To estimate the prognostic value of cathepsins B, H, L, D and stefins A and B in head and neck carcinoma, their concentrations in cytosols of primary tumours and adjacent normal tissue were measured (cathepsins B, D stefins A, B in 45, cathepsin L in 24 and cathepsin H in 21 patients). Median concentrations of cathepsins B, L, and D were significantly higher in tumour than in the adjacent normal tissue (B and D: p < 0.0001; L: p = 0.004); cathepsin H concentration was higher in normal tissue (p = 0.001). Concentrations of either stefin did not differ significantly between normal and tumour tissue. Concentrations of cathepsins B, H, L, and D were higher in laryngeal than in non-laryngeal normal and tumour tissues. The difference was statistically significant for cathepsin B in tumour tissue (p = 0.045), and marginally significant in normal tissue (p = 0.07). Early tumours had lower concentrations of stefins A and B than locally advanced tumours (stefin A: p = 0.04; stefin B: p = 0.07). Disease-free and disease-specific survival rates were better in patients with concentrations of cathepsin L in tumour tissue below or equal to the cut-off values (p = 0.035; p = 0.05), whereas for cathepsin B the difference was established only for disease-free survival (p = 0.07). The opposite was true for stefin A (p = 0.0002; p = 0.002) and stefin B (p = 0.009; p = 0.003), and in disease-free survival also for cathepsin H (p = 0.055). The concentration of cathepsin D did not correlate with survival. Our data indicate that cathepsins B, H, L and stefins A and B might have prognostic value in head and neck carcinoma.

In cytosols of tumour and normal tissue of 53 patients suffering from head and neck carcinoma cathepsins D, B, H and L were measured using quantitative immunoreactive assays (ELISA). The values of cathepsins D, B and L were significantly higher in tumour tissue, whereas cathepsin H concentration was lower in tumour than in normal tissue. Median cathepsin D values were 27 pmol (tumour tissue) vs. 12 pmol (normal tissue) per mg of total protein, median cathepsin B values were 1.25 micrograms/mg (tumour tissue) vs. 0.23 micrograms/mg (normal tissue) and median cathepsin L values were 39.8 ng/mg (tumour tissue) vs. 20.0 ng/mg (normal tissue). Median cathepsin H values were 1.05 micrograms/mg and 2.20 micrograms/mg for tumour and normal tissue, respectively. Additionally, stefin A and stefin B were measured in tumour and normal tissue samples. In contrast to the cathepsins, the concentrations of these inhibitors of cysteine proteinases was not significantly different between tumour and normal samples. The concentrations of cathepsins D, B, H and L and stefins A and B measured in head and neck tumours, were independent of standard clinical and histological prognostic factors. Significant correlation of tumour tissue values was observed between cathepsins B and L and between both stefins.

To determine the role of the cysteine proteinase inhibitor cystatin C in the invasive behavior of squamous cell carcinoma of the head and neck (SCCHN), Cystatin C protein level was measured in 82 pairs of primary tumour tissue and adjacent noncancerous mucosa, using the enzyme-linked immunosorbent assay. The median level of cystatin C in tumour tissue was 1.18 times lower than that in corresponding mucosa (P ¼ 0.031). In normal mucosa samples, the cystatin C level was influenced by the site of sampling: it was lower in nonlaryngeal tissue samples (oral cavity, oro-or hypopharynx) than in laryngeal samples (P ¼ 0.004). The tumour cystatin C level correlated inversely with pN-stage (P ¼ 0.047), whereas a trend of lower cystatin C levels was observed in the group with extranodal tumour extension compared to those with no extranodal spread (P ¼ 0.069). In univariate analysis, the patients with low tumour cystatin C levels exhibited poor disease-free survival (DFS, P ¼ 0.013) and disease-specific survival (DSS, P ¼ 0.013). In multivariate analysis, the most powerful predictor of survival was pN-stage (DFS: P ¼ 0.040, HR 2.78; DSS: P ¼ 0.011, HR 4.36,), followed by the cystatin C level (DFS: P ¼ 0.043, HR 0.22; DSS: P ¼ 0.067, HR 0.25). When comparing the prognostic strength of cystatin C to that of stefin A, another cysteine proteinase inhibitor, which emerged as the most significant prognosticator for survival in our previous study analysing the same cohort of patients, stefin A proved to be significantly more reliable predictor for both DFS and DSS than cystatin C. Our results indicate that cystatin C is implicated in the invasive behavior of SCCHN, and that there are variations in regulation of proteolytic pathways under nonmalignant conditions, inherent to individual subsites inside the upper aerodigestive tract. The correlation between high cystatin C levels and improved survival concurs with the concept of the protective role of high levels of cysteine proteinase inhibitors in tissue homogenates that has been previously suggested by the survival results in breast and lung carcinoma as well as SCCHN.

