Radiol Oncol 2004; 38(2): 93-100. review Cathepsins and their inhibitors as tumor markers in head and neck cancer Primož Strojan Institute of Oncology, Department of Radiation Oncology, Ljubljana, Slovenia The invasion and metastasizing of tumor cells is closely connected with the disintegration of basement membranes and extracellular matrix. The carriers of these processes are different proteolytic enzymes, among them cysteine and aspartic cathepsins B, H, L and D as well, a group of ubiquitous lysosomal proteases, and endogenous inhibitors of the former, cystatins. The aim of the present review was to collect the current knowledge on the predictive and prognostic value of cathepsins and their inhibitors in squamous cell carcinoma of the head and neck. In this particular tumor type, the UICC/AJCC TNM-classification system and histopatho-logical characteristics of the tumors were found inadequate to reliably predict either the response to therapy or patients’ survival. Moreover, to date, no factor within the wide spectrum of biochemical and histological factors has yet been identified as reliably predicting the natural course of the disease or its response to therapy. To construct a prognostically meaningful tumor profile, new markers are intensively investigated. Key words: head and neck neoplasms; carcinoma, squamous cell; cathepsins, cystatins; prognosis Introduction During the past decades it has been shown that proteases of various classes can act as tumor progression factors in all stages of malignant progression. They can create a local environment that is supportive to the growth, survival, and progression of a tumor through the Received 20 March 2004 Accepted 13 April 2004 Correspondence to: Primož Strojan M.D., Ph.D., Institute of Oncology, Department of Radiation Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 110; Fax: +386 1 5879 400; E-mail: pstrojan@onko-i.si This paper was presented at the “3nd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. modulation of growth factor pathways, cell-cell adhesions, and cell-matrix adhesions.1 Cathepsins are lysosomal proteolytic enzymes. With regard to their chemical composition, cathepsins are glycoproteins, and the majority of them belong to the group of endopep-tidases. In cancer, the most studied cathepsins are those of the cysteine and aspartic classes, cathepsins B, H, L and cathepsin D. Endogenous inhibitors of cysteine cathepsins belong to cystatins, which are subdivided into three families, i.e. stefins, cystatins, and kininogens, and thyropins, whereas the naturally occurring inhibitor of aspartic protease, cathepsin D, has not been found yet in men.2 Their contribution to the progression of breast, lung, and colorectal cancer has been most extensively investigated at a preclinical level and 94 Strojan P / Cathepsins and their inhibitors in head and neck cancer as markers predicting the response to various treatment regimens and prognosis.3,4 Head and neck cancer Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most prevalent cancer worldwide, with a global yearly incidence of 500,000.5 Despite the evolution and refinement of multimodal treatments for the head and neck cancer in the last 20 years, 5-year survival rates have not improved significantly, remaining at the 50% level.6 Patients grouping with the use of conventional UICC/AJCC TNM staging system and established histopathological characteristics of the primary tumor as well as its metastases on the neck in operated patients allow significant prognostic variation among the individuals within any of these groups. Compared to the carcinoma of the breast, lung or colorectum, the SCCHN falls into a far less investigated group of cancers. Apart from the studies focused on the activity or level of cathepsins and their inhibitors as determined in matched pairs of tumor tissue and normal mucosa 7, there is only a limited number of reports in literature assessing their predictive or prognostic value in this particular type of cancer. In the present review, clinical data on the predictive and prognostic