HENNI RUOKOLAINEN. Faculty of Medicine, Department of Oncology and Radiotherapy, University of Oulu Oulu University Hospital OULU 2005

Transcription

1 THE PROGNOSTIC ROLE OF MATRIX METALLOPROTEINASE -2 AND -9 (MMP-2, MMP-9) AND THEIR TISSUE INHIBITORS -1 AND -2 (TIMP-1, TIMP-2) IN HEAD AND NECK SQUAMOUS CELL CARCINOMA HENNI RUOKOLAINEN Faculty of Medicine, Department of Oncology and Radiotherapy, University of Oulu Oulu University Hospital OULU 2005

2 HENNI RUOKOLAINEN THE PROGNOSTIC ROLE OF MATRIX METALLOPROTEINASE -2 AND -9 (MMP-2, MMP-9) AND THEIR TISSUE INHIBITORS -1 AND -2 (TIMP-1, TIMP-2) IN HEAD AND NECK SQUAMOUS CELL CARCINOMA Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium 7 of Oulu University Hospital, on December 16th, 2005, at 12 noon OULUN YLIOPISTO, OULU 2005

3 Copyright 2005 University of Oulu, 2005 Supervised by Professor Taina Turpeenniemi-Hujanen Reviewed by Professor Reidar Grénman Professor Heikki Joensuu ISBN (nid.) ISBN (PDF) ISSN OULU UNIVERSITY PRESS OULU 2005

4 Ruokolainen, Henni, The prognostic role of matrix metalloproteinase -2 and -9 (MMP-2, MMP-9) and their tissue inhibitors -1 and -2 (TIMP-1, TIMP-2) in head and neck squamous cell carcinoma Faculty of Medicine, Department of Oncology and Radiotherapy, University of Oulu, P.O.Box 5000, FIN University of Oulu, Finland; Oulu University Hospital, P.O.Box 10, FIN OYS, Finland 2005 Oulu, Finland Abstract Traditional clinicopathological factors are not accurate enough to predict the behavior of head and neck squamous cell carcinoma (HNSCC). The most powerful indicator of prognosis is the stage of the disease. New prognostic markers have, however, been searched for in order to better identify patient groups in need of different treatments or follow-up. Gelatinases (MMP-2, -9) are endopeptidases associated with tumor invasion and angiogenesis, and their tissue inhibitors (TIMP- 1, -2) are also linked to cancer cell invasion and metastasis formation. In some cancer types they are even prognostic and relate with a more aggressive clinical course of the disease. In the present work the expression and the clinical significance of tumor tissue and circulating immunoreactive proteins for MMP-2, -9, TIMP-1 and -2 were assessed in HNSCC. The study group included 74 patients with HNSCC and 44 healthy controls. The expression of immunoreactive proteins was examined in paraffin-embedded tumor sections by immunohistochemical staining using specific antibodies, and the pretreatment serum levels of those proteins were quantitatively measured by ELISA assay. Immunohistochemical overexpression of MMP-9 in tumor was for the first time found to predict the prognosis for shortened survival in HNSCC, the cause-specific survival rates being 45% and 92% and relapse-free survival being 42% and 79% in MMP-9 positive or negative cases, respectively. Additionally, tissue TIMP-1, MMP-2 and TIMP-2 positivity were all also linked with poorer survival of patients with HNSCC. However, these differences remained less distinct than with MMP-9. The expression of gelatinases and their inhibitors in tumor tissue was also an indicator for later lymph node or hematogenic relapses in HNSCC patients. Circulating MMP-9 and TIMP-1 levels were significantly higher in HNSCC patients than in healthy controls. Further, the causespecific and relapse-free survival rates were lower among HNSCC patients with high MMP-9 and TIMP-1 serum levels compared to patients with low levels of circulating MMP-9 and TIMP-1. A significant correlation was shown between circulating MMP-9 and MMP-9 immunohistochemical staining in the corresponding tumors. No correlation was found between tissue or circulating levels of gelatinases or their inhibitors and the traditional clinical or histopathological factors, except for the association between tissue and circulating TIMP-1 and the size of the primary tumor. Taken together, these results suggest that tissue expression of gelatinases and their inhibitors as well as pretreatment circulating MMP-9 and TIMP-1 levels could be prognostic in estimation of the clinical course of HNSCC. The results indicate further studies are needed with larger patient materials. Keywords: head and neck squamous cell carcinoma, invasion, metastasis, prognostic marker, survival

5 To my family

6

7 Acknowledgements This thesis project was carried out at the Department of Oncology and Radiotherapy, University of Oulu, during the years I wish to express my sincere gratitude to my supervisor, Professor Taina Turpeenniemi-Hujanen, M.D., Ph.D., Head of the Department of Oncology and Radiotherapy, for introducing me to the challenging field of oncology and matrix metalloproteinases. I really appreciate her knowledge and the enthusiasm she has shown during the time of my research project. Her positive attitude towards life in general and her never failing optimism have supported and encouraged me during these years. It has been a privilege to work with you. I express my special appreciation to Docent Paavo Pääkkö, M.D., Ph.D., for his fruitful collaboration, encouragement and guidance in the field of pathology. It has been a great pleasure to work with you throughout the study period. I am most grateful to the reviewers of this work, Professor Reidar Grénman, M.D., Ph.D., and Professor Heikki Joensuu, M.D., Ph.D., for their careful revision of my thesis and for valuable comments and advice. I thank Anna Vuolteenaho, M.A., for the review of the language. I wish to thank Docent Ulla Puistola, M.D., Ph.D., and Professor Kirsi Vähäkangas M.D., Ph.D. for their support and for sharing their knowledge during this project. I wish to express my sincere thanks to my colleagues at the Department of Oncology and Radiotherapy for their friendship and encouragement during these years. I also warmly thank my investigator colleagues in the Research Laboratory of the Department of Oncology and Radiotherapy for creating a friendly and supportive working atmosphere. I wish to thank Ms. Kaisu Järvenpää and Ms. Anne Bisi for their skillful technical assistance and continuous support during this work. I would also like to thank Ms. Merja Matilainen, Ms. Annika Isohätälä, Ms. Liisa Kärki, Ms. Seija Leskelä, Ms. Toini Flygare and Ms. Katri Takalahti for their technical help and Risto Bloigu, M.Sc., for his guidance in the field of statistics. I want to thank my dearest friend Piritta Ruuska for her long-lasting friendship and her support during this study. Sincere thanks to all of my friends for encouragement. I am also grateful to the Blanco family for their support.

