1086 Vol. 11, 1086–1092, February 1, 2005 Clinical Cancer Research Matrix Metalloproteinase-12 Expression Correlates with Local Recurrence and Metastatic Disease in Non–Small Cell Lung Cancer Patients Hans-Stefan Hofmann,1 Gesine Hansen,2 the 169,000 new cases identified in the United States in 2002 Gu¨nther Richter,4 Christiane Taege,3 will lead to more deaths than breast, colon, prostate, and cervical Andreas Simm,1 Rolf-Edgar Silber,1 cancers combined (1). The pathogenesis of lung cancer remains highly elusive due to its aggressive nature and considerably and Stefan Burdach4 heterogeneity as compared with other cancers. Many lung cancer 1 2 3 Departments of Cardio-Thoracic Surgery and Paediatrics; Institute patients have distant metastases or occult hematogenous and of Pathology, Martin-Luther-University Halle-Wittenberg, Halle; and 4Cancer Center and Division of Pediatric Hematology/Oncology, lymphatic spread of tumor cells at diagnosis, thus accounting for Munich University of Technology, Munich, Germany poor prognosis of this disease (2). Metastasis is the final stage in tumor progression from a normal cell to a fully malignant cell. One of the initial steps in ABSTRACT the metastatic process involves degradation of different Purpose: Non–small cell lung cancer (NSCLC) is a very components of the extracellular matrix and requires the action common and aggressive malignancy. Survival after resection of proteolytic enzymes such as serine-, cysteinyl- and aspartyl- of tumor is especially determined by the occurrence of distant proteinases as well as matrix metalloproteinases (MMP). metastasis. Matrix metalloproteinases (MMP) support this MMPs are a family of more than 20 secreted or transmembrane metastatic process by degradation of the extracellular matrix. proteins that are capable of digesting extracellular matrix and Experimental Design: We used DNA microarray tech- basement membrane components under physiologic conditions nology to examine the expression of 22 MMPs in 89 surgically (3). According to their substrate specificity and structure, treated NSCLC patients. Validation of microarray results was MMPs are classified into five subgroups: collagenases (MMP- done using reverse transcription-PCR and immunohistology. 1, MMP-8, MMP-13), gelatinases (MMP-2, MMP-9), strome- Results: MMP-1, MMP-9, and MMP-12 expression was lysins (MMP-3, MMP-10, MMP-11), as well as metalloelastase significantly increased in tumors versus corresponding lung (MMP-12), the membrane-type MMPs (MMP14, MMP15), and tissues. MMP-12 expression significantly correlated with other MMPS (e.g., MMP-19, and MMP20; ref. 4). Reports local recurrence and metastatic disease. Multivariate Cox have shown correlations between the degradation of the regression analysis revealed MMP-12 expression as an basement membrane by MMPs and the metastatic potential independent prognostic factor for tumor relapse–free of tumor cells (5, 6). The clinical relevance of MMPs in non– interval. Gene expression analysis of 158 healthy tissues from 32 different organs revealed no MMP-12 expression in small cell lung cancer (NSCLC) is still under discussion. these organs and immunohistology identified MMP-12 Several MMPs, especially MMP-2 and MMP-9, seem to protein in NSCLC only in tumor cells. correlate with early cancer-related deaths in NSCLC (7, 8). Conclusions: MMP-12 might be not only a prognostic First, clinical trials have successfully investigated the efficacy marker, but also a valuable therapeutic target. of MMP inhibitors in advanced cancer (9). The present study was done to assess differentially expressed MMPs in NSCLC by DNA microarray technology INTRODUCTION and their impact on disease-free interval after surgical resection. Lung cancer is the leading cause of cancer deaths The aim was the evaluation of patients with high risk of early worldwide in men and in women. As 86% of the people who metastasis, who may provide candidates for neoadjuvant are diagnosed with lung cancer die of the disease within 5 years, chemotherapy or MMP-inhibitory therapy. MATERIALS AND METHODS Received 8/22/04; revised 11/4/04; accepted 11/4/04. Grant support: EOS Biotechnology, DFG SFB 610 TPB1, SFB 598 TP Patients and Samples. Tumor and control lung tissue A5, Deutsche Krebshilfe (70-2787-Bu 3), and BMBF (01-ZZ0109). The samples were obtained from 89 consecutive patients with NSCLC, sponsors of the study had no role in the data interpretation or writing of who underwent pulmonary resection surgery between 1999 and this report. 2001 (Table 1). The use of human tissues was approved by the The costs of publication of this article were defrayed in part by the local ethics committee and the patients gave informed consent. