Radiol Oncol 2006; 40(2): 107-13. review Cysteine cathepsins and their inhibitors in head and neck cancer: an overview of research activities at the Institute of Oncology Ljubljana and ENT Department at the Clinical Center Ljubljana Primož Strojan Department of Radiation Oncology, Institute of Oncology, Ljubljana, Slovenia. To determine the type and extent of the therapy needed for a successful treatment of cancer or to predict cli-nical outcome, an accurate risk stratification is required. The hypothesis on predictive and prognostic impli-cation of individual cathepsins and their inhibitors originated in their involvement in pericellular proteoly-sis that participates in virtually all aspects of normal life of a cell and is involved also in the degradation of extracellular matrix barriers during the invasion and metastasizing of tumor cells. The role of cathepsins and their inhibitors in cancer may be categorized as follows: screening markers for diagnosis; predictive markers for lymph node metastasis; predictive markers for response to therapy and for recurrent disease; markers for prognosis. Although the investigations on clinical utility of cathepsins and their endogenous inhibitors in he-ad and neck cancer are limited, the results warranted further evaluation. In the present review, we reported our experience and results gained during a decade of clinically oriented research and made comments on the-ir predictive and prognostic value for routine clinical setting. Key words: head and neck neoplasms; cysteine endopeptidases; cysteine proteinase inhibitors Introduction Head and neck cancer is the sixth most pre-valent cancer worldwide. Sites of tumor origin are the organs of the upper aerodige-stive tract, i.e. oral cavity, pharynx, larynx, Received 29 May 2006 Accepted 9 June 2006 Correspondence to: Assist. Prof. Primož Strojan, M.D., Ph.D., Department of Radiation Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 110; Fax: + 386 1 5879 400; E-mail: pstrojan@onko-i.si salivary glands, nasal cavity and paranasal sinuses. More than 95% of tumors are of epithelial origin, with alcohol and tobacco abuse being common etiological factors.1 At presentation, two thirds of patients have locally and/or regionally advanced tumors, and the 5-year survival rates have not improved significantly during the last deca-des, remaining at 50%.2 Conventional UICC/AJCC TNM staging system and esta-blished histopathological characteristics al-low us only an approximate insight into the inherent biological aggressiveness of indi-vidual tumor. At the moment, none of the 108 Strojan P et al. / Cysteine cathepsins and their inhibitors in head and neck cancer Table 1. Studies on cysteine cathepsins and their inhibitors in head and neck cancer: Ljubljana experience Study details Sample type Serum Cytosol Tissue section No. of patients 35, (1998, Ref. 6-8) Group 1: 45 (1995, Ref. 6, 9,10) 75 (2005)1 Group 2: 49 (1998, Ref. 11-13) Group 3: 93 (2005)1 Tumor type(s) OC,OP,HP,L OC, OP, HP, L OP Therapy SURG+RT SURG+RT RT+CT Analytical methods Sendwich ELISAs (KRKA dd & Institute Jožef Stefan Ljubljana, Slovenia) 1 Unpublished data. OC, Oral cavity; OP, Oropharynx, HP, Hypophaiynx; L, Larynx; SURG, Surgery; RT, Radiotherapy; CT, Chemotherapy. candidate markers within the wide spectrum of biochemical and histological fac-tors adds significantly to the prognostic information obtained from conventional pro-gnosticators. Cysteine cathepsins B, H and L are lyso-somal proteolytic enzymes. They are impli-cated in virtually all aspects of normal life of a cell as well as in the degradation of ex-tracellular matrix barriers during the invasion and metastasizing of tumor cells. En-dogenous inhibitors of cysteine cathepsins constitute a cystatin superfamily, subdivi-ded into several families (stefins, cystatins, kininogens, thyropins). In normal cells, the activation of proteolytic pathways is con-ducted in cascade manner and controlled by inhibitors. In the tumor tissue, the regulation of this cascade is altered as a result of the modulation of one or more mecha-nisms regulating the synthesis, transport and release of the involved enzymes and inhibitors.3,4 The predictive and prognostic value of cysteine cathepsins and their inhibitors