Oncogene (2004) 23, 2484–2498 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc Identification of genes associated with tumorigenesis and metastatic potential of hypopharyngeal cancer by microarray analysis Anne Cromer1,3, Annaı¨ ck Carles1,3,Re´ gine Millon2,3, Gitali Ganguli1, Fre´ de´ ric Chalmel1, Fre´ de´ ric Lemaire1, Julia Young1, Doulaye Dembe´ le´ 1, Christelle Thibault1, Danie` le Muller2, Olivier Poch1, Joseph Abecassis2 and Bohdan Wasylyk*,1 1Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/ULP, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch cedex, France; 2UPRES EA 34-30, Centre Paul Strauss, 3 rue de la Porte de l’Hoˆpital, 67085 Strasbourg, France Head and neck squamous cell carcinoma (HNSCC) is the Introduction sixth most common cancer among men in the developed world. There is a need, for both clinical and scientific Head and neck squamous cell carcinoma (HNSCC), reasons, to find markers to identify patients with which affects the oral cavity, the oropharynx, the larynx aggressive disease as early as possible, and to understand and the hypopharynx, is the sixth most common cancer the events leading to malignant transformation and among men in the developed world. Well-known susceptibility to metastasis. We report the first large- risk factors include tobacco and alcohol. Over the scale gene expression analysis of a unique HNSCC last decades, diagnosis and management have improved, location, the hypopharynx. Four normal and 34 tumour but not long-term survival rates. The prognosis of samples were analysed with 12 600 gene microarrays. HNSCC is influenced by many factors, such as TNM Clusters of differentially expressed genes were identified staging and pathological grading of differentiation. in the chromosomal regions 3q27.3, 17q21.2–q21.31, However, since these factors are not sufficient 7q11.22–q22.1 and 11q13.1–q13.3, which, interestingly, to evaluate outcome, there is a need to identify have already been identified by comparative genomic molecular biomarkers that will help to stage patients hybridization (CGH) as major regions of gene amplifica- in prognostic groups and to identify high-risk patients tion. We showed that six overexpressed genes (EIF4G1, who may benefit from different treatments. Some genes DVL3, EPHB4, MCM7, BRMS1 and SART1) located in have been suggested to be potential prognostic markers these regions are indeed amplified. We report 119 genes in HNSCC (Quon et al., 2001). However, molecular that are highly differentially expressed between ‘early’ markers do not yet contribute to the clinical decision- tumours and normal samples. Of these, we validated by making process. quantitative PCR six novel poorly characterized genes. Cancer appears to result from the progressive These genes are potential new markers of HNSCC. accumulation of genetic aberrations. Amplified chro- Comparing patients with relatively nonaggressive and mosomal regions may contain dominant oncogenes, aggressive tumours (without or with clinical evidence of whereas deleted regions may harbour tumour suppres- metastasis 3 years after surgery), we identified 164 sor genes. HNSCC is frequently associated with specific differentially expressed genes potentially involved in the chromosomal aberrations, including amplification of 3q, acquisition of metastatic potential. This study contributes 8q, 9q, 20q, 7p, 11q13 and 5p, and deletion of 3p, 9p, to the understanding of HNSCC, staging patients into 21q, 5q, 13q, 18q and 8p (Gollin, 2001). mRNA prognostic groups and identifying high-risk patients who expression levels often reflect these changes. However, may benefit from more aggressive treatment. the deregulation of gene expression in cancer is more Oncogene (2004) 23, 2484–2498. doi:10.1038/sj.onc.1207345 complex than a simple relationship between genomic Published online 15 December 2003 aberration and gene expression (Platzer et al., 2002; Pollack et al., 2002). Keywords: HNSCC; chromosomal location; prognosis; Expression-array profiling has recently been used to gene amplification distinguish between cancer subtypes (Golub et al., 1999) and stages of progression (Bittner et al., 2000). The capacity of tumour cells to metastasize could be acquired early in tumorigenesis (Bernards and Wein- berg, 