value of cathepsins B, H, L, and D, and their endogenous inhibitors stefin A, stefin B, and cystatin C in SCCHN have been assembled. The results of our own investigations are also presented and their applicability in routine clinical practice discussed. Clinical relevance of cathepsins and their inhibitors in squamous cell carcinoma of the head and neck Markers for diagnosis The largest pertinent study was reported by Kręcicki and Siewiński8, who measured Radiol Oncol 2004; 38(2): 93-100. serum cathepsin B-like activity in 110 samples from patients with laryngeal carcinoma. Enzyme activity was significantly higher in malignant samples compared to healthy controls, whereas no difference was found between the latter and the group with non-malignant, mainly infectious diseases. In cancer patients, no false-negative serum values of cathepsin B-like activity were obtained. The sensitivity of the assay was calculated to be 100% and the specificity 97.5%.8 The methodology used in this study was criticized by Bongers et al.9 They claimed that cathepsins B-like activity as determined by Kręcicki and Siewiński is better referred to as “serum-pro-tease-activity”. However, when serum-pro-tease-activity was compared between patients with SCCHN and non-cancer controls using the same methodology, and after the adjustment for alcohol and tobacco consumption, no difference was observed between the two groups. We used enzyme-linked im-munoassay (ELISA) and also found no alterations in serum cathepsin B between cancer patients and healthy controls, as was the case with cathepsin L.10 When the same group of patients was tested for cathepsins H and D, a significant increase of both enzymes was found in the sera of patients with cancer.11,12 The diagnostic value of the inhibitors of cysteine proteases was first studied by Siewiński et al.13 Using their method they were able to discriminate between total inhibitory activity (free molecules and enzymeinhibitor complexes) and that of active (enzyme-free) and latent (enzyme-inhibitor complexes) forms of specific papain-like cysteine inhibitors; however, the method had no potential to assess the contribution of individual inhibitors. A significantly higher total inhibitory activity (and of latent fraction) and lower activity of active fraction of the inhibitors were found in cancer patients compared to healthy controls or patients with inflammatory diseases. On the contrary, our ELISA measurements allowed quantification Strojan P / Cathepsins and their inhibitors in head and neck cancer 95 of specific inhibitors but were not able to recognize different forms of the inhibitor molecules. Significantly higher stefin A 10 and cys-tatin C 14 levels were measured in the patients’ sera than in controls, whereas levels of stefin B were significantly lower.10 Due to accompanying alterations of cathepsins and their inhibitors in non-malignant conditions1, it seems that their screening perspectives are quite limited. Furthermore, considerable overlap of their concentration ranges between patients and healthy controls or those with benign diseases further reduces their diagnostic strength. Predictive markers for lymph node metastasis The presence of lymph node metastases is the single most adverse independent prognostic factor in SCCHN that, in comparison to node negative patients, reduces a 5-year overall survival rate by about 50%.15 The use of tumor-related histopathological factors in predicting lymph node metastases or advanced imaging techniques is not a reliable method. Furthermore, one should be aware that up to two-thirds of clinically N0 necks are classified as node-free on histopathological examination after surgery (pN0) and that some morbidity resulted even after most strictly limited dissections on the neck, and vice versa, a substantial proportion of clinically positive necks are actually pN0.16 The predictive value for lymph node metastasis most often correlated with increased Cathepsin D immunohistochemical staining. Grandour-Edwards et al.17 reported on 34 patients with oral cavity, oropharyngeal