Matrix Metalloproteinase-12 Expression Correlates with Local Recurrence and Metastatic Disease in Non–Small Cell Lung Cancer Patients

1086 Vol. 11, 1086–1092, February 1, 2005 Clinical Cancer Research

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

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 by PCR with human h-actin as previously described (12–14). A detailed protocol is available primer 5V-CATCGTGATGGACTCCGGTG-3V and 5V- at www.affymetrix.com. Samples were hybridized to a custom GCTGGAAGGTGGACAGCGAG-3V, amplifying after 21 expression monitoring DNA microarray designed by Eos cycles a cDNA product of 610 bp in size. For analysis of Biotechnology, Inc. using Affymetrix GeneChip technology MMP-12 expression, primers specific for human MMP-12 (15) that contained essentially all expressed human genes in the transcript were designed (sense, 5V-GGCTGAAGGTTTCTGA- public domain at the time of design (EOS-K). Sequences GAGACC-3V; antisense, 5V-TTTGGTGATACGTTGGAG- included in the array were derived from human genomic TAGG-3V) enabling specific amplification of a 461-bp expressed mRNA and EST databases in Genbank (16). fragment. PCR amplification was run for 24 to 32 cycles Consensus sequences representing human expressed sequences (see RESULTS). were generated using the Clustering and Alignment Tool Immunohistology. To evaluate and localize MMP-12 software (DoubleTwist, Oakland, CA), and prediction of the expression in tumor tissue, immunohistology was done on expressed genome from the human genome sequence was done four NSCLC specimens with high MMP-12 expression using ab initio exon prediction (17). The 59,000 probesets on (adenocarcinoma, n = 2; squamous cell carcinoma, n =2) this microarray represent f45,000 mRNA and EST clusters and and their corresponding lung tissue. Four-micron-thick sec- 6,200 predicted exons. Data were used after gamma-distribution tions were prepared from formalin-fixed and paraffin-embed- normalization. ded tissues mounted on Star Frost adhesive-slides, dried for Gene Chip Analysis. For analysis of gene expression in 2 hours at 60jC and dewaxed in xylene and graded ethanols. native tumor samples and control tissues, biotinylated cRNA Nonspecific protein staining was blocked by preincubation for