8 I warmly thank Andreas Blanco for friendship, love and encouragement. I owe my deepest gratitude to my parents, Marja-Liisa and Hannu Ruokolainen, for their love, support and encouragement during my whole life and especially during this work. You have always been there for me and believed in my projects. This work was financially supported by the University of Oulu, the Cancer Society of Northern Finland, the Finnish Cancer Society and the Ida Montin Foundation, which are gratefully acknowledged. Oulu, October 2005 Henni Ruokolainen

9 Abbreviations AEC BM EBV ECM EGFR ELISA HNSCC HPV MMP MT-MMP mrna PA upa tpa TIMP TNM VEGF amino-ethyl-carbazole basement membrane Ebstein-Barr virus extracellular matrix epidermal growth factor receptor enzyme-linked immunoassay head and neck squamous cell carcinoma human papilloma virus matrix metalloproteinase membrane-type matrix metalloproteinase messengerrna plasminogen activator urokinase-type plasminogen activator tissue-type plasminogen activator tissue inhibitor of metalloproteinase Tumor-Node-Metastasis staging vascular endothelial growth factor

10

11 List of original articles This thesis is based on the following papers which are referred to in the text by their Roman numbers. I Ruokolainen H, Pääkkö P and Turpeenniemi-Hujanen T (2004) Expression of matrix metalloproteinase-9 in head and neck squamous cell carcinoma: a potential marker for prognosis. Clin Cancer Res 10: II Ruokolainen H, Pääkkö P and Turpeenniemi-Hujanen T (2005) Serum matrix metalloproteinase-9 in head and neck squamous cell carcinoma is a prognostic marker. Int J Cancer 116: III Ruokolainen H, Pääkkö P and Turpeenniemi-Hujanen T (2005) Tissue inhibitor of matrix metalloproteinase-1 is prognostic in head and neck squamous cell carcinoma comparison of the circulating and tissue immunoreactive protein. Clin Cancer Res 11: IV Ruokolainen H, Pääkkö P and Turpeenniemi-Hujanen T. Tissue and circulating immunoreactive protein for MMP-2 and TIMP-2 in head and neck squamous cell carcinoma tissue immunoreactivity predicts aggressive clinical course. Modern Pathology, in press.

12

13 List of figures Fig. 1. Clinical, pathological and molecular progression of head and neck cancer.. 21 Fig. 2. The role of matrix metalloproteinases in tumor progression and invasion.. 26 Fig. 3. Possible mechanisms of cellular signaling mediated by MMPs Fig. 4. Basic domain structure of the gelatinases Fig. 5. Correspondence of tissue and circulating TIMP-1 (a) and MMP-9 (b) in HNSCC patients Fig. 6. Association of the level of tissue MMP-9 expression to the site of first relapse in patients with HNSCC Fig. 7. Association between tissue TIMP-1 expression and the site of first relapse in HNSCC patients Fig. 8. Association of the level of tissue TIMP-2 expression with the site of first relapse in patients with HNSCC Fig. 9. The 5-year cause-specific survival (a) and relapse-free survival (b) rates for the HNSCC patients according to tissue MMP-9 and TIMP-1 immunoreactivity Fig. 10. The 5-year cause-specific survival (a) and relapse-free survival (b) rates of the HNSCC patients according to tissue MMP-2 and TIMP-2 immunoreactivity

14

15 List of tables Table 1 TNM classification. Lip cancer and cancer of the oral cavity are used as an example Table 2 Vertebrate MMPs and some of their substrates Table 3 Molecular characteristics of TIMPs Table 4 Reported findings of collagenases, stromelysins, matrilysins and membrane-type MMPs in head and neck squamous cell carcinoma Table 5 Clinical data of the head and neck squamous cell carcinoma patient material in this study Table 6 Antigens and their respective antibodies used in immunohistochemistry Table 7 Coating antibodies and secondary antibodies used in ELISA according to the measured immunoreactive proteins Table 8 The grades of the immunoreaction for MMP-2, MMP-9, TIMP-1 and TIMP-2 in head and neck squamous cell carcinoma tissue Table 9 Serum level (ng/ml) of MMP-2, MMP-9, TIMP-1, TIMP-2 immunoreactive protein in head and neck squamous cell carcinoma patients and in healthy controls Table 10 Correlation between the MMP-9 immunohistochemical staining results in primary tumors and the MMP-9 immunoreactive protein level in serum Table 11 The effect of tumor tissue immunoreactive protein staining intensity of MMP-2, MMP-9, TIMP-1 and TIMP-2 on the survival of patients with head and neck squamous cell carcinoma Table 12 The association of circulating immunoreactive protein level of MMP-9 and TIMP-1 with the survival of patients with head and neck squamous cell carcinoma

16

17 Contents Abstract Acknowledgements Abbreviations List of original articles List of figures List of tables Contents 1 Introduction Review of the literature Head and neck squamous cell carcinoma Epidemiology Diagnosis and staging Prognostic factors Treatment modalities in HNSCC Tumor invasion and metastasis Gelatinases and their inhibitors Matrix metalloproteinase (MMP) gene family Regulation of MMPs Matrix metalloproteinase Matrix metalloproteinase Tissue inhibitors of metalloproteinases Gelatinases and their tissue inhibitors in head and neck squamous cell carcinoma The prognostic role of gelatinases and their tissue inhibitors in cancer patients Aims of the present study Material and methods Patients and tissue and serum samples Immunohistochemistry Immunohistochemical staining Evaluation of the immunohistochemical stainings