and hypopharyngeal SCCs. In node negative group, 13/15 (87%) tumors were found to be cathepsin D negative, whereas 11/19 (58%) pN+ tumors stained positive for cathepsin D. When adjusted for tumor stage and grade, cathepsin D positivity was nearly twice as likely to be associated with node metastasis.17 In two other studies, including exclusively oral cavity tumors, cathepsin D immunoreac-tivity correlated significantly with the pN-stage of the disease 18,19, whereas in the study of Resnick et al, limited to laryngeal tumors, no such relationship was found.20 Of the other cathepsins, a statistically insignificant trend of higher levels of intensity of im-munoreactivity in pN+ group compared to pN0 group of oral cavity tumors was also reported for cathepsin B 18 and cathepsin L.21 The cathepsin inhibitors were studied by our team only. In the subgroup of patients with operable tumors (various subsites) and clinically positive neck nodes at presentation, stefin A and stefin B concentrations emerged as significant predictors of lymph-node involvement with tumor cells, i.e. pN-stage (Figure 1).22 Differentiating between the patients with nodes enlarged due to inflammation and those with metastatic nodes, a portion of patients could be spared more aggressive therapy and treatment-related side effects. On the other hand, in the patients with clinically undetectable nodes at diagnosis, stefins had no potential in predicting pN-stage of the disease. However, clinical relevance of this finding is limited if surgery is technically correctly performed because no adjuvant therapy is indicated in pN0 subgroup, whereas pN+ patients are highly curable with a moderate-dose of postoperative radiotherapy, i.e. ?95% cure rate at a dose of 50 Gy.23 Predictive markers for response to therapy and for recurrent disease When assessing the efficiency of particular therapy by monitoring the presence of tumor cells in the body, surgery-based therapies and non-surgical therapies should be evaluated separately regarding the differences in mechanisms of tumor cell eradication. Unfortunately, no study was found to have analyzed the predictive value of cathepsins and their inhibitors for response and for disease recurrence to non-surgical therapies, i.e. Radiol Oncol 2004; 38(2): 93-100. 96 Strojan P / Cathepsins and their inhibitors in head and neck cancer Figure 1. Distribution of tumor concentrations of stefins between patients with histopathologicaly determined negative and positive necks, as measured in a group with clinically palpable nodes at presentation. The top and the bottom of the box represent the 25th and 75th percentiles, respectively, and the ends of the bars represent the rang. The line in the box is the median value. n, number of samples. (Reproduced by kind permission of Radiology & Oncology from Strojan P et al., Radiol Oncol 2002; 36: 145-6.) radiotherapy and/or chemotherapy. For the patients successfully treated with surgery for larynx carcinoma, Kręcicki and Siewiński reported a constant decline in serum cathepsin B-like activity, which normalized within four months of the operation.8 In the subgroup of patients in whom the treatment failed, the mean serum values of cathepsin B-activity dropped in the first month after surgery, but rapidly increased in subsequent assays. The elevation occurred in all cases at least two months before clinical evidence of metastases or a recurrent tumor became apparent.8 According to our experience with more heterogeneous group of tumors treated with surgery and postoperative radiotherapy, cy-tosolic level of stefin A 24,25 and pretreatment serum level of cathepsin L 10 were found predictive for disease outcome, which, in turn, reflected the tumor response to applied therapy. In the same population the additional sample of serum was collected during regular follow-up visits 7 to 407 days (median, 59 days) after the completion of all therapies. No correlation was found between post-treatment concentrations of any of the studied cathepsins or inhibitors and the time of serum sampling. However, when