was widely investigated in breast, lung, and colorectal carcinoma, but not also in head and neck cancer.5 The main reasons Radiol Oncol 2006; 40(2): 107-13. are low incidence of the latter and its hete-rogeneity deriving from the diversity of possible primary sites inside the upper ae-rodigestive tract, each with its own natural history and treatment outcome. The aim of the present report was to summarize the results of our research work collected during the last decade in the field of cysteine proteases and their inhibitors in head and neck cancer. Ljubljana experience Our experience originated in 1995. During a decade of systematic research, we tested the prognostic and predictive role of cyste-ine cathepsins and their inhibitors in seve-ral independent groups of patients and in different types of biological samples, i.e. serum, tissue cytosols, and recently also tis-sue sections (Table 1).6-13 We gained the most extensive experien-ce from the studies on cytosols prepared from the tumor tissue of operable tumors, treated with surgery and postoperative radi-otherapy. However, the main drawback of these studies was a relative heterogeneity of Strojan P et al. / Cysteine cathepsins and their inhibitors in head and neck cancer 109 Figure 1. Cathepsin B immunohistochemistry is strongly positive in the cells of moderately differenta-iated invasive squamous cell carcinoma. the included primary tumors; one half of them were laryngeal tumors, whereas the others originated from the oral cavity, oro-pharynx or hypopharynx. From this viewpoint, much more homogenous was a group of patients from our recent study on tissue sections in which only those with inoperable carcinoma of the oropharynx were in-cluded; they were treated uniformly with irradiation and concomitant chemotherapy with Mitomycin C and Bleomycin. Highly positive and extensive immunohistochemi-cal reaction in tumor cells (more than 50% of the cells with positive cytoplasmic reacti-on) was observed in the case of cathepsin B (Figure 1) and stefin A, whereas cathepsin L and stefin B immunohistochemistry was less pronounced (minimal, ?10% positive cells) or modest (10-50% positive cells). Analyzing non-malignant stromal cells in the tumors, reactivity to the cathepsins and stefins were recognized also in lymphocytes and ductal cells. The immunohistochemical reaction in the former case was scored as modest and in the latter case as minimal (unpublished data). Furthermore, in all groups of patients, the same kits for biochemical determination of studied cathepsins and stefins were used, i.e. the commercially available ELISEs developed at the Jožef Stefan Institute. Be-cause the tests kits have been modified du-ring the years as has also been the metho-dology for tissue cytosol preparation, the results of measurements in individual gro-ups are not directly comparable. Prediction of lymph node metastasis The possibility to predict cervical lymph node infiltration with tumor cells from a primary tumor biopsy specimen would be of critical importance for treatment optimi-zation. The presence of lymph node meta-stases is the single most adverse prognostic factor in head and neck cancer, reducing 5-year overall survival rate up to 50% compa-red to node negative patients.14 Primary tu-mor-related histopathologic factors (site, T-stage, grade, growth pattern, thickness, pe-rineural infiltration, and others) are not re-liable enough in predicting lymph node metastases. Consequently, up to one third of clinically node negative necks at presen-tation are bearing lymph nodes infiltrated with tumor cells and, vice versa, a signifi-cant proportion of patients with palpable neck nodes or radiologically determined neck disease were actually disease free on the neck. In the latter case, nodal enlarge-ment is caused by inflammatory processes in the affected node(s).15 The results of immunohistochemical stu-dies published so far, analyzing the potential of cysteine cathepsins and their inhibitors for predicting tumor cell infiltration of regional lymphatics, were not conclusi-ve.16-18 We observed the same in our series for cathepsin L and both stefins. On the contrary, comparing the pattern of cathe-psin B immunostaining between N0-1 and N2-3 subgroups (but not between N0 and N+ subgroups!) in the patients with inoperable oropharyngeal cancer treated with conco-mitant chemoradiotherapy, the difference Radiol Oncol 2006; 40(2): 107-13. 