18 4.3 Enzyme-linked immunoassay (ELISA) Statistical analyses Ethical aspects Results The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in head and neck squamous cell carcinoma tissue (I IV) The pretreatment serum levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 immunoreactive protein (II IV) Comparison of tumor tissue and circulating immunoreactive protein levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 (II IV) Correlation of MMP-2, MMP-9, TIMP-1 and TIMP-2 immunoreactive proteins with traditional clinicopathological markers Tissue immunoreactive proteins (I, III, IV) Circulating immunoreactive protein (II, III, IV) The prognostic value of tumor tissue gelatinases and their tissue inhibitors in head and neck squamous cell carcinoma (I-IV) Tissue MMP-2 and MMP-9 as prognostic markers (IV, I) Tissue TIMP-1 and TIMP-2 as a prognostic marker (III, IV) The prognostic value of pretreatment serum level of MMP-2, MMP-9, TIMP-1 and TIMP-2 in HNSCC (II-IV) MMP-2 and MMP-9 (IV, II) TIMP-1 and TIMP-2 (III, IV) Discussion Expression of gelatinases as well as TIMP-1 and TIMP-2 in head and neck squamous cell carcinoma Circulating MMP-2 and -9 and their tissue inhibitors in HNSCC patients and in healthy controls Evaluation of correspondence with tissue and circulating immunoreactive protein of gelatinases and their tissue inhibitors in HNSCC patients The prognostic value of gelatinases and their tissue inhibitors in HNSCC MMP-2 and MMP TIMP-1 and TIMP Comparison of the clinical relevance of gelatinases and their tissue inhibitors in HNSCC Conclusions References

19 1 Introduction Head and neck cancers consist of a heterogeneous group of neoplasias, 90% of these tumors representing a squamous cell carcinoma (HNSCC). Globally, more than 500,000 new cases are diagnosed each year. The incidence of HNSCC has increased in Western countries during the last few years, partly because of the increased use of tobacco and alcohol, which are widely documented risk factors of HNSCC (Sanderson & Ironside 2002). Although patients presenting with early stage disease have rather high cure rates, those diagnosed with locoregionally advanced disease have had poorer prognosis and limited therapeutic options. The last decade has seen the integration of chemoradiotherapy and some improvements in the survival in HNSCC patients. The major cause of death among these patients is a local-regional recurrence. At present, the only prognostic factor that is commonly used in clinical work is the stage of the disease, particularly the presence of lymph node metastases (Ensley et al. 2002, Cummings et al. 2004). It is recognized that the prognosis of patients in each stage group of the disease may also differ greatly, suggesting that some other, possibly biological, factors may also play a role in the progression of HNSCC. Therefore, valuable markers associated with biological aggressiveness of the tumors are needed to predict the outcome of the disease and to better recognize patient groups that need more aggressive treatment options. Additionally, the discovery of new prognostic markers could promote entirely new treatment possibilities, especially in adjuvant treatment. Nowadays also molecularly targeted therapies are in clinical use in locally advanced and metastatic HNSCC, and there is an obvious need of markers that could be used as selection criteria for these novel therapies. The capability of tumors to infiltrate the surrounding tissue is one of the main characteristics of a malignancy. This process is based on the tumor cells ability to destroy the extracellular matrix (ECM), including the basement membrane. Matrix metalloproteinases form a family of zinc dependent endopeptidases that are capable of degrading most components of the extracellular matrix (ECM) (Stetler-Stevenson et al. 1993, Nagase & Woessner 1999, Stetler-Stevenson & Yu 2001). Two of these MMP enzymes, gelatinase A (MMP-2) and gelatinase-b (MMP-9), are known to be able to degrade several extracellular matrix proteins including type IV collagen, which is an essential part of basement membranes. Gelatinases have thus been linked very strongly to cancer cell invasion and the process of metastasis (Stetler-Stevenson 2001). Tissue inhibitors of matrix metallo-

20 18 proteinases (TIMPs) are known to have the ability to inhibit the catalytic activity of matrix metalloproteinases. TIMP-1 and TIMP-2 are inhibitors of MMP-9 and MMP-2, respectively. In addition to TIMPs inhibitory role, they can also take part in the activation of MMPs (Gomez et al. 1997). The TIMPs also seem to have anti-angiogenic activity and they are able to act as growth factors (Fassina et al. 2000). There are growing amount of data suggesting that expression of gelatinases as well as their tissue inhibitors are associated with clinical course in some cancer types (Turpeenniemi-Hujanen 2005). Additionally, circulating levels of gelatinases and TIMPs could be valuable in assessing prognosis or diagnosing a relapse during follow-up (Endo et al. 1997, Ylisirniö et al. 2000, Laack et al. 2002, Ranuncolo et al. 2003). The present study was designed to study the expression and localization of gelatinases and their tissue inhibitors in head and neck squamous cell carcinoma, with specific focus on whether they correlate with traditional clinico- and histopathological prognostic markers. Further, the expression of immunoreactive protein of gelatinases and their inhibitors in tumor tissue and in serum was compared, as were the serum levels of HNSCC patients and healthy controls. Finally, the tumor tissue and pretreatment serum levels of MMP-2, MMP-9, TIMP-1 and TIMP-2 were correlated with the occurrence and type of later metastases as well as with cause-specific and relapse-free survival.