only those patients with a time interval of more than 45 days (n = 14) from the completion of therapy to the serum sampling were considered, a trend of gradual decline in stefin B (unpublished data) and cys-tatin C concentrations was observed with the increasing time delay (Figure 2).14 It appears that the decrease in enzyme and inhibitor activity in the treated area after the resection of the gross tumor burden was followed by a transient elevation of their serum levels, likely due to the inflammatory response of the tissues confined in the irradiation field during postoperative radiotherapy. The cutoff time of 45 days concurs well with the duration of ra-diomucositis as seen in clinics, gradually subsiding in 4–6 weeks following the end of a radiotherapy course.26 Markers for prognosis The prognostic relevance of cathepsin D was studied most frequently (Table 1). It showed a universal trend of higher survival probability in the patients with low cytosolic or serum levels of the enzyme 24,27-29, and low intensity level of immunoreactivity.19 The prognostic reliability of cathepsin D was proved in three 27,30,31 out of four 32 studies that utilized mul-tivariate analysis. The same relation between enzyme expression and survival was ob- Radiol Oncol 2004; 38(2): 93-100. Strojan P / Cathepsins and their inhibitors in head and neck cancer 97 Figure 2. Relationship between post-therapy concentration of stefin B and cystatin C, and the time interval from the completion of therapy to serum sampling in non-relapsed patients with a time delay of more than 45 days (n = 14). served for cathepsin B 10,25,33 and cytosolic levels of cathepsin L 24,25, whereas higher levels of cathepsin L in the serum was identified as prognostically superior 10, as was the case in cathepsin H.11,24 However, due to univari-ate setting of survival analysis, the prognostic information collected from these studies leaves much to be desired. Table 1. Prognostic relevance of cathepsins and their endogenous inhibitors in squamous cell carcinoma of the head and neck Author (Ref.) No.of patients Tumor site Method Prognostic significance Cathepsin D Maurizi et a.27 63 Lazaris et al.30 64 Seiwerth et al.31 61 Kawasaki et al.19 78 Cathepsin B Russo et al.33 68 Cathepsin H Strojan et al.11 18 Cathepsin L Budihna et al.24 23 Strojan et al.10 35 Stefin A Strojan et al.25 90 Stefin B Strojan et al.25 90 Cystatin C Strojan et al.34 82 Larynx Larynx Larynx Oral cavity IRA IHC IHC IHC Ś1, MVA Ś1\ MVA 'h, MVA 'h, UVA Larynx EA Ś1\ UVA All sites ELISA -J/, UVA All sites All sites ELISA ELISA Ś1\ UVA -J/, UVA All sites ELISA -J/, MVA All sites ELISA -Is, MVA All sites ELISA -Is, MVA IRA, immunoradiometric assay; IHC, immunohistochemical analysis; EA, enzyme activity; ELISA, enzyme-linked immunosorbent assay; MVA, multivariate analysis; UVA, univariate analysis; 'h, correlation of high levels with poor prognosis; ^, correlation of low levels with poor prognosis. Radiol Oncol 2004; 38(2): 93-100. 98 Strojan P / Cathepsins and their inhibitors in head and neck cancer STEFIN A & CYSTATIN C, CYTOSOL DISEASE-SPECIFIC SURVIVAL p<0.0001 100-,------„ \ t LR-group 80- & 60- I 40- HR-group 20- 0 2 4 6 8 10 Time (years) Figure 3. Prognostic significance of the combination of cystatin C and stefin A concentrations: disease-free survival and disease-specific survival. LR, Low-risk group (high stefin A and high cystatin C, n=23); MR, Medium-risk group (high stefin A and low cystatin C or low stefin A and high cystatin C, n=41); HR, High-risk group (low stefin A and low cystatin C, n=18). The results on the prognostic value of cathepsin inhibitors were provided by our team only.22,24,25,28,34 In operable SCCHN, higher cytosolic concentrations of stefin A, stefin B and cystatin C strongly correlated with longer survival probability in univariate survival analysis, which concurs with the concept of protective role of high levels of cysteine protease inhibitors in tissue ho-mogenates. In multivariate analysis, only stefin A and cystatin C retained their independent prognostic information. However, when