110 Strojan P et al. / Cysteine cathepsins and their inhibitors in head and neck cancer STEMM A STEFINB 2400 i 2000 Z 1600 g < 1200 800 400 P=0.03 1000 1 750 500 i 250 8 o P=0.02 pN-negative (n-15) pN-positive (n=42) pN-neg ative (n-15) pN-positive (n-42) Figure 2. Distribution of tumor concentrations of stefins A and B between patients with histopathologically deter-mined negative (pN0-stage) and positive (pN+-stage) necks, as measured in a group with clinically palpable nodes at presentation (cN+-stage). The top and the bottom of the box represent 25th and 75th percentiles, respectively and the end of the bars represents the rang. The line in the box is the median value. N, Number of patients. Re-produced by kind permission of Radiology & Oncology from Strojan P et al, Radiol Oncol 2002; 36: 145-6. reached the level of statistical significance (Fisher exact test, P=0.03; unpublished data). However, as the key question is how to differentiate node-negative from node-po-sitive necks, we have concluded that cathe-psin B immunohistochemistry of primary tumor biopsy sample has no clinical impli-cations in predicting the presence of meta-stases in the cervical lymphatics. More encouraging were the results from the tissue cytosols. In the operated patients with clinically positive neck nodes at pre-sentation (i.e. before surgery), a statistically significant difference in stefin A and stefin B cytosolic concentrations was calculated between the subgroup of patients who we-re actually disease-free in the neck and tho-se with metastases confirmed on histopa-thological examination of the resected spe-cimen (Figure 2).12 This observation poin-ted out the ability of stefins to differentiate between the nodes enlarged due to inflam-mation and those with deposits of tumor cell, and raised a possibility of spearing a portion of cN+ patients from more aggressive therapy and treatment related side ef-fects. On the other hand, in the patients with clinically undetectable nodes at dia-Radiol Oncol 2006; 40(2): 107-13. gnosis, stefins had no potential to predict pN-stage of the disease. Prediction of response to therapy Tumor regression during external beam ra-diotherapy course is an independent pre-dictive factor of local control in head and neck carcinomas. However, the regression is sequential: the maximum clearance rates for the primary were recorded during the treatment, whereas they were delayed for the nodes, with the maximal complete regression rate at about two months after ir-radiation.19,20 One of the important mecha-nisms underlying tumor regression after io-nizing radiation or chemotherapy is cell disintegration via apoptotic pathways in which cysteine cathepsins and their inhibitors have also been suggested to participa-te actively.21 According to our experience, only cathe-psin B immunostaining showed some potential for predicting treatment failure (Fi-sher exact test, P=0.034; unpublished data). The latter was defined as no tumor respon-se or only partial response (less than 100% Strojan P et al. / Cysteine cathepsins and their inhibitors in head and neck cancer 111 Table 2. Cysteine cathepsins as markers for prognosis in head and neck cancer: review of literature Studies Cathepsin B Cathepsin H Cathepsin L Positive1 Russo et al, 1995 (Ref. 23) Oblak et al, 20053 Strojan et al, 1999 (Ref. 6) Budihna et al, 1996 (Ref. 9) Negative2 Budihna et al, 1996 (Ref. 9) Kawasaki et al, 2002 (Ref. 17) Russo et al, 1995 (Ref. 23) Strojan et al, 2000 (Ref. 11) Strojan et al, 2000 (Ref. 11) KawasaM et al, 2002 (Ref. 17) Kawasaki et al, 2002 (Ref. 17) Strojan et al, 20053 Oblaketal,20053 Strojan et al, 20053 No. of 404 96 387 patients 'Prognostic value of cysteine cathepsins was confirmed. 