21 2 Review of the literature 2.1 Head and neck squamous cell carcinoma More than 90% of cancers in the head and neck region are squamous cell carcinomas. HNSCC consist of a heterogeneous group of neoplasias arising from different anatomical locations in the oral cavity, pharynx, larynx, nose, lips and paranasal sinuses. (Forastiere et al. 2001, Ensley et al. 2002). In the pharynx the area is bordered by the cricopharyngeal muscle in the esophagus and in the larynx it is bordered at the cricoid cartilage to the trachea. HNSCC tumors have the capacity to invade adjacent tissues and metastasize locoregionally (Ensley et al. 2002). The clinical behavior of tumors of the head and neck and their responses to oncological treatment vary and may be partly associated with the biological heterogeneity of these tumors. As distant metastases are quite rare, head and neck squamous cell carcinomas represent malignancies where locoregional control is the most important factor determining survival, indicating that local-regional recurrence is the most important cause of death among these patients (Cerezo et al. 1992, Forastiere et al. 2001). Unfortunately, the long-term survival rates among HNSCC patients have increased only marginally during the last decade, despite the fact that diagnostic methods and treatment strategies have improved Epidemiology Squamous cell carcinoma of the head and neck is one of the most common cancers, constituting 4% of all newly diagnosed cancers in the United States and 5% in the United Kingdom. Worldwide more than 500,000 new cases are diagnosed annually (Ensley et al. 2002). In Finland around six hundred cases appear annually (Cancer Registry in Finland, The incidence rates are higher among male compared with female population. In general, the incidence of laryngeal cancer in women is lower than in males, whereas the difference is not that significant in oral and pharyngeal cancer. In Finland the incidence of larynx cancer in males has decreased during the last four

22 20 decades from 7.6 to 2.2 per , whereas in females it has remained stable (0.3) (Cancer Registry in Finland, The median age at diagnosis for HNSCC is about 60 years. The most important risk factors for HNSCC are tobacco and excessive alcohol consumption. Alcohol potentiates the tobacco-related carcinogenesis and is also an independent risk factor. The relative risk for head and neck carcinoma among tobacco and alcohol abusers is 20 times that of nonsmokers and nondrinkers (Blot et al. 1988, Lewin et al. 1998). Epidemiologic studies also suggest a strong association between most types of smokeless tobacco and oral carcinogenesis (Gupta et al. 1996, Hoffmann & Djordjevic 1997). Occupational risk factors include exposure to hard wood dust, concrete dust and textile fibers (Decker & Goldstein 1982, Brown et al. 1988, Muscat & Wynder 1992, Dietz et al. 2004). A growing amount of evidence suggests that some viruses contribute to the cause of head and neck cancer. DNA from human papillomavirus has been detected in cancer tissue of head and neck using immunoperoxidase staining, polymerase chain reaction, Southern blotting and in situ hybridization, and the most common subtypes detected are type 16 and 18 (Syrjänen et al. 1983a, 1983b, Miller & Johnstone 2001, Paz et al. 1997, McKaig et al. 1998, Mineta et al. 1998b, Gillison et al. 2000, Gillison & Shah 2001). According to Syrjänen (2005), the latest meta-analyses of epidemiological studies as well as case-control studies have confirmed HPV as an independent risk factor for oral cancer, particularly for tonsillar carcinoma and HPV-16 being the most prevalent subtype. Seropositivity for HPV-16 has in particular been found to be an obvious risk factor for HNSCC (Mork et al. 2001). There is also evidence that tumors in the oropharyngeal area and tumors in nonsmokers are most frequently HPV positive (Fouret et al. 1997, Gillison et al. 2000, Dahlström et al. 2003). Infection with the Epstein-Barr virus is connected particularly with endemic nasopharyngeal cancer, whereas no clear association has been found with sporadic nasopharyngeal carcinomas and EBV (Zong et al. 1992, Atula et al. 1997, Lin et al. 2001). Additionally, some dietary items such as maize have been linked with HNSCC (Franceschi et al. 1990). It is known that HNSCC emerges after the accumulation of genetic changes in epithelial cells exposed to carcinogenesis. This hypothesis consists of three basic principles: 1) neoplasms are the result of inactivation of tumor-suppressor genes and/or activation of proto-oncogenes; 2) there exists a defined order of genetic events leading to the development of tumor phenotype; and 3) there could be variations in the order of genetic events, but it is eventually the net accumulation of genetic alterations that determines the malignant phenotype (Kim & Califano 2004) (Fig. 1). The opportunity of a genetic predisposition to cause head and neck cancer is suggested by its sporadic occurrence in some cases in young adults, nonsmokers and nondrinkers. Information is accumulating on the genetic basis of a possible multistep process of carcinogenesis. Deletions of chromosomes 9p21-22, 3p, 17p13 and other non-random deletions and rearrangements of chromosomes have been identified in patients with HNSCC (Jin et al. 1988, Cowan et al. 1992, Latif et al. 1992, Kim & Califano 2004). Molecular changes include amplification and overexpression of the receptor for epidermal growth factor and amplification of int-2, bcl-1, cyclin D1 and other oncogenes (Zhou et al. 1988, Berenson et al. 1989, Ishitoya et al. 1989, Weichselbaum et al. 1989, Somers et al. 1990, Callender et al. 1994). In addition, p53 and p16 mutations have been described (Brachman et al. 1992, Reed et al. 1996, Lang et al. 1998, Miracca et al. 1999). There are findings of frequent deletions of chromosome 18