2002), and patterns of gene expression can predict the metastatic outcome of patients with breast (van’t Veer et al., 2002) and prostate (Singh et al., 2002) cancer. Microarray analysis of gene expression has been *Correspondence: B Wasylyk; E-mail: [email protected] reported for HNSCC (Leethanakul et al., 2000; Alevizos 3These authors contributed equally to this work Received 22 July 2003; revised 22 October 2003; accepted 11 November et al., 2001; Al Moustafa et al., 2002; El-Naggar 2003 et al., 2002; Mendez et al., 2002), but did not concern Hypopharyngeal cancer transcriptome A Cromer et al 2485 well-defined stages of tumour evolution in a precise head tumours with similar initial stage and histopathological and neck site. features but with different clinical outcomes. Here we describe the first large-scale gene expression analysis of the hypopharynx, a localization associated with particularly aggressive behaviour (Genden et al., 2003). The aim of our study was to identify genes Results and discussion involved in tumorigenesis, as well as gene expression patterns that will distinguish tumours that will metas- We determined with Affymetrix HG-U95A microarrays tasize from those that will not. We have identified by the expression profiles of 34 hypopharyngeal cancer microarray analysis genes whose expression differs samples (including 31 individual tumours, two pools of between tumours and normal tissues, as well as between two and one pool of three additional tumours) and four Table 1 Patient and sample characteristics Class Patient Sample Localiz. T N M Diff Sex Age Treatment after Evolution Actual Overall Disease free surgery (4) state survival (5) survival (5) Normal N pool(1)* N U N 1047(2)* N U N (3) 1102* N U N (3) 1107* N U ‘Early’ tumours Pool_E PE1* T H 2 0 0 2 M 53 RX 0 D 15 11 PE2* T H 1 0 0 2 M 61 N 0 D 25 15 PE3* T H 2 0 0 2 M 44 N 0 A 41 41 E 1047(2)* T H 2 0 0 1 M 68 RX 0 A 12 12 E 435* T H 1 0 0 1 M 72 RX SC A 92 91 E 834* T H 1 0 0 2 M 52 N SC D 61 42 ‘Late’ tumours LR 118 T H 4 2b 0 1 M 51 RX LR D 39 35 LR 429 T H 3 3 0 2 M 58 RX LR D 77 37 LR 684 T H 3 1 0 3 M 65 RX LR D 40 29 LR 702 T H 3 2c 0 3 M 70 RX LR D 5 4 Pool_NM PNM1* T H 3 2b 0 3 M 66 RX 0 D 58 54 PNM2* T H 3 2b 0 2 M 52 RX SC A 44 39 NM 065* T H 4 2c 0 1 M 67 RX 0 A 118 118 NM 103 T H 3 1 0 2 M 55 RX 0 A 143 95 NM 167 T H 4 2c 0 2 M 61 RX 0 A 140 86 NM 190* T H 1 2b 0 1 M 70 RX 0 A 63 39 NM 279* T H 3 2c 0 1 M 53 RX 0 A 104 104 NM 357 T H 3 2c 0 2 M 45 RX 0 A 101 101 NM 409 T H 4 1 0 2 M 43 RX 0 A 116 116 NM 423 T H 2 2b 0 2 M 64 RX 0 A 83 83 NM 847 T H 2 1 0 3 F 63 RX 0 A 67 67 NM 856* T H 3 2b 0 1 M 59 RX 0 A 66 66 Pool_M PM1* T H 2 2c 0 3 M 54 RX M D 25 16 PM2* T H 3 2b 0 2 M 54 RX M D 7 5 M 135* T H 3 2c 0 2 M 51 RX M D 84 14 M 165 T H 4 2c 0 2 M 71 RX M D 22 9 M 203 T H 3 2b 0 2 M 54 RX M D 20 11 M 209* T H 3 2c 0 2 M 62 RX M D 18 8 M 215 T H 4 3 0 2 M 43 RX+CT M D 12 3 M 218 T H 3 3 0 2 M 56 RX M D 12 8 M 330 T H 3 2b 0 3 M 63 RX M D 7 7 M 408 T H 1 2c 0 3 M 49 RX+CT M A 16 1 M 621 T H 3 3 0 3 M 41 RX+CT M D 34 28 M 629 T H 3 3 0 2 F 57 RX+CT M D 8 8 M 744 T H 3 2c 0 2 M 51 RX M D 9 8 M 829 T H 4 3 0 2 M 69 RX M D 5 1 M 888* T H 3 2b 0 2 M 64 RX M D 26 21 M 933* T H 2 2b 0 2 M 70 RX M D 23 22 Sample type: T ¼ tumour, N ¼ normal; localization (localiz.): H ¼ hypopharynx, U ¼ uvula; T, N and M reflect the TNM nomenclature for tumour stage; evolution: 0 ¼ no evolution, LR ¼ local recurrence, M ¼ metastasis, SC ¼ secondary cancer; last follow-up status: D ¼ dead, A ¼ alive; *samples from batch 1. (1) The N pool contains the normal samples corresponding to the tumours in the TE, TNM and TM pools, the E pool consists of three tumours (PE1, PE2 and PE3), the NM pool two (PNM1 and PNM2) and the M pool two (PM1 and PM2); (2) 1047T and 1047N are tumour and normal samples from the same patient; (3) samples were macrodissected to avoid nonepithelial cells; (4) RX ¼ radiotherapy treated, RX+CT ¼ radiotherapy and chemotherapy treated, N ¼ no treatment; (5) in months Oncogene Hypopharyngeal cancer transcriptome A Cromer et al 2486 normal samples (including one pool of seven normal samples are less similar among themselves than the tissues from patients whose tumours are in the tumour normal samples, as represented by the length of the pools).