The purpose of this study was to determine the prognostic significance of a high pretreatment serum CYFRA 21-1 level (a cytokeratin 19 fragment) adjusted for the effects of well-known co-variables in non-small-cell lung cancer (NSCLC). This meta-analysis based on individual updated data gathered comprehensive databases from published or unpublished controlled studies dealing with the prognostic effect of serum CYFRA 21-1 level at presentation in NSCLC of any stage (nine institutions, 2063 patients). Multivariate regression was carried out with the Cox model. The proportional hazard assumption for each of the selected variables retained in the final model was originally checked by log minus log plots baseline hazard ratio. The follow-up ranged from 25 to 78 months. A total of 1616 events were recorded. In the multivariate analysis performed at the 1-year end point, a high pretreatment CYFRA 21-1 level was an unfavourable prognostic determinant in all centres except one (Hazard ratio (95% confidence interval): 1.88 (1.64 -2.15), Po10 À4 ). Other significant variables were stage of the disease, age and performance status. Within the first 18 months, the procedure disclosed a nearly similar hazard ratio for patients having a high pretreatment serum CYFRA 21-1 level (1.62 (1.42 -1.86), Po10 À4 ). For patients who did not undergo surgery, the hazard ratio during the first year of follow-up was 1.78 (1.54 -2.07), Po10À4 . Finally, in the surgically treated population, at the 2-year end point, a high pretreatment CYFRA 21-1 and a locally advanced stage remained unfavourable prognostic determinants. In conclusion CYFRA 21-1 might be regarded as a putative co-variable in analysing NSCLC outcome inasmuch as a high serum level is a significant determinant of poor prognosis whatever the planned treatment.

The erythrocyte glycoprotein-bound N-acetylneuraminic acid which determines the blood groups MN and the autoantigens Pr,/Pr, was modified and investigated with regard to antigen activities.We confirmed the results of Lisowska and Morawiecki, who inactivated MN antigenicity by acetylating the 6-amino group of lysine of erythrocyte glycoproteins, suggesting that electrostatic interactions between carboxyl groups of N-acetylneuraminic acid and side-chain lysyl amino groups ensure stable MN antigens. Further support for this concept was provided by our observation that amidation of the N-acetylneuraminic acid carboxyl groups also abolished MN antigenicity.A further differentiation between the autoantigens Pr, and Pr, was elucidated by oxidation of polyhydroxy side chains of N-acetylneuraminic acid.The immunodominant component of the myxovirus receptors, the MN blood groups [l] and the Pr,/Pr, antigens corresponding with cold agglutinins, described by Roelcke [2,3], is N-acetylneuraminic acid which is linked via a 2-0x0 to the nonreducing end of the oligosaccharide side chains of red-cellmembrane glycoproteins.According to the terminology by Reeves [4], the preferred conformation of N-acetylneuraminic acid is l C in which the large substituents are located in equatorial position (Fig. 1).The influence of the carboxyl group ((3-1) and of the polyhydroxy side chain (at (3-6) on the antigen activity of the different receptors was investigated by chemical modifications of these groups. Untreated N-acetylneuraminic acid and oxidized analogue of N-acetylneuraminic acid were determined by gas-liquid chromatography as trimethylsilyl derivatives. The samples were silylated with bis(trimethylsily1)trifluoroacetamide in acetonitril. Arabitol was used as internal standard. The chromatographic separation was performed with the liquid phase methylphenylsilicone (OV 17) 20/, (w/w) on Chromosorb G.The 6-amino groups of lysine of the glycoproteins were acetylated with acetanhydride in 0.1 M phos-