23 21 q in patients with HNSCC, suggesting that other tumor-suppressor genes may also be involved (Cowan et al. 1992). There is evidence of a high frequency of second primary tumor development in patients whose first primary tumor in head and neck area has been definitively treated (Söderholm et al. 1994). Tumors are called second primary tumors if they exhibit a different histology than the first primary tumor, if they show similar histology but they occur more than three years after therapy for the first primary, or if they are separated from the initial primary tumor by more than 2 cm (Warren & Gates 1932). Nowadays, the validity of this definition has been examined through molecular classification of the first and second tumors (León et al. 2002). Second primaries are divided into three categories according to their time of diagnosis; simultaneous, synchronous (when diagnosed within six months after the first primary) and metachronous (when diagnosed more than six months after the first primary) (Brownson et al. 1973, Shapsay et al. 1980). Patients having a first primary tumor in the head and neck region have at least a 40% chance of developing a second upper aerodigestive tract tumor in their lifetime at a rate of approximately 3.5 4% per year. It is notable that also nonsmokers show a defined rate of second primary tumor development. However, the risk is lower than that found associated with current or former tobacco smokers. (Khuri et al. 2001). Fig. 1. Clinical, pathological and molecular progression of head and neck cancer (oral cancer is used as an example). Clinical progression shows a typical clinical presentation of oral cancer. Benign squamous hyperplasia can often appear similar to normal mucosa. The progression from normal-appearing mucosa to invasive cancer is presented in Histological progression. Normal-appearing mucosa already harbors early genetic changes ( Molecular progression ), often with loss of 9p21 and inactivation of p16. Further clinical progression through dysplasia is associated with further genetic changes. Carcinoma in situ often harbors most of the genetic changes described in invasive carcinoma. LOH = loss of heterozygosity. (Modified from Forastiere et al. 2001).

24 Diagnosis and staging The signs and symptoms of HNSCC patients vary with the location of the primary anatomical site and the stage of the tumor. Staging of head and neck tumors is usually done according to the International Union Against Cancer s (UICC) classification system (Hermanek et al. 1992) (Table 1). The TNM (tumor, node, metastasis) classification system puts together clinical information, including that obtained by endoscopy, imaging and cytological evaluation. The tumor stage has different specifications for each primary site. In general, designations of T1 to T3 indicate the increasing size of the primary lesions; a designation of T4 indicates the involvement of an adjacent structure. The staging strategy of lymph-node involvement varies depending of the primary site. Together, the T and N classifications determine the overall clinical stage (I, II, III or IV). Patients with distant metastases are classified as having a stage IV disease. The risk of nodal metastasis in HNSCC is related to the size and location of the primary tumor. Table 1. TNM classification. Lip cancer and cancer of the oral cavity are used as an example. TNM class T0 T1 T2 T3 T4 N0 N1 N2a N2b N2c N3 M0 M1 Definition No evidence of primary tumor Tumor 2cm in greatest dimension Tumor > 2 cm but 4 cm Tumor > 4 cm Tumor invades adjacent structures No regional lymph nodes Metastasis to single ipsilateral lymph node ( 3 cm) Metastasis to single ipsilateral lymph node (> 3 cm but 6 cm) Metastases to multiple ipsilateral lymph nodes ( 6 cm) Metastases to bilateral or contralateral lymph nodes ( 6 cm) Metastasis to a lymph node (> 6 cm) No evidence of distant metastasis Distant metastasis Prognostic factors It is known that in addition to the general conditions of patients, the clinical stage at the time of the diagnosis is the major effector of the prognosis. The higher the clinical stage, the poorer the prognosis of the HNSCC patient. The size of the primary tumor has prognostic value, but the nodal status has been found to be the most significant prognostic factor for locoregional recurrence and survival (Cerezo et al. 1992, Leemans et al. 1994, Mamelle et al. 1994). The number and the size of the lymph nodes are important, as is the location of lymph node metastasis. Survival rates are further decreased in patients with extracapsular spread of lymph node metastasis (Myers et al. 2001, Greenberg et al. 2003, Cummings et al. 2004). The extent of extracapsular spread could be measured from the

25 23 capsular margin to the farthest perinodal extension, and extracapsular spread could be defined as representing either the macro- or microscopic form or a combination of the two. Microscopic extracapsular tumor spread has in particular been found to be in clear relation to local recurrence and distant metastasis (Ferlito et al. 2002). Furthermore, other important factors are the location and the anatomical factors of the tumor, such as localization of the lymph and blood vessels (Cerezo et al. 1992). The importance of tumor localization is not only due to late diagnosis and more complex operations in some areas, but also due to the differences in clinical behavior of tumors in different areas. There is evidence that carcinoma in the larynx area has been found to have better prognosis than carcinomas in the oral cavity or pharynx (Vokes et al. 1993, Kowalski et al. 1995, Moe et al. 1996). Nasopharyngeal carcinoma has been found to differ from other HNSCC carcinomas, one characteristic being a high incidence of distant metastases (Perez et al. 1992, Tang et al. 2000, Forastiere et al. 2001). Histologically HNSCC tumors are graded into well differentiated (grade 1), moderately differentiated (grade 2) and poorly differentiated (grade 3) carcinoma (Shanmugaratnam et al. 1991). The association between histological grade and prognosis is not as obvious in HNSCC as in some other cancers. There is evidence that histological grade has some prognostic relevance in HNSCC patients prognosis when combined with other prognostic factors. Moreover, other histological parameters like perineural spread, capillary density and apoptotic as well as mitotic indexes have been characterized in some studies as indicators of aggressive behavior of the tumor. (Németh et al. 2005). EGFR overexpression can be detected in approximately 90% of all HNSCC tumors and it correlates strongly with aggressive tumor behavior and poor outcome (Grandis et al. 1998). There is also some evidence that p53 mutations are associated with locoregional treatment failure and poor survival in HNSCC patients (Koch et al. 1996, Mineta et al. 1998a). It is recognized that the prognosis of patients in each stage group of the disease and with the same localization may differ greatly, suggesting that some other, possibly biological, factors may also play a role in the progression of HNSCC. However, useful markers associated with biological aggressiveness are needed to predict the outcome of the disease. As a matter of fact, quite a few biological prognostic factors have been investigated as prognostic indicators among HNSCC patients. None of them have, however, been accepted as a routine clinical marker Treatment modalities in HNSCC In Finland, at the time of diagnosis approximately one third of patients present with locally limited T1-T2 disease. The majority of others, about two thirds, present with locally or regionally advanced disease. In developing countries the patients are as a rule diagnosed at a more advanced stage of the disease. In Finland, the outcome results among patient with localized disease are quite good, especially in the case of lip and laryngeal carcinomas, the 5-year survival rates being over 90% and over 70%, respectively. In the cancers of the pharynx and oral cavity the five-year survival rates in all stage categories vary from 41 to 53% and even for patients with diagnosed localized disease, the five-year survival numbers are relatively low (50 to 58%). (Dickman et al. 1999). The primary treat-

