Gene expression profiling in sinonasal adenocarcinoma

Dominique Tripodi 1 2 Sylvia Qumner 2 Karine Renaudin 0 6 Christophe Ferron 5 Olivier Malard olivier.malard@chu- 5 Isabelle Guisle-Marsollier 4 Vronique Sbille-Rivain 3 Christian Verger 8 Christian Graut 1 Catherine Gratas-Rabbia-R 2 7 0 Service d'Anatomie Pathologique , CHU de Nantes, Nantes, F-44093 , France 1 Service de Medecine du Travail et des Risques Professionnels , CHU de Nantes, Nantes, F-44093 , France 2 Inserm, UMR 892, Nantes , F-44007 , France; Universite de Nantes, UFR Medecine et Techniques Medicales , Nantes, F-44000 , France 3 Universite de Nantes, UFR Medecine et Techniques Medicales, Laboratoire de Biomathematiques-Biostatistiques , Nantes, F-44000 , France 4 Universite de Nantes, UFR Medecine et Techniques Medicales, Plateforme Puces a ADN- OGP , Nantes, F-44000 , France 5 Service ORL , CHU de Nantes, Nantes, F-44093 , France 6 Universite de Nantes, UFR Medecine et Techniques Medicales , EA Biometadys, Nantes, F-44093 , France 7 Service de Biochimie , CHU de Nantes, Nantes, F-44093 , France 8 Consultation des Pathologies Professionnelles, CH Hotel-Dieu , Rennes, F-35000 , France Background: Sinonasal adenocarcinomas are uncommon tumors which develop in the ethmoid sinus after exposure to wood dust. Although the etiology of these tumors is well defined, very little is known about their molecular basis and no diagnostic tool exists for their early detection in high-risk workers. Methods: To identify genes involved in this disease, we performed gene expression profiling using cancerdedicated microarrays, on nine matched samples of sinonasal adenocarcinomas and non-tumor sinusal tissue. Microarray results were validated by quantitative RT-PCR and immunohistochemistry on two additional sets of tumors. Results: Among the genes with significant differential expression we selected LGALS4, ACS5, CLU, SRI and CCT5 for further exploration. The overexpression of LGALS4, ACS5, SRI, CCT5 and the downregulation of LGALS4 and CLU, that were significantly differentially expressed in tumors compared to normal tissue. A further evaluation on a new set of tissues, including precancerous stages and low grade tumors, is necessary to evaluate the possibility of using them as diagnostic markers. - were confirmed by quantitative RT-PCR. Immunohistochemistry was performed for LGALS4 (Galectin 4), ACS5 (Acyl-CoA synthetase) and CLU (Clusterin) proteins: LGALS4 was highly up-regulated, particularly in the most differentiated tumors, while CLU was lost in all tumors. The expression of ACS5, was more heterogeneous and no correlation was observed with the tumor type. Conclusion: Within our microarray study in sinonasal adenocarcinoma we identified two proteins, Background Sinonasal adenocarcinoma is a rare cancer which usually develops in the ethmoid sinuses. It mainly develops amongst 30 to 85 year old men, with a high frequency around 60. The incidence of this type of cancer was estimated by the IARC (International Agency for Research on Cancer) at 0.7/100 000 in China to 1.4/100 000 in USA and 1.5/100 000 in France, and it has been reported to account for 3% of head and neck tumors [1,2]. This cancer is recognized as an occupational cancer. In fact, it is well confirmed today that sinonasal adenocarcinoma is highly correlated with duration and level (3.5 mg/m3) of wood dust exposure [3,4]. As such, woodworkers have very high risks of nasal cancer (Standard Mortality Ratio: 310, 95% CI, 160-560) [5,6]. Other suspected risk factors include exposure to leather dust [7,8], metals such as chromium or nickel [9,10], and formaldehyde, although the epidemiological data regarding this chemical are partly conflicting [4,11]. In contrast to most other head and neck cancers, alcohol and tobacco do not seem to be risk factors [12]. Although the etiology of sinonasal adenocarcinoma is well-defined, its wood-related pathogenesis is not clearly understood [13]. From a morphological and histopathological point of view, these tumors are mainly intestinal-type adenocarcinomas [14,15] and demonstrate characteristic changes, such as gland formation, seen in adenocarcinomas at other anatomic sites. The most common clinical symptoms (nosebleeding, rhinitis and nasal obstruction) are not specific and this explains the delay in the diagnosis and the frequency of advanced stages. The conventional treatment includes local surgery [16] associated with radiotherapy. The survival rate at 5 years is only about 50% and it is important to point out that secondary effects are considerable due to the location of these tumors [17]. Therefore, early detection and alternative treatments are necessary. This requires, however, better knowledge of the molecular mechanisms involved in the development of these tumors. Although many reports on epidemiological studies and risk factors of sinonasal adenocarcinomas have been published, only a small number of reports have been made so far on their molecular biology. As reviewed recently by Llorente et al [13], several groups have proceeded with molecular studies of sinonasal adenocarcinomas. However these focused on specific genes, such as ERBB1, CCND1, ERBB2, TP53, K-ras, COX-2 or APC, involved either in other head and neck tumors or in colorectal cancer because of morphological similarities [13,18,19]. Two groups reported comparative genomic hybridization in ethmoid sinus adenocarcinomas and revealed hot spots of chromosomal imbalances [20-22]. Global genetic modifications (micronuclei and chromosomal aberrations) were also found in buccal epithelial cells and blood lymphocytes of wood furniture workers [23]. The conclusion of all these investigations is that ethmoid sinus adenocarcinomas have their own molecular development pathway. Thus, to identify genes involved in this pathway, we pioneered a gene expression profiling study of 9 sinonasal adenocarcinomas versus their matched normal tissue. We found 186 genes with significant differential expression. The further evaluation of several selected genes by reversetranscription quantitative real-time-PCR (RT-qPCR) and immunohistochemistry (IHC), on two additional validation samples, confirmed the microarray data. We have hereby opened up a new field of investigation into biomarkers of this tumor type and have identified two promising candidate genes: LGALS4 and CLU. Methods Subjects Our study included 26 patients. A first set of 19 male patients undergoing surgery for ethmoid sinus adenocarcinomas were initially included between 2004 and 2006. Following this, a second set of 7 patients whose samples were collected from 2006 to 2007 was used to complete the immunohistochemistry study. This project was approved by the Clinical Board of the Centre Hospitalo-Universitaire of Nantes and all included patients provided written informed consent in accordance with French regulations and the Declaration of Helsinki. All patients answered a codified questionnaire regarding occupational exposures, addictive consumptio (...truncated)