Prognostic value of FHIT, CTNNB1, and MUC1 expression in non-–small cell lung cancer

Summary

Comprehensive expression analysis using microarrays has identified a number of differentially expressed genes in smoke-exposed bronchial epithelium and non–small cell lung cancers (NSCLCs). To evaluate the prognostic relevance of these proteins in NSCLCs, we used immunohistochemistry to investigate the expression of β-catenin (CTNNB1), dickkopf, Xenopus, homolog of 3 (DKK3 gene), fibroblast growth factor receptor 3 (FGFR3), fragile histidine triad (FHIT), tumor protein p53 (TP53), mucin1 (MUC1), topoisomerase II α (TOP2A), and glutathione S-transferase-Pi (GST) in a cohort of patients (n = 125). We correlated the expression data with clinicopathologic features and clinical outcome. In addition, SNaPshot multiplex assays (Applied Biosystems, Darmstadt, Germany) and restriction fragment length polymorphism analysis were used to screen for activating point mutations at the hot spots of FGFR3 in a cohort of 30 samples of NSCLC. Using Kaplan-Meier analysis, we observed significantly better overall survival in adenocarcinomas compared with squamous cell cancers (P = .049). Loss of FHIT expression showed a strong association with shorter overall survival in both histologic types of NSCLC (squamous cell cancers, P < .001; adenocarcinomas, P = .001). In adenocarcinomas, the cytoplasmic expression of β-catenin was associated with shorter survival (P = .012); MUC1 expression was associated with worse prognosis in patients with squamous cell cancers (P = .002). The nuclear staining of TP53 (P = .008) and TOP2A (P = .059) was associated with cancers without lymphonodal metastases. A correlation with positive staining of TOP2A (P = .03) and FGFR3 positivity (P = .057) was found in adenocarcinomas of male patients. Positive MUC1 stainings were associated with squamous cell cancers of male patients (P = .03). DKK3 expression did not show any significant association with clinical outcome or pathologic features. The screening of the FGFR3 sequence in lung cancers showed only wild-type sequences and did not detect mutations in the known hot spots for FGFR3 mutations. We conclude that the immunohistochemical loss of FHIT expression and the positivity for β-catenin and MUC1 in NSCLC are useful prognostic markers, whereas the variable expression of TP53, TOP2A, and FGFR3 in relation to the different histologic types of NSCLC and sex of the patients is suggestive for different underlying molecular pathways.

Introduction

The main carcinogens such as polycyclic aromatic hydrocarbons, N-nitrosamines, and aromatic amines responsible for most lung cancers are contained in tobacco smoke [1]. However, the exact mechanisms leading to molecular alterations in bronchial epithelium in smokers and subsequent lung cancer development are still poorly understood. In previous studies, we were able to identify in smokers an overlapping signature of differentially expressed genes and allelic losses in tumor tissues and histologic inconspicuous lung tissues [2], [3]. These new studies gave new insights into the molecular mechanisms of lung carcinogenesis and support the hypothesis that in smokers, the imbalance between oxidative stress and xenobiotic protective systems is responsible for the accumulation of DNA damages [4], [5]. The 8 antibodies used detect the expression of gene products on the protein level, which are involved in the tobacco-induced carcinogenesis of lung cancers (β-catenin [CTNNB1]; dickkopf, Xenopus, homolog of 3 [DKK-3 gene]; fibroblast growth factor 3 [FGFR3]; fragile histidine triad [FHIT]; tumor protein p53 [TP53]; mucin1 [MUC1]; topoisomerase II α [TOP2A]; and glutathione S-transferase-Pi [GST]). GST plays an important role in detoxification by catalyzing the conjugation of hydrophobic and electrophilic compounds with reduced glutathione and was down-regulated in lung cancers. TOP2A catalyzes the relaxation of supercoiled DNA and is associated with DNA proliferation and DNA repair [6], [7]. Both GST and TOP2A are involved in molecular protective systems of the cells [8]. During the smoking history, the imbalance between the smoke-induced oxidative stress and weakness of xenobiotic cellular protective systems leads to the accumulation of irreparable DNA damages [9]. The genomic locus of the FHIT gene is one of the most fragile sites of the human genome and is localized on the chromosomal region 3p14.2 [10]. Previous studies by us [11] and others found allelic loss on 3p14.2 in patients with long-term smoking history most frequently in bronchial epithelium and corresponding lung cancers [12]. Although the exact biological function of FHIT protein is still unknown, it is conceivable that FHIT plays a role in cell proliferation and is considered as an early molecular lesion in smokers. Various other studies have shown an association between the amount of cigarettes smoked and p53 mutations [13], [14]. In the course of carcinogenesis, the p53 gene mutations appear early but subsequent to the genetic loss of 3p in histologically visible preneoplastic lesions and cancer tissues [15]. Gene expression profiles have also shown members of the Wnt pathway dysregulated. The Wnt pathway may occur at different levels, including extracellular components of the signaling cascade [16]. Recently, we were able to find WIF I [17], a component of the Wnt pathway, down-regulated in lung cancers. Dickkopf 3 is another protein that also has the ability to bind Wnt proteins. DKK3 is localized at the chromosomal region 11p15. It is known as an inducer in the development of amphibian head structures and mediates its effect by antagonizing the Wnt signaling. In non–small cell lung cancer (NSCLC), allelic losses and abnormal promoter methylation were detected at the chromosomal region 11p15, where the DKK3 gene is localized [18], [19], [20]. β-catenin is a member of the Wnt signaling cascade and is related to cadherin-mediated cell-cell adhesion systems. In lung cancers, the immunohistologic loss of membranous staining and positive cytoplasmic or nuclear staining of β-catenin was described [21]. In addition, gene expression profiles found FGFR3 up-regulated in smoke-exposed lung tissue [2] and lung cancers; and recently, various studies have shown that oxidants in cigarette smoke mediate cell signaling in airways and pulmonary epithelium that regulate the expression pattern of mucins [22].