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The hypothesis was tested that a specific pattern in the cysteine cathepsin/inhibitor ratio is associated with the development of more aggressive tumor cell phenotypes in squamous cell carcinoma of the head and neck (SCCHN). For this purpose commercially available ELISAs were used to determine the concentrations of cysteine cathepsins B and L and their inhibitors, stefins A and B, in cytosols of nontumorous mucosa and primary tumors from 92 patients. Using the stefin A concentration difference in matched pairs of tissue samples as a stratifying variable, 53 cases were found to be upregulated (higher concentrations in tumor samples than in nontumorous mucosa) and 39 cases downregulated. Disease recurrence was more frequent in the downregulated group than in the upregulated group (35.9% vs 11.3%, p=0.009), which resulted in significantly different 5-year disease-free survival rates (61.2% vs 88%, p=0.004). The consistency of these results was confirmed by repeating the analysis in an independent group of patients (the reference group). The presented results suggest that in patients with SCCHN, specific patterns in the proteolytic profile of cysteine proteases and their inhibitors are associated with the development of distinctly aggressive tumor cell phenotypes and are of prognostic value.

Izhodišče: test ELF (angl. enhanced liver fibrosis test) predstavlja kombinacjo direktnih označevalcev fibroze jeter, ki tvorijo algoritem, s katerim je mogoče oceniti prisotnost in blago, zmerno ter težjo stopnjo fibroze jeter. Test vključuje tri biološke označevalce: hialuronsko kislino (HA), amino-terminalni del prokolagena tipa III (PIIINP) in tkivni inhibitor metaloproteinaze-1 TIMP-1). HA in PIIINP sta označevalca nastajanja depozitov matriksa – fibrogeneze, TIMP-1 pa predstavlja razgradnjo matriksa – fibrolizo. Raziskave so pokazale, da lahko našteti trije označevalci ali test ELF služijo kot uporaben presejalni test pri zgodnjem diagnosticiranju biokemične poškodbe jeter pri avtoimunskih hepatitisih, pri hepatitisu C in drugih okvarah jeter, npr. okvarah zaradi alkoholizma. Namen naše raziskave je bil določiti vrednosti testa ELF pri treh različnih skupinah preiskovancev: kontrolni skupini, pri skupini s potrjeno diagnozo alkoholizma in pri skupini, ki akutno pije alkohol. Rezultate meritev ELF testa smo primerjali z rezultati uveljavljenih biokemičnih označevalcev alkoholizma. Metode: V raziskavo smo vključili 113 preiskovancev, (71 moških, 42 žensk). Povprečna starost preiskovancev je znašala 43 let. Razdelili smo jih v tri skupine: MDPŠ so predstavljali preiskovanci (N = 39), ki so prišli na pregled v ambulanto medicine dela, prometa in športa, AZA je bila skupina 31 preiskovancev z akutnim alkoholnim opojem in DOA skupina 43 preiskovancev, ki so se zdravili zaradi diagnoze odvisnosti od alkohola. V serumskih vzorcih vseh treh skupin preiskovancev smo izmerili povprečni volumen eritrocitov (MCV), katalitične aktivnosti aspartat amino transferaze (AST), alanin amino transferaze (ALT), gama glutamilne transferaze (GGT) in parametre novejšega testa ELF za oceno stopnje z alkoholom povzročene jetrne fibroze. Blaga stopnja jetrne fibroze je pri vrednostih ELF pod 7,7, zmerna pri vrednostih od 7,7 do 9,8 in huda stopnja fibroze pri vrednostih nad 9,8. Za statistično analizo podatkov smo uporabili program SPSS 21.0 za Windowsovo okolje (SPSS, Inc. Chicago, USA). Rezultati: Izmerjene povprečne vrednosti uveljavljenjih bioloških označevalcev alkoholizma pri skupinah MDPŠ, AZA in DOA znašajo za MCV : 91,9; 90,9 in 95,3 fL, mediane katalitične aktivnosti za AST pa 0,30; 0,33 in 0,42 µkat/L, za ALT 0,41; 0,34 in 0,56 µkat/L in za GGT 0,37; 0,34 in 0,92 µkat/L. Kruska-Wallisov test je pokazal statistično značilne razlike med skupinami preiskovancev za AST, GGT in MCV (p < 0,002), medtem ko se aktivnost ALT (p = 0,052) med skupinami razlikuje le na meji statistične značilnosti. V skupini MDPŠ je vrednost mediane testa ELF znašala 7,99 (6,99–10,18), v skupini DOA pa 9,47 (6,98–14,73). V skupini DOA smo dokazali statistično značilno korelacijo med AST, ALT, GGT in testom ELF (r = 0,524; 0,306 in 0,632), v skupini MDPŠ pa je bila statistično značilna samo za MCV (r = 0,327). Zaključek: Rezultati meritev so pokazali, da so v skupini DOA statistično značilno višje vrednosti uveljavljenih bioloških označevalcev alkoholizma (MCV, AST, ALT in GGT) kot v kontrolni skupini MDPŠ. Mediana testa ELF pri skupini DOA kaže na prisotnost zmerne fibroze jeter, ob tem da območje vrednosti od 6,98 do 14,73 kaže, da so v skupini tudi posamezniki s hudo fibrozo. Iz rezultatov je razvidno, da povišane vrednosti jetrnih encimov AST, ALT in GGT ter korelacije med njimi kažejo na poškodbo hepatocitov, vrednosti testa ELF pa opredeljujejo stopnjo in obseg fibroze jeter. Za oceno fibroze jeter se uporabljajo različni indeksi, med katerimi ima test ELF po izsledkih tujih raziskovalcev visoko diagnostično uporabnost. ELF ima kot neinvazivni test tudi vse možnosti za uporabo na primarni ravni zdravstvene obravnave bolnikov z alkoholno in nealkoholno okvaro jeter.