comparing the prognostic strength of stefin A with that of cystatin C, the latter lost its significant prognostic power. In addition, the combination of the two inhibitors, stefin A and cystatin C could further stratify the risk of adverse event (Figure 3).34 No prognostic information was provided from the serum levels of any of the studied in-hibitors.10,14 the presence of tumor cells in the body, and for prognosis. In future studies, larger numbers of patients with clinically more homogenous characteristics should be included and a comparison between various analytical procedures should be focused on. It seems, however, that clinical value of tumor marker profiling in SCCHN patients would be further improved by combining the predictive information from several markers with unrelated or mutually opposing biological roles. Acknowledgment The author thanks to Professor Janko Kos for his critical reading of the manuscript. The work was supported by the Ministry of Education, Science and Sport of the Republic of Slovenia Grant J3-4308. Conclusions References Although the clinical utility of cathepsins and their endogenous inhibitors in the management of SCCHN remains open to investigation, it is evident that they exhibit potential in the clinical setting, particularly as markers for lymph node metastasis, for monitoring 1. Bank U, Krüger S, Langner J, Roessner A. Review: Peptidases and peptidase inhibitors in the pathogenesis of diseases. Adv Exp Med Biol 2000; 477: 349-78. 2. Strojan P. Cathepsins and their endogenous inhibitors in clinical oncology. Radiol Oncol 1996; 30: 120-33. Radiol Oncol 2004; 38(2): 93-100. Strojan P / Cathepsins and their inhibitors in head and neck cancer 99 3. Kos J, Lah TT. Cysteine proteinases and their endogenous inhibitors: target proteins for prognosis, diagnosis and therapy in cancer (review). Oncol Rep 1998; 5: 1349-61. 4. Kos J, Werle B, Lah T, Brunner N. Cysteine pro-teinases and their inhibitors in extracellular fluids: Markers for diagnosis and prognosis in cancer. Int J Biol Markers 2000; 15: 84-9. 5. Vokes EE, Weichselbyum RR, Lippman SM, Hong WK. Head and neck cancer. N Engl J Med 1993; 328: 184-94. 6. Parker SL, Tong T, Bolden S, Wingo PA. Cancer statistics. CA Cancer J Clin 1997; 47: 5-27. 7. Kos J, Šmid L, Krašovec M, Svetic B, Lenarčič B, Vrhovec I, et al. Lysosomal proteases cathep-sins D, B, H, L and their inhibitors stefins A and B in head and neck cancer. Biol Chem Hoppe-Seyler 1995; 376: 401-5. 8. Kręcicki T, Siewiński M. Serum cathepsin B-like activity as a potential marker of laryngeal carcinoma. Eur Arch Otorhinolaryngol 1992; 249: 293-5. 9. Bongers V, Konings CH, Grijpma AM, Steen I, Braakhuis BJM, Snow GB. Serum proteinase activities in head and neck squamous cell carcinoma patients. Anticancer Res 1995; 15: 2763-6. 10. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I, Škrk J. Cathepsin B and L and stefin A and B levels as serum tumor markers in squa-mous cell carcinoma of the head and neck. Neoplasma 2001; 48: 66-71. 11. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I, Kos J, et al. Cathepsin H in squa-mous cell carcinoma of the head and neck. Radiol Oncol 1999; 33: 143-51. 12. Strojan P, Budihna M, Šmid L, Vrhovec I, Škrk J. Cathepsin D in tissue and serum of patients with squamous cell carcinoma of the head and neck. Cancer Lett 1998; 130: 49-56. 13. Siewiński M, Kręcicki T, Jarmułowicz J, Berdowska I. Cysteine proteinase inhibitors in serum of patients with head and neck tumors. Diagn Oncol 1992; 2: 323-6. 14. Strojan P, Svetic B, Šmid L, Kos J. Serum cys-tatin C in patients with head and neck carcinoma. Clin Chim Acta 2004; 344: 155-61. 15. Rodrigo JP, Suarez C, Ferlito A, Devaney KO, Petruzzelli GJ, Rinaldo A. Potential molecular prognostic markers for lymph node metastasis in head and neck squamous cell carcinoma. Acta Otolaryngol 2003; 123: 100-5. 16. Pitman KT, Johnson JT, Edington H, et al. Lymphatic mapping with isosulfan blue dye in squamous cell carcinoma of the head and neck. Acta Otolaryngol Head Neck Surg 1998; 124: 790-3. 