26 24 ment modality for HNSCC is surgery and radiotherapy alone or used concomitantly with chemotherapy (Forastiere et al. 2001). Frequently, surgery followed by postoperative radiotherapy is used in patients with resectable disease. In some cases patients are unable to undergo surgery, and concomitant chemotherapy and radiotherapy are currently used in this situation. There are also some studies showing the effectiveness of postoperative chemoradiotherapy after surgical resection (Bernier et al. 2004, Cooper et al. 2004). In surgical treatment major improvements in function and cosmetic results have been achieved with improved reconstruction techniques using microvascular transfers and organ sparing and with technical development of lasers and other equipments (Cummings et al. 2004). In spite of improved treatment techniques approximately 40 50% of patients relapse locoregionally. Concomitant chemoradiotherapy has recently improved these figures (Forastiere et al. 2001). Side effects, such as grade 3 4 mucositis, weight loss and pain are common acute reactions to the intensified treatment, but they are manageable with adequate nutrition, pain control and local treatment. However, the long-term side effects such as xerostomia, loss of organ function and problems with speech and deglutination have not increased. In the case of metastatic or recurrent disease the general treatment option is chemotherapy. It is mainly palliative, the main goal being symptom reduction. Traditional therapies may cause problems with speech, swallowing as well as cosmetic morbidity. Therefore, it is evident that prognostic markers could also serve in developing new treatment strategies in this group of cancers. The treatment of HNSCC has very recently witnessed the introduction of molecular targeted agents based on disease biology and target discovery. There are several compelling targets in HNSCC based on the molecular biology of the disease. The most well-known novel therapeutic targets include the epidermal growth factor receptor (EGFR) inhibitors, which are currently in phase III trials. Similarly, hypoxic cell sensitizers are also in phase III trials. In addition, the molecular biology of HNSCC suggests that the specific pathways are relevant for the development or progression of this disease. These contain growth factors and their receptors, signal transducers, cell cycle regulators, p53, apoptotic proteins, nuclear factor kappa B, hypoxia-related proteins and angiogenesis. At the moment, refined patient selection criteria are required for optimizing the clinically available therapies. This has led to the search for biomarkers that might introduce new strategies, making it possible to better identify the patient groups in need of more aggressive treatment modalities or other treatment options. 2.2 Tumor invasion and metastasis Tumor invasion and the process of metastasis formation are characteristic for malignant neoplasia. The metastatic spread of tumors continues to be the main barrier to successful treatment of malignant tumors. After neoplastic transformation, tumor-host interactions promote coordinated molecular and cellular processes underlying a diversity of steps that characterize metastatic spread (Aznavoorian et al. 1993, Nelson et al. 2000). Metastasis means the migration of cells from the primary tumor to regional lymph nodes, distant sites and other organs, and the basic steps of that process are similar for all tumor types

27 25 (Liotta et al. 1991). The process of metastasis is composed of the degradation of ECM as well as a process of cell adhesion and angiogenesis (Liotta et al. 1980, Turpeenniemi- Hujanen et al. 1985, Tryggvason et al. 1993, Mignanti & Rifkin 1993). Specific sequential steps are necessary for the formation of metastasis and they include tumor cell attachment, neovascularization for further growth of tumor, disruption of the basement membrane with subsequent invasion of malignant cells into the host stroma, intravasation into the blood or lymphatic circulation, survival and transport within the circulation, extravasation at distant sites and growth within the new location (Fidler & Hart 1982, Fidler & Balch 1987, Nelson et al. 2000). Proteinases are required by the malignant cell to invade through the basement membrane and its underlying connective tissue and then subsequently through the basement membrane of the small blood vessels and lymphatics. These processes are then repeated upon extravasation of tumor cells from the vessel. The extracellular matrix (ECM) is a structure of heterogeneous biomolecules offering structural support to cells and tissues as well as supporting adhesion of cells, binding and storing growth factors and transmitting signals to adhesion receptors (Gustafsson & Fässler 2000, Ziober et al. 2001). Basement membranes (BM) are highly specialized layers constructed of components of proteins of the extracellular matrix dividing organ cells, epithelia and endothelia from each other and from interstitial connective tissue, thus being responsible for tissue compartmentalization and the maintenance of the structural design of tissues (Timpl & Dziadek 1986, Gustafsson & Fässler 2000). BM is composed of numerous different kinds of components containing collagen, glycoproteins and proteoglycans, and type IV collagen is the main constituent of basement membranes (Weber 1992, Timpl & Brown 1996). The destruction of BM allows tumor cell migration in the local and regional environment (Tryggvason et al. 1987, Boyd 1996). The proteolytic degradation of ECM and BMs has been observed as an essential event for tumor invasion and the metastatic potential of tumors (Liotta et al. 1980, Matrisian 1992, Birkedal- Hansen 1995b, Kähäri & Saarialho-Kere 1999, Nelson et al. 2000). There are four main groups of proteinases that are significant in protein degradation: aspartate and cysteine proteinases that are mainly involved in the intracellular proteolysis, and serine and metaldependent proteinases that are responsible for extracellular proteolysis (Curran & Murray 1999). Metal-dependent enzymes are better known as the family of matrix metalloproteinases. Serine proteinases include plasminogen activators such as urokinase plasminogen activator-receptor (upa) and tissue-type plasminogen activator (tpa), which are able to activate serum protein plasminogen to plasmin (Mazzieri et al. 1997). MMPs and serine proteinases are able to form cascades in which they can activate each other, indicating that there is clear interaction and collaboration between these enzymes (Birkedal- Hansen et al. 1993, Hewitt & Dano 1996, Werb 1997). Moreover, proteinases play extended roles in several interactions between the extracellular matrix and cancer (Fig. 2).

