Abnormality of the hepatocyte growth factor/MET pathway in pulmonary adenocarcinogenesis

Abstract

Background

Signaling mediated by hepatocyte growth factor (HGF)/MET promotes multiple biological activities, including cell proliferation, motility, invasion, angiogenesis, and morphogenesis. Overexpression of HGF and MET and an increase of the MET gene copy number have recently been found in various cancers that had a poor outcome. Here we investigated the copy number of the MET gene and expression of MET and HGF in small pulmonary adenocarcinomas.

Methods

Tumor tissues were obtained from 106 pulmonary small adenocarcinomas 2 cm or less in diameter. MET gene copy number, and the expression of MET and HGF, were analyzed using fluorescence in situ hybridization (FISH) and immunohistochemistry, respectively.

Results

MET FISH-positive signals were observed in 11 (10.4%) of 106 cases. One case (0.9%) showed gene amplification and 10 (9.4%) exhibited high polysomy. High immunoreactivity for MET and HGF in tumor cells was found in 30 (28.3%) and 19 cases (17.9%), respectively. HGF was also expressed in stromal cells in 32 cases (30.2%). No cases of non-invasive adenocarcinoma (adenocarcinoma in situ, localized bronchioloalveolar carcinoma) showed MET FISH-positive signals or high expression of HGF in the tumor cells. Expression of both MET and stromal HGF was stronger in invasive than in non-invasive adenocarcinoma. MET FISH-positive signals and high immunoreactivity for MET and HGF in tumor cells were associated with factors indicative of poor prognosis such as pleural invasion, vascular invasion, lymphatic permeation, lymph node metastasis, and nuclear grading. Univariate and multivariate analyses that included these factors showed that all statuses except for MET and HGF immunoreactivity were significantly associated with an increased risk of death. However, multivariate analysis revealed no independent factors related to poor prognosis.

Conclusion

Our results suggest that abnormality of the HGF/MET pathway occurs during the course of progression from non-invasive to invasive pulmonary adenocarcinoma. An increased MET gene copy number is indicative of a poor outcome in patients with small pulmonary adenocarcinomas.

Introduction

Lung carcinoma is the leading cause of cancer death worldwide [1], and one of the most common histologic types is adenocarcinoma, which has been showing a continuous increase in incidence in both Japan and Western countries. In particular, more cases of small peripheral adenocarcinoma are now being found as a result of technological advances in computed tomography [2]. Surgical resection remains the standard treatment for non-small cell lung carcinoma (NSCLC), including small peripheral adenocarcinomas. Noguchi et al. [3] examined a number of surgically resected small adenocarcinomas of the lung, and demonstrated that there are definite cases showing a very favorable outcome. According to their criteria, localized bronchioloalveolar carcinoma (LBAC, type A) and LBAC with alveolar collapse (type B) are defined as non-invasive adenocarcinoma and show a 100% 5-year survival rate. Therefore, type A and B tumors can be candidates for reduction surgery. On the other hand, LBAC with foci of active fibroblastic proliferation (type C) is a type of adenocarcinoma showing lepidic growth, but it represents a large category containing both minimally invasive and invasive adenocarcinomas. Some patients with type C tumors who have no detectable metastasis at the time of surgery die of their disease because type C tumors include adenocarcinomas with various prognoses. Therefore, there is a definite need for prognostic markers that can be used to determine which patients can be treated with reduction surgery or those who require the standard operation.