5 minutes with 1:10 diluted Normal Horse Serum (Vector of the eligible 70 patients had tumor relapse. Local recurrence Laboratories). The slides were incubated with 1:10 diluted occurred in 5 (7.1%) and distant metastases in 20 (28.6%) monoclonal anti-human MMP-12 hemopexin-like domain patients, 6 (8.6%) patients suffered from both. There were no antibody Clone 4D2 (RD Systems, Wiesbaden, Germany) significant correlations between MMP-1, -9, -10, and -11 for 30 minutes at 37jC. After addition of biotinylated expression and the incidence of tumor relapse (Table 2B). secondary antibody (DCS, Hamburg, Germany) the slides However, among the 56 patients with high MMP-12 expression were incubated with Avidin-Biotin Complex reagent (DCS) in the tumor tissues, 29 (51.7%) developed local recurrence and/ for another 10 minutes. Antibody localization was visualized or distant metastases, whereas only 2 (14.2%) of 14 with low with 3-amino-9-ethyl-carbazol and H2O2, followed by a final MMP-12 expression in the tumor tissues had tumor relapse. counter-staining with hematoxylin. All slides were rinsed with Interestingly, MMP-12 expression level significantly correlated phosphate-buffered saline after each step of the procedure, with tumor relapse (P = 0.04). except after application of the normal serum. Controls were Matrix Metalloproteinase-12 Expression Significantly prepared without primary antibody. Correlated with Relapse-Free Survival. Kaplan-Meier ana- Statistical Analysis. Differences in the frequency of lysis of tumor relapse-free survival, comparing patients with MMP-positive sections insubgroups of patients were ana- MMP-12 low expressed (n = 14) to MMP-12 high expressed lyzed by Pearson’s v2 test. For analysis of follow-up data, tumors (n = 56), showed a significant correlation (P = 0.02) life table curves were calculated with Kaplan-Meier between high MMP-12 expression and an unfavorable outcome methods, and survival distributions were compared by use (Fig. 1). The 3-year tumor relapse-free rate in patients with high of log-rank statistics. The Cox proportional hazards model MMP-12 Expression was 32% and 85% in patients with low were applied for multivariate analysis using the Statistical MMP-12 tumor expression. Expression of MMP-12, sex, age, Package for the Social Sciences software program (SPSS, Inc., histology, tumor-node-metastasis staging, and grading were Chicago, IL). Only patients with confirmed post-operative R0 tested for independence of a possible prognostic value (Table status were admitted to the analysis of the disease-free 3). The Cox proportional hazards model showed that MMP-12 survival. The threshold for statistical significance was chosen expression was a significant (P = 0.04) independent prognostic as P = 0.05. predictor for disease-free survival in patients with resected NSCLC. The relative risk for tumor relapse in all R0-resected RESULTS patients was 4.8-fold higher in patients with high expression of Relative Expression of Matrix Metalloproteinases in MMP-12 in the tumor tissue. The other possible risk factors did Non–Small Cell Lung Cancer and Control Lung. Compa- not significantly correlate to tumor recurrence. rison of expression profiles from control lung and NSCLC No Coexpression of Matrix Metalloproteinase-12 in revealed changes in gene expression in a total of 344 (0.6%) Healthy Tissue. To examine the prevalence of MMP-12 in genes according to the chosen levels of significance (P < 8.0 Â other healthy organs, we compared our MMP-12 expression 10À7) and fold-change (cutoff of 2.0). Most of the 22 values to data of an EOS-Biotechnology own body map list. investigated MMPs were not differentially expressed in control Except for two samples (colon and lymph node) all other tissues lung and tumor tissue (Table 2A). Only MMP-1, MMP-9, and showed no MMP-12 expression (Fig. 2). MMP-12 were differently expressed. MMP-10 and MMP-11 Verification of Array Data by Reverse Transcription- expression achieved only one of the quality objectives for PCR and Immunohistology. To verify the results of our differentially expressed genes, level of significance or fold- microarray studies, reverse transcription-PCR and immunohis- change. Lung tissues showed no or only minimal expression of tology were done. MMP-1, MMP-10, and MMP-12, whereas MMP-9 and MMP- Differences in expression patterns for MMP-12 could be 11 were also coexpressed at a high level in lung tissue. The confirmed in 23 NSCLC and 6 control lungs by reverse highest fold-change (4.1) and the highest level of significance transcription-PCR (Fig. 3A). All patients with tumor relapse (1.4 Â 10À20) was reached by MMP-12. Because the had moderate to high levels of MMP-12 expression. As expression values of these MMPs were highly variable in graphically depicted in Fig. 3B, there was a high correlation the tumor samples, we individually determined cutoffs for the (KK = 0.83) between reverse transcription-PCR and oligonu- classification into groups of low and high expression. The cleotide array results for all 23 tumor probes. All samples cutoffs were defined by an expression level of about 150% without tumor relapse were focused in the left lower corner of of the controls (cutoffs for MMP-1, -10, and -12 was 100 the graph. relative expression units; for MMP-11, 300 relative expression Immunostaining for MMP-12 showed a weak to units; and for MMP-9, 400 relative expression units, moderate, sometimes patchy positive reaction of the tumor respectively). tissue. The staining was restricted to the cytoplasm of the Matrix Metalloproteinase-12 Predicts Tumor Relapse. tumor cells only (Fig. 4). Concerning the intensity of the Of the 75 R0-resected patients, two died within 30 days (hospital immunoreaction, we did not notice remarkable differences mortality), two succumbed to cancer-unrelated diseases and one between adenocarcinomas and squamous cell carcinomas. woman died of a second primary tumor. This resulted in 70 R0- There was an invariable weak to moderate positive immunos- resected patients for the analysis of tumor relapse. Tumor relapse taining of alveolar macrophages and bronchiolar epithelia of was defined as local recurrence, distant metastasis, or both. the surrounding lung tissue, serving as an internal positive Within the follow-up period (median 26.3 months), 30 (42.8%) control.