17. Grandour-Edwards R, Trock B, Donald PJ. Predictive value of cathepsin-D for cervical lymph node metastasis in head and neck squamous cell carcinoma. Head Neck 1999; 21: 718-22. 18. Vigneswaran N, Zhao W, Dassanayake A, Muller S, Miller DM, Zacharias W. Variable expression of cathepsins B and D correlates with highly invasive and metastatic phenotype of oral cancer. Hum Pathol 2000; 31: 931-7. 19. Kawasaki G, Kato Y, Mizuno A. Cathepsin expression in oral squamous cell carcinoma: relationship with clinicopathologic factors. Oral Surg Oral Med Pathol Oral Radiol Endod 2002; 93: 446-54. 20. Resnick MJ, Uhlman D, Niehans GA, Gapany M, Adams G, Knapp D, et al. Cervical lymph node status and survival in laryngeal carcinoma: prognostic factors. Ann Otol Rhinol Laryngol 1995; 104: 685-94. 21. Nikitakis NG, Rivera H, Lopes MA, Siavash H, Reynolds MA, Ord RA, et al. Immuno-histochemical expression of angiogenesis-relat-ed markers of oral squamous cell carcinoma with multiple metastatic lymph nodes. Am J Clin Pathol 2003; 119: 574-86. 22. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I, Kos J, et al. Cysteine protease inhibitors stefin A and stefin B in operable carcinoma of the head and neck. Radiol Oncol 2002; 36: 145-51. 23. Withers HR, Peters LJ, Taylor JMGP. Dose-response relationship for radiation therapy of subclinical disease. Int J Radiat Oncol Biol Phys 1995; 31: 353-9. 24. Budihna M, Strojan P, Šmid L, Škrk J, Vrhovec I, Župevc A, et al. Prognostic value of cathep-sins B, H, L, D and their endogenous inhibitors stefins A and B in head and neck carcinoma. Biol Chem Hoppe-Seyler 1996; 377: 385-90. 25. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I, Kos J, et al. Prognostic significance of cysteine proteinases cathepsins B and L and their endogenous inhibitors stefins A and B in patients with squamous cell carcinoma of the head and neck. Clin Cancer Res 2000; 6: 1052-62. Radiol Oncol 2004; 38(2): 93-100. 100 Strojan P / Cathepsins and their inhibitors in head and neck cancer 26. Cooper JS, Fu K, Marks J, Silverman S. Late effects of radiation-therapy in the head and neck region. Int J Radiat Oncol Biol Phys 1995; 31: 1141-64. 27. Maurizi M, Almadori G, Cadoni G, Scambia G, Ottaviani F, Ferrandina G, et al. Cathepsin D concentration in primary laryngeal cancer: correlation with clinico-pathological parameters, EGFR status and prognosis. Int J Cancer (Pred Oncol) 1996; 69: 105-9. 28. Šmid L, Strojan P, Budihna M, Škrk J, Vrhovec I, Žargi M, et al. Prognostic value of cathepsins B, D and stefin A and B in laryngeal carcinoma. Eur Arch Otorhinolaryngol 1997; 254: 150-3. 29. Strojan P, Budihna M, Smid L, Vrhovec I, Skrk J. Urokinase-type plasminogen activator, plas-minogen activator inhibitor type 1 and cathep-sin D: and lysis of their prognostic significance in squamous cell carcinoma of the head and neck. Anticancer Res 2000; 20: 3975-82. 30. Lazaris AC, Lendari I, Kavantzas N, Kandiloros D, Adamopoulos G, Davaris P. Correlation of tumor markers p53, bcl-2 and cathepsin-D with clinicopathologic features and disease-free survival in laryngeal squamous cell carcinoma. Pathol Int 2000; 50: 717-24. 31. Seiwerth S, Štambuk N, Konjevoda P, Mašić N, Bura VM, Klapan I, et al. Immunohistochemical analysis and prognostic value of cathepsin D determination in laryngeal squamous cell carcinoma. J Chem Inf Comput Sci 2000; 40: 545-9. 32. Leto G, Tumminello FM, Gebbia N, Bazan V, Tomasino RM, Dardanoni G, et al. Differential expression levels of urokinase-type plasmino-gen activator and cathepsin D in locally advanced laryngeal squamous cell carcinoma: clinical implications. Int J Biol Markers 2001; 16: 245-9. 33. Russo A, Bazan V, Gebbia N, Pizzolanti G, Tumminello FM, Dardanoni G, et al. Flow cyto-metric DNA analysis and lysosomal cathepsins B and L in locally advanced laryngeal cancer. Cancer 1995; 76: 1757-64. 34. Strojan P, Oblak I, Svetic B, Šmid L, Kos J. Cysteine proteinase inhibitor cystatin C in squamous cell carcinoma of the head and neck: relation to prognosis. Br J Cancer 2004; 90: 1961-8. Radiol Oncol 2004; 38(2): 93-100.