The immunohistochemical expression of 2 of those genes (DKK3, FGFR3) was completely unknown in lung cancers. The further used 6 genes showed conflicting results in the literature. Only one single immunohistochemical study found an increased expression of GST corresponding to increasing tumor volume and progressive cellular dedifferentiation [5]. In contrast to that finding, our previous study [2] has shown dysregulated RNA levels for GST in early lesions of carcinogenesis and smoke-exposed lung tissue. Our own [11] and other studies [23], [24], [25], [26] support that molecular alterations on the FHIT region are a further early target for tobacco smoke–induced carcinogenesis. However, conflicting results still exist about the clinical and prognostic relevance of loss of FHIT in lung cancers. The loss or marked reduction in FHIT expression has shown variations between tumor types, but several studies did not show an association to worse survival from lung cancers [27], [28]. In contrast to that, others reported about the negative prognostic value of loss of FHIT [29]. Conflicting results were also given for p53 [30], [31], [32]. MUC1 [33], [34] and CTNNB1 [35], [36], [37] were described as worse prognostic markers. The aim of our study was to clarify the prognostic relevance of those genes in the lung cancers of our cohort.

Our study was designed to clarify the clinical and prognostic relevance of differentially expressed genes in 125 tissue samples of NSCLC by immunohistochemistry on the protein level (78 men, 47 women; mean age at diagnosis, 67 years; range, 43-87 years) of patients who underwent surgical therapy at the University Hospital Regensburg, Germany (1993-2003), or at the Department of Thoracic Surgery, Hospital Berlin-Buch, Berlin, Germany (2001-2003). Tumor stage, histologic type, and grade were assigned according to the International Union Against Cancer and World Health Organization grading. Sixty-eight percent (85/125) of the patients had a strongly positive smoking history (mean pack-years, 51). In 40 patients, exact information about smoking history was not available. No patient was clinically labeled as a nonsmoker. The recruitment period was from 1994 to 2005; the clinical follow-up data were documented by the regional clinical tumor registries in Regensburg and Berlin, and clinical follow-up controls were documented in the Department of Thoracic Surgery, Chest Hospital Berlin-Buch. The clinicohistopathologic data are listed in Table 1. In squamous cell cancers, the median overall survival was 28.6 months (men, 29.8 months; women, 17.1 months; median, 19 months; range, 12.9-23.0 months). In adenocarcinomas, the median overall survival was 35.2 months (men, 36.2 months; women, 32.2 months; median, 37 months; range, 2-74.7 months). With 60 squamous cell cancers, 44 adenocarcinomas, and 21 lung cancers with other differentiation, the distribution of the different histologic tumor types reflects approximately the epidemiological frequency of different histologic types of NSCLC. Because of the extensive immunohistochemical sections and limited tumor tissue for mutation analysis of FGFR3, we used paraffin-embedded tumor tissue blocks from 30 additional NSCLCs (14 squamous cell carcinomas, 16 adenocarcinomas; 23 men, 7 women; mean age at the diagnosis, 60 years; smoking history, mean 49 pack-years) collected from 2000 to 2003 at the University Hospital Regensburg. Independent histologic examination of the tumor tissue was performed according to the World Health Organization criteria to define the tumor-node-metastasis stage.