Table 2 MMP gene expression (A) Gene expression data from all investigated MMPs Gene name Expression (lung) Expression (tumor) t test (P) Fold-change (tumor/lung) Matrix metalloproteinase 1 60.0 220.0 1.9 Â 10À11 3.7 Matrix metalloproteinase 2 789.0 807.0 0.7 1.0 Matrix metalloproteinase 3 60.0 64.5 0.04 1.0 Matrix metalloproteinase 7 228.0 386.5 0.004 1.7 Matrix metalloproteinase 8 81.0 99.0 0.004 1.2 Matrix metalloproteinase 9 252.0 530.0 5.5 Â 10À10 2.1 Matrix metalloproteinase 10 60.0 185.6 3.1 Â 10À6 3.0 Matrix metalloproteinase 11 213.9 342.8 7.9 Â 10À8 1.6 Matrix metalloproteinase 12 60.0 248.7 1.4 Â 10À20 4.1 Matrix metalloproteinase 13 60.0 85.0 0.002 1.4 Matrix metalloproteinase 14 319.0 413.2 0.001 1.3 Matrix metalloproteinase 15 65.8 71.6 0.08 1.0 Matrix metalloproteinase 16 60.0 60.0 1.0 1.0 Matrix metalloproteinase 17 139.8 163.9 0.01 1.1 Matrix metalloproteinase 19 93.9 61.7 0.02 0.6 Matrix metalloproteinase 20 60.0 60.0 1.0 1.0 Matrix metalloproteinase 23B 402.5 308.0 0.007 0.7 Matrix metalloproteinase 24 306.0 314.0 0.4 1.0 Matrix metalloproteinase 25 213.5 163.5 0.02 0.7 Matrix metalloproteinase 26 60.0 60.0 1.0 1.0 Matrix metalloproteinase 27 60.0 60.0 1.0 1.0 Matrix metalloproteinase 28 209.5 212.5 0.73 1.0 (B) Differently expressed MMPs in correlation to tumor relapse (local recurrence and/or distant metastasis) Gene Expression n No recurrence Local recurrence Distant metastasis Both P MMP-1 Low expression 34 21 (61.8%) 3 (8.8%) 7 (20.6%) 3 (8.8%) 0.53 High expression 36 18 (50.0%) 2 (5.6%) 13 (36.1%) 3 (8.3%) MMP-9 Low expression 18 12 (66.7%) 1 (5.6%) 3 (16.7%) 2 (11.1%) 0.57 High expression 52 27 (51.9%) 4 (7.7%) 17 (32.4%) 4 (7.7%) MMP-10 Low expression 47 24 (51.1%) 3 (6.4%) 16 (34.0%) 4 (8.5%) High expression 23 15 (65.2%) 2 (8.7%) 4 (17.4%) 2 (8.7%) 0.53 MMP-11 Low expression 38 21 (55.3%) 3 (7.9%) 10 (26.3%) 4 (10.5%) 0.89 High expression 32 18 (56.3%) 2 (6.3%) 10 (31.3%) 2 (6.3%) MMP-12 Low expression 14 12 (85.7%) 1 (7.1%) 0% 1 (7.1%) 0.04 High expression 56 27 (48.2%) 4 (7.1%) 20 (35.7%) 5 (8.9%)