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Summary In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its protein levels were determined in 148 pairs of lung tumour tissue and adjacent non-tumourous lung parenchyma using the enzyme-linked immunosorbent assay technique. Additionally, Cath H levels were determined in sera of 171 patients with malignant tumours, 34 patients with benign lung diseases and 47 healthy controls. The median level of Cath H in tumour tissue was 0.64 times that in the corresponding lung parenchyma. Relating tumour levels with histological type we found higher Cath H levels in small-cell and adenocarcinomas and lower levels in squamous cell carcinoma, large-cell carcinoma and secondary tumours. A significant difference in Cath H level between lung tumour tissue and non-tumourous lung parenchyma was associated with the group of cigarette smokers (156 vs 263 ng mg -1 protein, P < 0.001). For this group of patients Cath H tumour levels correlated with the survival rate, while for the entire patient population this was not the case. Smokers with high tumour levels of Cath H experienced poor survival. Cath H was significantly higher in sera of patients with malignant and benign lung diseases than in control sera (P < 0.001). The increase was significant for all histological types, being the highest in small-cell and squamous cell carcinomas. Our study reveals that in lung tumours there is different behaviour of Cath H compared with other cysteine peptidases, e.g. cathepsin B and cathepsin L. Variations between tissue and serum levels of Cath H indicate either reduced expression or enhanced secretion of this enzyme in lung tumours.

We investigated the appearance and activity of the cysteine proteinase cathepsin B and its physiological inhibitors, stefii A and B, at the cellular level in human tumor cell lines HS-24, derived from a primary lung tumor (squamous cell), and SB-3, derived from a metastasis (lung adenocarcinoma). In addition to cathepsin B, these tumor cells also expressed the immunologically and hctionally related cathepsin L, but not cathepsin H. Stefin A was found in HS-24 but not in SB-3 cells; stefm B was found in both cell types. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized and quantified. Thus, the cellular cathepin B kinetics for the synthetic substrates ZArgArg-4MPNA and Z-Val-Lys-Lys-Arg-4M~NA, its pH dependence and inhibition by E64, stefii A and B, and cystatin C could be determined. From these measurements it appeared that the enzyme exhibited different cleavage rates for these substrates in the different cell types, showed considerable cleavage activity at neutral pH, which was stable under these conditions for extended time periods, and was highly sensi- IntroductionThe cysteine proteinase cathepsin B (cath B: EC 3.4.22.1) is a ubiquitous enzyme. It is involved in lysosomal proteolysis (6) and protein processing (20,26,53,75). After detection and biochemical characterization it was described as a lysosomal enzyme with optimal activity in a slightly acidic (pH 5-6) environment (4). Subsequent investigations also revealed secreted forms ofthe enzyme under physiological (66) and malignant (32,37,44,52) conditions. Recently, interest focused on the appearance of elevated levels of the enzyme in tumors and tumor cells, particularly those derived from metastases or with metastatic character (29,33,57,(60)(61)(62)(63). The ability of the Supported in part by a grant from the Tumor Zcntrum Heidelber Mannheim.'-Correspondence tO: Dr. Eberhard Spiess, Biomedizinische Suukturforschung 0195, Deutsches Krebsforschungszentrum, PO Box 101949, D 69009 Heidelberg, Germany. tive to the inhibitors E64 and cystatin C but was considerably less sensitive to stefim, particularly stefim A. By conventional light microscopy, confocal laser scanning microscopy, and electron microscopy the enzymatic activity was localized in lysosomes, as expected, but also in the endoplasmic reticulum, nuclear membrane, and plasma membrane. The endoplasmic reticulum is a site at which only pre-mature enzyme forms exist, which are usually not active. The appearance of enzymatic activity at the plasma membrane confiims earlier biochemical and immunofluorescence microscopic investigations. The different sites of localization within the cells make it likely that di&ent forms ofthe enzyme are expmsed simultaneously, which follow alternate ways of processing and sorting. Taken together, the results support an involvement of the enzyme under extracellular conditions in degradative proasses. (JHistochem Cyrochem 4291 7-929,lW) KEY WORDS: Cathepsin B Stefin; Activity; Tumor cell; Localization; Endoplasmi...