2Prognostic value of cysteine cathepsins was not confirmed. Unpublished data. regression) to applied chemoradiotherapy evaluated locally and regionally two months after finishing all therapies. We fo-und low cathepsin B immunostaining being uniformly predictive (10/10 cases) for favo-rable clinical response; however, a substan-tial proportion of patients with highly positive CB staining were also complete respon-ders (41/65 patients). It seems that cathe-psin B immunohistochemistry per se is not specific enough and should not be used as a predictive marker of the tumor response to applied therapy independently from oth-er markers. Evaluation in combination with other candidate markers is warranted. The observation on low cathepsin B im-munostaining being predictive for the favo-rable response of the tumor to radiochemo-therapy directly contradicts the recognition of cathepsins as promoters of apoptosis which, in turn, leads into the reduction of cell number and, finally, the volume of the tumor. It seems that other, cathepsin B inde-pendent molecular mechanisms are invol-ved in the irradiation induced apoptotic pathways. On the other hand, because com-plete tumor regression was recorded in a substantial proportion of patients with strongly positive cathepsin B tumors as well, we hypothesized that the ratio between the cathepsins and their inhibitors may also play a decisive role. For example, an evalua-ted inhibitor expression, blocking the intrin-sic cathepsin B activity, was shown to rescue the tumor cells from TNF-induced apoptosis in experimental setting of the cell lines deri-ved from primary and metastatic lesions of oropharyngeal squamous cell carcinoma.22 Markers for prognosis In head and neck cancer, the prognostic va-lue of cysteine cathepsins was studied much less extensively than in breast, lung, or colorectal carcinoma (Table 2).6,9,11,17,23 With the exception of cathepsin H, the trend of higher survival probability correla-tes with lower levels of cathepsin B and ca-thepsin L. In the studies on tissue cytosols, however, no strong relationship with pro-gnosis was established. As an immunohi-stochemical marker, only cathepsin B sho-wed some association with the outcome of the disease; the latter was not confirmed on multivariate analysis (unpublished results). We recognized stefins A and B and cystatin C as the most influential progno-Radiol Oncol 2006; 40(2): 107-13. 112 Strojan P et al. / Cysteine cathepsins and their inhibitors in head and neck cancer i 100 80 En 60 E 40 ¦ H W ž 20 S o 3rdterti]e (1eyent/31 pts) H 2nd tertile (7 ev ents/31 pts) lsttertüe (12 ev ents/31 pts) TERTI LES [S A], No.of 5yi-DFS ng/mgp PTS 1st L506 31 59% 2nd »506 -1143 31 74% 3rd >1143 31 97% 4 6 UME, YEARS Figure 3. Disease-free survival as a function of stefin A status. sticators in tumor cytosols. In our first two data sets from 1995 and 1998, higher cyto-solic concentrations of any of the two ste-fins as well as those of cystatin C correlated significantly with longer disease-free interval on univariate survival analysis.9,11,13 In multivariate model, only stefin A and cystatin C retained their independent pro-gnostic information. However, when com-paring the prognostic strength of the latter two, cystatin C lost its significant progno-stic power for both survival endpoints un-der evaluation, disease-free survival and di-sease-specific survival.13 The prognostic strength of stefin A con-centration as determined in tumor cytosol was reconfirmed recently on an indepen-dent dataset of 93 patients with operable head and neck cancer (unpublished re-sults). After stratifying the patients accor-ding to stefin A concentration in 3 subgro-ups, we recognized an obvious pattern of improved survival probability with the in-creasing levels of stefin A (Figure 3). The maximal difference in survival rates betwe-en low and high stefin A subgroups was calculated at approximately 400 ng/mgp, which classified 29% of tumors as stefin A low and the rest as stefin A high. It is inte-resting that, in both historical data sets, the optimal cut-off concentration fell into the Radiol Oncol 2006; 40(2): 107-13. same range of measured values as it was the case in our recent group, i.e. around 30th percentile. On multivariate analysis, stefin A appeared as the strongest indepen-dent predictor of a disease-free survival in the model, irrespective of whether it was tested as continuously or categorically variable. Conclusions The results presented in this overview warranted further evaluation of cysteine cathe-psins and their inhibitors as predictive and prognostic