DISCUSSION Tumor invasion and metastasis require controlled degradation of extracellular matrix. MMPs play a crucial role in this breakdown of collagen and basement membrane components. In the present study, we investigated the gene expression of MMPs in NSCLC and the lung. Whereas previous studies (7, 8, 18) compared expression of a few members of the MMP family by immunohistology, genome-wide DNA microarray analysis enabled us to investigate 22 MMPs simultaneously. In our study, MMP-1, MMP-9, and MMP-12 were expressed more strongly in the NSCLCs of both squamous epithelial and adenoid morphology than in the corresponding nonmalignant lung tissues, where expression of MMP-1 and MMP-12 was generally low or absent. The strong MMP-1 and MMP-9 expression in NSCLC (8, 18, 19), as well as in SCLC (20), has been previously described. The present study shows for the first time that MMP-12 is highly expressed in NSCLC and strongly correlates with local recurrence as well as metastatic disease in patients with resected NSCLC. MMP-12 was primarily identified as an elastolytic metalloproteinase secreted by inflammatory macrophages (21). This Fig. 1 Kaplan-Meier plot of disease-free survival for R0 resected enzyme has broad substrate specificity, including extracellular NSCLC patients (n = 70). Overall relapse-free survival predicted by low matrix proteins such as type I gelactin, fibronectin, laminin, versus high MMP-12 expression.

Table 3 Multivariate Cox regression analysis of potential risk factors for tumor relapse in patients with R0 resected NSCLC RR 95% Confidence interval P Sex Male 0.77 0.28-2.10 0.61 Female Age (y) V65 0.75 0.36-1.56 0.44 >65 Histology Squamous cell carcinoma 1.77 0.77-4.05 0.17 Adenocarcinoma Tumor-node-metastasis stage I 0.23 II 0.79 0.28-2.25 0.67 III 1.82 0.76-4.36 0.17 Grading well/moderately well differentiated 0.85 0.35-2.06 0.73 poorly differentiated/undifferentiated MMP-12 Low expression 4.86 1.06-22.26 0.04 High expression

vitronectin, proteoglycans, and fibrin and it can also produce enous staining in the tumor cells, whereas no significant staining angiostatin from plasminogen. Expression of MMP-12 in vivo was seen in the lung or esophagus. Therefore, it is postulated has previously been described in hepatocellular carcinoma that the tumor cell–related MMP-12 expression might be (22), colorectal carcinoma (23), vulva carcinoma (24), renal correlated with tumor growth, invasion, and metastasis (24). In cell carcinomas (25), skin cancer (26), pancreatic cancer (27), fact, Ding et al. (28) found a direct significant correlation and in esophageal carcinoma (28). However, the role of between MMP-12 mRNA expression and the depth of MMP-12 in tumor pathogenesis seems to depend on the type esophagus wall invasion as well as the frequency of lymph of tissue involved. In contrast to our study with NSCLC, node metastasis in esophageal squamous cell carcinomas. MMP-12 overexpression in hepatocellular as well as in Corresponding with our data, a positive correlation of MMP- colorectal carcinoma closely correlated with better prognosis, 12 expression and tumor aggressiveness has been shown for which was explained by the antiangiogenic function of MMP- vulva carcinoma and skin cancer. The level of MMP-12 12 on the basis of the generation of angiostatin from expression correlated with epithelial dedifferentiation and plasminogen. Angiostatin inhibits endothelial cell proliferation, histologic aggressiveness (24, 26). MMP-12 expression is also thereby possibly leading to a reduction of metastatic potential (22, 29). On the other hand, the macrophages of MMP-12 À/À mice had a markedly diminished capacity to degrade extracellular matrix components and were also essentially unable to penetrate reconstituted basement membranes in vitro and in vivo (30). However, MMP-12 is not only required for macrophage- mediated extracellular matrix proteolysis. Immunochemical investigations of MMP-12 in the present NSCLC as well as in a esophageal squamous cell carcinoma study showed homog-

Fig. 3 A, reverse transcription-PCR analysis of MMP-12 gene expression in 23 NSCLC tissues with (+) or without tumor relapse and Fig. 2 MMP-12 expression of NSCLC and corresponding lung in 6 normal lung tissues. h-Actin expression level is used as a control. B, correlation to MMP-12 expression data from 158 tissues of healthy correlation analysis of MMP-12 expression determined by reverse organs (body map). transcription-PCR and oligonucleotide array (KK = 0.83).