28 26 Fig. 2. The role of matrix metalloproteinases in tumor progression and invasion. Classically MMPs have been thought to contribute to tissue destruction required for cancer cells to invade, intravasate, extravasate and migrate. Recent studies suggest that MMPs could also play a major role in the growth of benign neoplasms into malignant tumors, angiogenesis and nonstop growth of metastatic tumors. (Modified from Nelson et al. 2001). 2.3 Gelatinases and their inhibitors Matrix metalloproteinase (MMP) gene family The matrix metalloproteinases are a family of zinc-dependent endoproteases. These enzymes are capable of proteolytic degradation of the extracellular matrix (ECM). Currently, 24 different MMPs have been identified among vertebrates; 23 of them have been found in humans (Visse & Nagase 2003). The members of the MMP family have similarities in their structure. All MMPs have the zinc-binding motif in the catalytic domain. In addition, all MMPs have an N-terminal domain (predomain) followed by the propeptide domain to maintain the latency. The majority of MMPs also have additional domains, such as hemopexin domain. These additional domains are important in substrate recognition and in inhibitor binding. MMPs can be divided into subgroups according to their structure and substrate specificity (Woessner 1991, Birkedal-Hansen 1995a, Sternlict & Werb 2001, Visse & Nagase 2003). These subfamilies include gelatinases, collagenases, stromelysins, matrilysins, membrane-type MMPs and other MMPs (Table 2). MMPs are

29 27 involved in a variety of physiological and pathological processes. They are linked to ovulation, blastocyst implantation, embryonic development and tissue morphogenesis. They also play important roles in tissue repair, wound healing, nerve growth, mammary gland development as well as angiogenesis and apoptosis. Table 2. Vertebrate MMPs and some of their substrates (modified from Sternlict & Werb 2001, Visse & Nagase 2003, Overall 2002). MMP Common name(s) Some substrates MMP-1 Collagenase-1 collagen III>I>II, VII, VIII, X, XI, gelatin, entactin, perlecan, laminin, casein, pro-mmp-1, prommp-2, prommp-9 MMP-2 Gelatinase-A gelatin, collagen I, III, IV, V, VII, X, XI, elastin, fibrinogen, laminin, aggregan, vitronectin, decorin, plasminogen MMP-3 Stromelysin-1 Transin-1 aggregan, laminin, gelatin, fibronectin, collagen III, IV, V, IX, X, XI, XVIII MMP-7 Matrilysin fibronectin, laminin, gelatin, aggregan, collagen I, IV, V, IX, X, XI, XVIII MMP-8 Collagenase-2 collagen I>II>III, VII, X, gelatin, entactin, Neutrophil collagenase aggregan, tenascin, pro-mmp-8 MMP-9 Gelatinase-B gelatin, collagen I, IV, V, VII, X, XI, XVIII, Elastin, laminin, fibronectin, vitronectin, prommp-2, prommp-9 MMP-10 Stromelysin-2 collagen I, III, IV, IV, gelatin, elastin, prommp-1,-8 and -10 Transin-2 MMP-11 Stromelysin-3 fibronectin, laminin. aggregan, gelatin MMP-12 Metalloelastase elastin, collagen I, IV, fibronectin, laminin, Macrophage elastase proteoglycans, fibrinogen MMP-13 Collagenase-3 collagen II>III>I, VII, X, XVIII, gelatin, entactin, tenascin, aggregan MMP-14 MT1-MMP collagen I, II, III, gelatin, laminin, aggregan, prommp-2, -13 MMP-15 MT2-MMP proteoglycans, prommp-2 MMP-16 MT3-MMP collagen III, fibronectin, prommp-2 MMP-17 MT4-MMP gelatin, fibrinogen, prommp-2 MMP-18 Collagenase-4 collagen, I, II, III, gelatin MMP-19 Stromelysin-4 collagen I, IV, gelatin, laminin, tenascin MMP-20 Enamelysin amelogenin, aggregan, laminin, MMP-21 XMMP(Xenopus) gelatin MMP-22 CMMP(Chicken) MMP-23 Cysteine array (CA)MMP gelatin MMP-24 MT5-MMP fibronectin, gelatin, prommp-2 MMP-25 MT6-MMP collagen IV, gelatin, prommp-2, -9 MMP-26 Matrilysin-2 collagen IV, gelatin, prommp-9 MMP-27 MMP-28 Epilysin casein

30 28 Furthermore, MMPs are associated with many pathological conditions including arthritis, fibrotic lung and liver diseases, aortic aneurysms, the rupture of atherosclerotic plaque, gastric ulceration and tumor cell invasion and metastasis (Kähäri & Saarialho- Kere 1997, Nagase & Woessner 1999). Additionally, increased expression of MMPs has been found in many human malignant tumors and cell lines (Stetler-Stevenson et al. 1993, Birkedal-Hansen 1995a, Chambers & Matrisian 1997, Kähäri & Saarialho-Kere 1999, Nelson et al. 2000). Among all the proteolytic enzymes potentially associated with tumor invasion, the members of the MMP family have reached an important position due to their ability to degrade the ECM and basement membranes, allowing cancer cells to migrate and invade from their primary site (Johansson et al. 2000, Nelson et al. 2000, Nabeshima et al. 2002). Even though the mechanistic process of ECM degradation mediated by MMPs had been the main focus of study, recent studies have illustrated that the role of MMPs in cancer progression is much more multifaceted than their direct degradative action on ECM components (Egeblad & Werb 2002, Freije et al. 2003, Hojila et al. 2003). The latest description of new MMPs substrates like growth-factor receptors, cell adhesion molecules, chemokines, apoptotic ligands and angiogenic factors as well as the generation of genetically modified animal models of gain or loss of MMP function, have shown the significance of MMP activities in the early stages of cancer development (Coussens et al. 2002, Overall & Lopez-Otin 2002, Folgueras et al. 2004) (Fig. 3) The gelatinases are particularly able to degrade type IV collagen, the main component of the extracellular matrix. Gelatinase-A (MMP-2) and gelatinase-b (MMP-9) are thought to play critical roles in tumor invasion and the process of angiogenesis, in addition to participating in cancer progression in several neoplasias. The basic structure of gelatinases is illustrated in Fig. 4. Fig. 3. Possible mechanisms of cellular signaling mediated by MMPs (modified from Sternlict & Werb 2001).