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to Only patients with clear histologic classification as NSCLC indicate this fact. (adenocarcinoma or squamous cell carcinoma) and without Note: H-S. Hofmann and G. Hansen contributed equally. neoadjuvant chemotherapy or radiotherapy were admitted to the Requests for reprints: Hans Stefan Hofmann, Department of Cardio- study. Immediately following resection, the tumor tissue and Thoracic Surgery, Martin-Luther-University Halle-Wittenberg, Ernst- Grube-Strasse 4, 06097 Halle, Germany. Phone: 49-345-557-2793; Fax: tumor distant lung tissue from the same patient were snap-frozen 49-345-557-2802; E-mail: [email protected]. and stored in liquid nitrogen until use. Tumor histology and stages D2005 American Association for Cancer Research. were classified according to the WHO classification (10) and the Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Clinical Cancer Research 1087 Table 1 Clinical and pathologic characteristics of patients and was prepared from total RNA by in vitro transcription after their tumors synthesis of double-stranded cDNA using standard protocols. No. of patients 89 After cRNA-fragmentation and hybridization with microarrays Mean age (y) 65.5 (EOS-K), signals were detected with streptavidin-phycoerythrin. Male/female ratio 71/18 Signal enhancement was done using biotinylated goat anti- Smoking/nonsmoking ratio 60/29 Surgical procedure streptavidin antibodies. Arrays were washed and stained with the PE/diagnostic thoracotomy 6 (6.8%) GeneChip Fluidics Station 400 and scanned with a GeneArray Segment resection 5 (5.5%) Scanner. Primary image analysis was done by using Microarray Lobectomy 64 (72%) Suite 5.0. Altogether, 49 squamous cell carcinomas, 40 Bilobectomy 2 (2.2%) Pneumonectomy 12 (13.5%) adenocarcinomas and 15 corresponding control lung samples Histology were analyzed. All expression values below 60 were set to 60. Squamous cell carcinoma 49 (55%) To identify specific genes that were differentially expressed in Adenocarcinoma 40 (45%) Tumor-node-metastasis staging tumors as compared with lung tissue, we used a criterion that I 31 (34.8%) marks differential gene expression at an approximate signifi- II 23 (25.8%) cance level (determined by Bonferroni method) of 8.0 Â 10À7 III 29 (32.6%) using Student’s t test and a fold-change cutoff of 2.0 and 0.5 for IV 6 (6.8%) Grading up-regulated and down-regulated genes, respectively. Calcula- Well/moderately well differentiated 25 (28%) tion of fold-change was done by dividing the mean expression Poorly differentiated/undifferentiated 64 (72%) level of a gene in the tumor samples by the mean expression Residual tumor situation level of the same gene in the lung samples. R0 75 (84.2%) R1 6 (6.8%) Body Map. The evaluation of MMP-12 expression in R2 8 (9%) other healthy organs was done with an EOS-Biotechnology own body map list, containing expression data collected by the same microarray technology. This gene expression database encloses expression values of 158 healthy tissues (brain, larynx, tumor-node-metastasis system for staging cancer staging system lip, pharynx, salivary gland, heart, aorta, breast, thymus, of the Unio Internationale Contra Cancrum, respectively (11). The esophagus, omentum, stomach, intestine, small bowel, colon, median follow-up duration was 24.4 months (range 14-43 rectum, liver, pancreas, lien, kidney, adrenal gland, bladder, months). ureter, urethra, cervix, ovary, skin, muscle, diaphragm, and RNA Preparation. Total RNA was prepared from lymph node). cryopreserved lung cancer samples or control lung tissue with Reverse Transcription-PCR. Validation of microarray acid phenol/chloroform extraction (TRIzol; Invitrogen, Karls- results was done on 23 tumor and 6 lung samples initially ruhe, Germany) followed by purification with RNeasy Mini Kit used for gene chip analysis. For evaluation of predictive (Qiagen, Hilden, Germany) according to the manufacturer’s value, previously uncharacterized tumor probes were included instructions and was quantitated spectrophotometrically. The (see RESULTS). Tissue was lysed in TRIzol (Life Technol- quality of the purified RNA was assessed by visualization of 18S ogies), RNA prepared and converted to first-strand cDNA by and 28S RNA bands after electrophoresis through agarose gels use of oligo (dT) primers and Moloney murine leukemia virus and staining with ethidium bromide. reverse transcriptase (Promega Corporation, Madison, WI, Microarray Expression Analysis. A total of 10 Ag RNA USA) according to the manufacturer’s instructions. Success of from each sample was used to prepare biotinylated target cRNA cDNA synthesis was monitored