markers in head and neck can-cer. In particular, this is the case when analyzing the stefin A concentrations from tissue cytosols. The latter confirmed its prognostic value in three independent data sets, given identical results in all three in-stances. In future, larger numbers and more homogenous (in regard to primary tumor site) populations of patients and standardi-zation of analytical methods should be con-sidered more rigorously to obtain maxi-mally informative results applicable also to routine clinical practice. Acknowledgement The study was supported by the Slovenian Research Agency, Grant P3-0307 and pre-sented at the 4th Conference on Experimen-tal and Translational Oncology, Kranjska gora, Slovenia, March 22-26, 2006. The au-thor thanks to Professor Nina Gale for pro-viding photograph on cathepsin B immuno-histochemical staining. Strojan P et al. / Cysteine cathepsins and their inhibitors in head and neck cancer 113 References 1 Vokes EE, Weichselbyum RR, Lippman SM, Hong WK. Head and neck cancer. N Engl J Med 1993; 328: 184-94. 2 Parker SL, Tong T, Bolden S, Wingo PA. Cancer statistics. 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Oral Oncol 2003; 39: 559-68. 23 Russo A, Bazan V, Gebbia N, Pizzolanti G, Tum-minello FM, Dardanoni G, et al. Flow cytometric DNA analysis and lysosomal cathepsins B and L in locally advanced laryngeal cancer. Cancer 1995; 76: 1757-64. Radiol Oncol 2006; 40(2): 107-13. 136 Slovenian abstracts Radiol Oncol 2006; 40(2): 95-105. Odpornost na komplement ovira onkološko zdravljenje Konatschnig T, Geis N, Scultz S, Kirschfink M Izhodišča. Različne in vitro raziskave, ki so bile narejene v zadnjih dveh desetletjih, jasno kažejo, da je odpornost človeških tumorskih celic na avtologni komplement pogojena z na membrano vezanimi regulatornimi proteini komplementa (mCRP). Takšna proteina sta CD55 in CD46, najpomembnejšo vlogo pa ima CD59. Ta imunska dogajanja zelo vplivajo na potek bolezni, kar potrjujejo novejše klinične raziskave. Odpraviti odpornost na komplement obeta izboljšanje zdravljenja bolnikov z različnim rakom, s tem pa tudi izboljšanje napovedi izhoda bolezni. V pričujočem kratkem preglednem članku podrobneje predstavljamo: (1) nevtrali-zacijo proteinov mCRP z monoklonskimi ali rekombinantnimi protitelesi in (2) strategijo »utišanja« genov za proteine mCRP z delovanjem na nivoju RNA ob uporabi siRNA. Zaključki. Ker so proteini mCRP prisotni v vseh normalnih tkivih endotelnih celic paren-himskih organov (jetra, ledvica, itd...) in v krvnih celicah, je zelo pomembno, da je blokiranje delovanja proteinov mCRP selektivno in da tako ne prizadene zdravega tkiva. Čeprav so prvi rezultati ohrabrujoči, je vplivanje na delovanje proteinov MCRP, da bi izboljšali imunoterapijo, še vedno velik izziv v klinični praksi. Radiol Oncol 2006; 40(2): 107-13. Katepsini cisteinske skupine in njihovi inhibitorji pri raku glave in vratu: pregled raziskovalnega dela na Onkološkem inštitutu Ljubljana in Kliniki za otorinolaringologijo Kliničnega centra Ljubljana Strojan P Za odločitev o vrsti in intenzivnosti terapije, potrebne za uspešno ozdravitev raka, kot tudi za napoved izida bolezni je potrebna natančna ocena agresivnosti bolezni. Hipoteza, ki predpostavlja napovedni in prognostični pomen posameznih katepsinov in njihovih inhibitorjev, temelji na vpletenosti enih in drugih v obcelične proteolitične procese. Ti so sestavni del večine aktivnosti, povezanih z življenjem normalne celice, kot tudi procesov, povezanih z razgradnjo zunajceličnega matriksa med procesom invazije in zasevanja tu-morskih celic. Vlogo katepsinov in njihovih inhibitorjev pri raku lahko razčlenimo na naslednje skupine: markerji za presejanje; markerji za napoved prisotnosti zasevkov v področnih bezgavkah; markerji za napoved odgovora na zdravljenje in ponovitev bolezni; prognostični markerji. Čeprav je raziskav s področja katepsinov in njihovih endogenih inhibitorjev pri raku glave in vratu malo, rezultati opravičujejo nadaljna preučevanja. V pričujočem pregledu smo predstavili naše izkušnje in rezultate iz desetletnega obdobja klinično usmerjenega razsikovalnega dela in podali mnenje o njihovi napovedni in prognostični vlogi za potrebe vsakodnevne klinične prakse. Radiol Oncol 2006; 40(2): 133-8.