31 29 Fig. 4. Basic domain structure of the gelatinases (modified from Sternlict & Werb 2001 and Visse & Nagase 2003) Regulation of MMPs The MMPs are strongly regulated on many levels (Sternlict & Werb 2001). The MMP genes are transcriptionally reactive to a great variety of oncogenes, growth factors, cytokines and hormones. The regulation also occurs at the protein level. MMPs are secreted as latent enzymes and this process can be achieved by activators and inhibitors. Transcriptional MMP regulation is a consequence of the binding of various cytokines, growth factors, hormones and other soluble or solid cell surface mediators to receptors. It is characteristic of these mediators to act by paracrine or direct cell-cell interactions with target receptors. Several oncogenes and viruses induce MMP expression in malignant cell lines (Turpeenniemi-Hujanen et al. 1986, Birkedal-Hansen et al. 1993). Moreover, interferons α and γ have been shown to modulate the expression of MMP-2 and MMP-9 in cultured melanoma cell lines (Hujanen & Turpeenniemi-Hujanen 1991, Hujanen et al. 1994). In some MMPs the effect of different cytokines and growth factors is mediated by regulatory elements, for example via the regulative phorbol ester-responsive element (TRE) and its activator protein-1 (AP-1). AP-1 binds dimers of the Fos and Jun transcription factor families, which are oncogene proteins, and they are able to stimulate the transcription of AP-1 reactive genes (Angel & Karin 1991, Sternlict & Werb 2001, Vincenti 2001). An AP-1 binding sequence has been identified in MMP-9, but not in MMP-2 (Sternlict & Werb 2001). All MMPs are produced as inactive proenzymes, and most of them are secreted into the extracellular milieu as latent proforms that need to be further processed to become active proteases. The activation of MMPs can be achieved through three diverse mechanisms: by stepwise activation, by intracellular activation or activation on cell surface. The latent prommps can be activated either by chemical activation in stepwise model of

32 30 MMP activation or by proteolysis mechanism (Sternlict & Werb 2001, Visse & Nagase 2003). The chemical activation may be induced by thiol-modifying agents, oxidized glutathione or reactive oxygens, and low ph and heat treatment can also lead to the activation process. The chemical activation process relies on modification of the cysteine switch sulfhydryl resulting in partial activation of the MMP and intramolecular cleavage of the propeptide. The elimination of the residue of the propeptide by intermolecular process leads to full activity. The process of activation by proteinases is mediated by cleavage of the bait region in the propeptide leading to an MMP intermediate, in which the catalytic site zinc is separated from the sulfhydryl group of the cysteine residue in the propeptide. After that another MMP-molecule degrades the rest of the propeptide producing an active MMP. In the final step, equally to the chemical activation, another MMP eradicates the rest of the propeptide (Sternlict & Werb 2001, Visse & Nagase 2003). Some MMPs can be activated intracellularly by furin or other proprotein convertases (PCs). MMP-11, -21, -23, -28 and MT-MMPs have a furin recognition motif and they can be secreted as active enzymes (Pei & Weiss 1995, Visse & Nagase 2003). There is some evidence to suggest that the cell surface connection may be critical for optimal MMP function. Activation of MMP-2 is an example of cell-membrane associated MMP activation. ProMMP-2 can be activated on the cell surface by MT-MMPs, MT4-MMP being the only one that is not known to activate pro-mmp-2 (Sternlict & Werb 2001). Among the other MMPs, also MMP-14, -15, -16, -24 and -25 are able to activate prommp-2 (Sato et al. 1996, Velasco et al. 2000). It is interesting that the activation process via MT1-MMP needs the support of TIMP-2, while activation via MT2-MMP is independent of TIMP-2 (Strongin et al. 1995, Morrison et al. 2001, Sternlict & Werb 2001). The activity of MMPs can be inhibited by endogenous and exogenous inhibitors. Endogenous inhibitors originate from different human cells, and the tissue inhibitors of metalloproteinases (TIMPs) constitute the main group. Several other proteins have also been reported to inhibit MMPs and they include α2-macroglobulin, tissue factor pathway inhibitor 2 (TFPI-2), type I collage C-proteinase enhancer protein (PCPE), membrane bound β amyloid precursor protein and GPI-anchored glycoprotein called RECK (reversion-including cysteine-rich protein with Kazai motifs) (Sternlict & Werb 2001, Visse & Nagase 2003). Synthetically MMPs can be inhibited with chelating agents like EDTA as well as novel drugs designed as MMP inhibitors such as marimastat and batimastat. Moreover, some older drugs like doxycycline, chemically modified tetracyclines, bisphosphonates and statins seems to have MMP-inhibitory properties (Birkedal-Hansen et al. 1993, Chambers & Matrisian 1997, Sternlict & Werb 2001, Visse & Nagase 2003) Matrix metalloproteinase-2 MMP-2 was first identified and purified from metastatic murine tumors (Liotta et al. 1979, Salo et al. 1983) and cultured human melanoma cells (Höyhtyä et al. 1988). It is secreted by many connective tissue cells such as endothelial cells, fibroblasts, keratinocytes and osteoblasts, in addition to macrophages and a wide variety of tissues including heart, lung, kidney, placenta and muscles as well as by many malignant cell lines (Salo & Oikarinen 1985, Salo et al. 1991, Brown et al. 1990, Pyke et al. 1992, 1993, Birkedal-