In the 2004 World Health Organization (WHO) classification [1], major adenocarcinoma was divided into five subtypes, including bronchioloalveolar carcinoma (BAC), acinar, papillary, solid with mucin production, and mixed subtypes. However, more than 90% of adenocarcinomas fall into the mixed subtype. In addition, although the term BAC is defined as non-invasive adenocarcinoma, it is used loosely for a broad spectrum of tumors showing BAC-type spread. In order to resolve these issues, a multidisciplinary adenocarcinoma classification has been recently proposed by the International Association for the Study of Lung Cancer in 2011 [4]. In this new classification, adenocarcinomas were fundamentally classified into three categories based on progression: (1) preinvasive lesions including atypical adenomatous hyperplasia (AAH) and adenocarcinoma in situ (AIS), (2) minimally invasive adenocarcinoma (MIA), and (3) invasive adenocarcinoma including lepidic-predominant, acinar-predominant, papillary-predominant, micropapillary-predominant, and solid-predominant with mucin production. AIS is considered to be synonymous with BAC, according to the 2004 WHO classification, and with type A and B tumors in the Noguchi classification. MIA is a small, solitary adenocarcinoma with a predominantly lepidic pattern and showing less than 5 mm of invasion in greatest dimension. Therefore, like AIS, MIA is expected to show an extremely favorable outcome. MIA is thought to include a proportion of adenocarcinomas of mixed subtype with BAC (the 2004 WHO classification) and also type C tumors (Noguchi classification). On the other hand, adenocarcinoma of mixed subtype with BAC (2004 WHO classification) includes any adenocarcioma subtype that includes a lepidic pattern (2011 proposed new classification) and type C adenocarcinoma (Noguchi classification).

Hepatocyte growth factor (HGF), also known as scatter factor, was originally and independently isolated as a hepatic regeneration factor [5]. HGF is a pleiotropic cytokine, whose biological effects are mediated by activation of the MET proto-oncogene tyrosine kinase receptor [6]. HGF is currently considered to be both an autocrine and a paracrine mediator produced by mesenchymal cells, including fibroblasts [7], [8], [9]. The MET oncogene was originally isolated from a human osteogenic sarcoma cell line that had been subjected to chemical mutagenesis in vitro [10]. MET-receptor tyrosine kinase is activated by its cognate ligand HGF, and receptor phosphorylation activates the downstream pathways [11]. Signaling mediated by HGF/MET promotes multiple biological activities, including cell proliferation, motility, invasion, angiogenesis, and morphogenesis in a wide variety of normal and neoplastic cells [12], [13], [14], [15].

Alteration of the MET gene, including amplification, overexpression, and mutation, has been described in various cancers [16], [17]. Expression of MET and HGF proteins assessed by immunohistochemistry has been reported to predict poor outcome in patients with resected lung cancer [18], [19], [20], [21]. Recently, MET amplification has been identified as one of the mechanisms of acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) [22], [23], and has been found not only in tumors with acquired resistance to EGFR-TKI but also in primary untreated NSCLC [23]. Several studies have demonstrated that an increase in the MET gene copy number is associated with poor outcome in patients with NSCLC [24], [25], [26], [27]. However, all of the patients analyzed in these studies had undergone resection of advanced NSCLC, and no previous investigations have focused on HGF/MET in small adenocarcinomas, including both non-invasive and invasive cases.

Besides HGF and MET abnormalities, a number of studies have demonstrated various gene abnormalities during the progression of adenocarcinoma [28], [29], [30], [31], [32], [33], [34]. These include mutation analyses of the EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), and p53, and copy number amplifications of genes including EGFR, thyroid transcription factor (TTF-1), MYC, and KRAS. Some chromosomal alterations and deletions have also been examined. These abnormalities were shown to be added during the course of malignant progression, although extremely early-stage tumors (AIS, MIA, etc.) were not examined.

In the present study, we examined both the copy number of the MET gene by fluorescence in situ hybridization (FISH) and the expression of MET and HGF using immunohistochemistry in small adenocarcinomas. We demonstrated that an increased MET gene copy number and high immunoreactivity for MET and HGF in tumor cells were significantly associated with factors indicative of a poor prognosis, such as lymphatic permeation and nuclear grading. An increased MET gene copy number was particularly associated with a poor outcome. Our results also suggest that abnormality of the HGF/MET pathway occurs during the course of progression from non-invasive to invasive adenocarcinoma.