upon immunohistochemical examination in the AIS and MIA groups. Figure 3 shows the differences in TGF-β1 immunostaining between AIS and MIA scored using the Allred method. The rate of TGF-β1 positivity was significantly (P <0.05) higher in the MIA group (65.2%; 15/23) than the AIS group (27.3%; 6/22). Similarly, the median Allred score for TGF-β1 expression was significantly higher (P = 0.0017) in the MIA group than the AIS group (3.0 (range 0–6) vs. 0.5 (range 0–5)).

One instance of lymph node metastasis was detected in the MIA group using HE and IHC staining with AE1/AE3 antibodies. In addition, isolated tumor cells were found in a second patient diagnosed with MIA (Figure 4). This patient was staged as pT1aN0(i+)M0. The overall survival rates differed somewhat between AIS and MIA patients (100% vs. 89.5%), but that difference did not reach statistical significance (P = 0.093).

The results of the present study suggest that patients with MIA show greater expression of TGF-β1 than patients with AIS, as indicated by the significantly greater TGF-β1 positivity rate and Allred score. In addition, HE and IHC staining revealed that lymph node metastasis and isolated tumor cells were only present in the MIA group. Thus, TGF-β1 expression by adenocarcinomas appears to be an important factor associated with clinicopathological microinvasion in patients with small lung adenocarcinoma.

TGF-β increases the invasiveness of cancer cells by increasing their proteolytic activity and promoting their binding to cell-adhesion molecules [15]. In an earlier study, we demonstrated that tumor-derived TGF-β1 induces dendritic cell apoptosis within lymph nodes in non-small cell lung cancer [9], and that overexpression of TGF-β1 by tumor cells promotes lymph node metastasis in mice [11]. Consistent with those findings, the effects of TGF-β1 on angiogenesis, stroma formation and immune function appear to further support tumor progression and invasion [16, 17]. AIS is a small localized adenocarcinoma in which growth is restricted to neoplastic cells along preexisting alveolar structures without fibrous stromal invasion. In MIA, by contrast, tumor cells have infiltrated the myofibroblastic stroma. In many diseases, excessive TGF-β contributes to a pathological excess of tissue fibrosis that compromises normal organ function, a topic that has been the subject of several reviews [18‐20]. However, we provide no direct evidence of the mechanism by which TGF-β1 increases tissue fibrosis in MIAs.

The Noguchi classification [21] is predictive of outcome in patients with small adenocarcinomas, with type D, E, and F tumors showing a worse outcome than the other tumor types. However, the majority of small adenocarcinomas are classified as type C, so that subclassification of type C tumors does seem necessary. Tumors that meet the criteria for AIS were formerly classified as bronchioloalveolar carcinoma based on the strict definition of type A and type B adenocarcinomas from the 1995 Noguchi classification [21]. MIAs are tumors showing lepidic growth and a small (≤5 mm) area of invasion. The MIA invades areas of stromal fibrosis in an acinar pattern. Nearly all type C adenocarcinomas can be classified as MIAs. The outcome of patients with adenocarcinomas with diameters of 2 cm or less can be predicted using the following scoring system based on three histological criteria: 1) the tumor is lymphovascular invasion-positive; 2) the non-bronchioloalveolar carcinoma component is >10 mm in diameter; and 3) the percentage of the solid, cribriform, and/or papillary components is ≥30% of the entire tumor volume [22]. Patients whose MIAs have a non-bronchioloalveolar carcinoma component ≤5 mm show 100% survival [22, 23]. Consistent with that finding, no difference in survival was observed between AIS and MIA in the present study. However, nodal involvement was observed in 2 of 23 patients with MIA. We therefore suggest that it would be problematic to conduct limited surgery for MIA patients with micrometastasis or isolated tumor cells, though some MIAs may be candidates for limited surgery.

In conclusion, our findings suggest that patients with MIAs have both a significantly greater rate of TGF-β1 positivity and higher TGF-β1 Allred scores than patients with AIS. However, because we do not provide direct evidence of the mechanism by which TGF-β1 increases tissue fibrosis in MIAs, and we examined only a limited number of patients, further studies will be needed to more precisely define the role played by tumor-derived TGF-β1 in determining the invasiveness of adenocarcinoma of the lung.