Expression of hypoxia-inducible factor-1 alpha and its prognostic significance in small-sized adenocarcinomas of the lung

Abstract

Recent advancement in radiographic techniques, such as high-resolution computed tomography, has enabled us to detect small-sized adenocarcinomas of the lung [1,2]. Early detection may eventually lead to a decrease in disease mortality. However, the prognosis of patients with lung carcinoma is generally poor, even in patients with small-sized adenocarcinomas [3]. Noguchi et al. have conducted research to elucidate the histopathologic prognostic factors for small-sized adenocarcinomas of the lung. They showed that lung adenocarcinomas with active fibroblastic proliferation are more likely to metastasize and have a less favorable prognosis [4]. However, the molecular mechanisms underlying the aggressive behavior of small-sized adenocarcinomas of the lung are poorly understood. To improve the prognosis of patients with small-sized adenocarcinoma of the lung, new therapeutic strategies based on a better understanding of tumor behavior are needed. The transcription factor hypoxia-inducible factor-1 (HIF-1) consists of HIF-1 alpha and HIF-1 beta subunits. HIF-1 alpha reflects HIF-1 activity, because it is expressed rapidly in response to hypoxic conditions, while HIF-1 beta is expressed constitutively [5–7]. HIF-1 alpha protein has a very short half-life under normoxic conditions due to its continuous ubiquitination and proteosome-mediated degradation. Hypoxia stabilizes HIF-1 alpha protein by inhibiting ubiquitination and degradation [8]. Previous studies described that HIF-1 alpha plays a critical role in angiogenesis [9–11]. Therefore, we hypothesized that the expression of HIF-1 alpha may have prognostic value in patients with small-sized adenocarcinomas of the lung. In this study, we investigated the prognostic significance of HIF-1 alpha and its relationship with clinicopathological factors and angiogenesis factors, such as vascular endothelial growth factor-A (VEGF-A) and VEGF-C. Tumor specimens were obtained from 78 patients who had small-sized adenocarcinoma of the lung (maximum tumor dimension ≤ 2 cm). All patients were treated by standard surgical therapy alone at the Fukuoka University School of Medicine (Fukuoka, Japan) between 1993 and 2002. Informed consent was obtained from each patient. Disease stage was determined based on the tumor, node, metastasis classification of the International Union Against Cancer [12]. All patients underwent contrast-enhance computed tomography scans within 1 month prior to surgery. Histological typing was determined according to the World Health Organization classification [13]. The smoking index was defined as the product of the number of cigarettes smoked per day and the number of years of smoking. We defined cN2 as mediastinal lymph node(s) greater than 1.0 cm in shortest dimension, as determined by preoperative conventional computed tomography. Cases were classified as either localized bronchioloalveolar carcinoma (LBAC) or conventional adenocarcinoma according to the major morphologic pattern. The LBAC group consisted of patients with bronchioloalveolar growth pattern tumors that had no evidence of vascular, pleural, or stromal invasion. The conventional adenocarcinoma group consisted of patients with invasive adenocarcinoma, encompassing mixed subtype, acinar, papillary, and solid adenocarcinoma. Lymphatic invasion and vascular invasion indicated tumor cells identifiable in the lymphatic and vascular vessel lumen, respectively. Scar grades were classified as follows: grade 1, tumor had foci of alveolar collapse with resulting condensation of elastic fibers but no or minimal fibroblastic tissue with collagen; grade 2, tumor had fibroblastic tissue with a small amount of collagen fibers; grade 3, tumor had fibroblastic tissue with moderate or abundant amount of collagen fibers; and grade 4, tumor showed hyalinization [14]. The following clinicopathologic factors were analyzed to determine if they impacted patient survival: age, gender, smoking index (<400 vs ≥400), serum carcinoembryonic antigen (CEA) level (<5.0 mg/ml vs ≥5.0 mg/ml), clinical T factor (cT) (cT2-4 vs cT1), clinical lymph node status (cN) (mediastinal node involvement as cN2 vs less extensive as cN0-1), histological type of tumor (LBAC vs conventional), pleural involvement (p0-1 vs p2-3), lymphatic invasion (positive vs negative), vascular invasion (positive vs negative), pathologic N status (pN: pN2-3 vs pN0-1), and degree of fibrotic scarring (scar grade 1–2 vs grade 3–4). Formalin-fixed and paraffin-embedded tissues were deparaffinized. Tissues used for immunohistochemical staining were subsequently microwaved in citrate buffer. Tissues were preincubated with 0.3% hydrogen peroxide and protein blocking solution. For HIF-1 alpha (H-206), a 1:200 dilution of rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the sections. For VEGF-A (clone JH121), a 1:100 dilution of mouse monoclonal antibody (Uptake, New York, NY, USA) was added to the sections. For VEGF-C, a 1