Fig. 4 Immunhistochemical staining of adenocarcinoma (A) with corresponding lung (B) and squamous cell carcinoma (C) with corresponding lung (D) showing positive MMP-12stainingintumor cellsaswellaspositive MMP-12 expression in alveolar macrophages in the normal lung; Â100.

inversely correlated with prognosis. Survival of patients with ACKNOWLEDGEMENTS pancreatic cancer overexpressing MMP-12 mRNA was signif- We thank Anke Graul and Dr. Hannes Bahn for their technical icantly shorter as compared with patients with a tumor that did assistance. not overexpress MMP-12 (27). Our body map data demonstrating low or missing expression REFERENCES of MMP-12 in healthy tissues support the concept of adjuvant 1. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA therapy with MMP inhibitors in NSCLC. However, the first phase Cancer J Clin 2002;52:23–47. III studies with broad-spectrum MMP inhibitors revealed no 2. Passlick B, Kubuschok B, Izbicki JR, Thetter O, Pantel K. Isolated significant differences in survival in a NSCLC prinomastat study tumor cells in bone marrow predict reduced survival in node-negative non-small cell lung cancer. Ann Thorac Surg 1999;68:2053–8. as well as in a SCLC marimastat trial (31). Selective MMP 3. Chambers AF, Matrisian LM. Changing views of the role of matrix inhibitors might be of more benefit, because BAY 12-9566 for metalloproteinases in metastasis. J Natl Cancer Inst 1997;89:1260–70. instance, a potent inhibitor of MMP-2, -3, and -9 prolonged 4. Westermarck J, Kahari VM. Regulation of matrix metalloproteinase progression-free interval of NSCLC patients in an adjuvant expression in tumor invasion. Faseb J 1999;13:781–92. therapy (9). ONO-4817, a third-generation MMP inhibitor against 5. Liotta LA, Abe S, Robey PG, Martin GR. Preferential digestion of MMP-2, -8, -9, -12, and -13, suppressed progression of lung basement membrane collagen by an enzyme derived from a metastatic murine tumor. Proc Natl Acad Sci U S A 1979;76:2268–72. micrometastasis of MMP-expressing tumor cells in nude mice in 6. Liotta LA, Tryggvason K, Garbisa S, et al. Metastatic potential combination with docetaxel (32). Phase III clinical trials with correlates with enzymatic degradation of basement membrane collagen. Neovastat, a multifunctional antiangiogenic agent against vascu- Nature 1980;284:67–8. lar endothelial growth factor, MMP-2, -9, and -12 are currently 7. Passlick B, Sienel W, Seen-Hibler R, et al. Overexpression of matrix under investigation in patients with unresectable NSCLC (33). metalloproteinase 2 predicts unfavorable outcome in early-stage non- small cell lung cancer. Clin Cancer Res 2000;6:3944–8. In summary, we show for the first time that MMP-12 8. Sienel W, Hellers J, Morresi-Hauf A, et al. Prognostic impact of is highly expressed in NSCLC tumors but not in control lung matrix metalloproteinase-9 in operable non-small cell lung cancer. Int J tissueandthatMMP-12expressioninNSCLCtumor Cancer 2003;103:647–51. correlates positively with the metastatic potential as well as 9. Rigas JR, Denham CA, Rinaldi DA, et al. Randomized placebo- shortened relapse-free interval in patients with NSCLC. controlled trials of the matrix metalloproteinase inhibitor (MMPI), Therefore, MMP-12 expression might be suitable for identifi- BAY12-9566 as adjuvant therapy for patients with small cell and non- small cell lung cancer. Proc Am Soc Clin Oncol 2003;22:628. cation of patients at high risk for early tumor relapse that 10. Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. might benefit from adjuvant therapy. Furthermore, the present Histological typing of lung and pleural tumours. 3rd ed. Berlin: study might support the concept of selective MMP inhibitor Springer; 1999. therapy in NSCLC patients with high expression of MMP-12 11. Mountain CF. Revisions in the international system for staging lung in tumor tissue. cancer. Chest 1997;111:1710–7.

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Hans-Stefan Hofmann, Gesine Hansen, Günther Richter, et al.

Clin Cancer Res 2005;11:1086-1092.

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