A number of clinical studies have been performed to evaluate the diagnostic and prognostic significance of high levels of cysteine proteases cath B and L in tumor cytosols (12
). It has been shown that patients with higher content or increased proteolytic activity of cath B in primary lung tumors exhibited significantly higher risk of recurrence or death than patients with a low content of the enzyme (13
). In breast cancer, the prognostic impact of cath L was shown to be comparable to that of lymph node status (2
). However, the role of cathepsins in the serum of cancer patients remains unclear.
An increased expression and release of cath B has been detected in various tumor tissues as well as in the sera of tumor patients (14
). Retrospective immunohistochemical analysis revealed that patients who had cath B-positive tumors are characterized by a significantly shorter survival compared to patients who had cath B-negative tumor tissues (4
). Moreover, results of another study showed that lung cancer patients with an up-regulation of cath B tended to have higher rates of haematogenous and intrapulmonary metastases (15
). One reason for this poor prognosis may be the high concentration of cath B at the periphery of the tumor cell foci, indicating that these areas were possibly involved in the process of tumor cell invasion. In addition, a significant increase of the cath B activity in tumor-infiltrated lymph nodes was observed compared to non-infiltrated regional lymph nodes, which also impaired patient survival (16
). Consistent with these data, results of our study showed that the levels of cath B were significantly increased in the sera of lung cancer patients as compared to those in healthy controls. Non-small cell lung cancer (NSCLC) patients with high levels of cath B had a more frequent metastasis compared to patients with low levels of cath B. Our results showed that cath B is markedly involved in the mechanism of tumor cell invasion and metastasis.
Compared to the control serum levels, cath L was also elevated in primary lung cancer patients, whereas increased cath L levels did not correlate with any of the clinicopathological parameters. This finding is consistent with the immunohistochemical study of Cordes et al
), which showed the weakest expression and staining intensity for cath L in lung cancer among the cathepsins. These findings support the hypothesis that the role of cath L in lung cancer progression and metastasis is less important than that proposed for other tumors (15
It has been suggested that in cancer, the increased levels of cysteine protease activity, which are not balanced by a corresponding increase of cysteine protease inhibitors, are involved in the remodelling and degradation of extracellular matrix proteins, a proteolytic event associated with tumor spread, invasion and metastasis. An enhanced expression of cystatins is expected to diminish the tumor-associated proteolytic activity and evidence indicates a suppressive role of tumor-associated cystatins in various types of cancer (18
). In the present study, the levels of cystatin C were elevated in primary lung cancer patients. However, increased cystatin C levels did not correlate to any of the clinicopathological parameters. These observations are in contrast to previous findings in other types of cancer, in particular in colorectal cancer, in which the level of cystatin C is an independent prognostic factor of relapse-free survival (19
The analysis of the relationship of the variables with the survival probability using Kaplan-Meier models revealed the most significant prognostic value for cath B. Patients with high cath B serum levels were associated with significantly lower survival probability rates as compared with patients with low cath B levels. A strong prognostic impact of cath B is consistent with previous findings, confirming the association of its overexpression with the metastatic potential of lung cancer cells. Results of this study did not show any prognostic value for the levels of cath L and cystatin C. For cystatin C, the median survival of patients with low levels is apparently longer than that of patients with high levels, but the difference is not statistically significant. The lack of cystatin C and cath L prognostic impact in the serum is contrary to results obtained in tissue cytosols of the head and neck (7
) and breast tumors (2
). This event has yet to be elucidated.
The levels of u-PA and u-PAR in malignant tumors have been found to be correlated with patient prognosis (8
). For example, in breast cancer high u-PA levels are independent and significant prognostic markers for poor relapse-free and overall survival. Furthermore, a high level of u-PAR is associated with a poor prognosis. In lung cancer, the role of the u-PA system is less clear. u-PA was found to be a prognostic marker when detected with immunohistochemistry, and to be correlated with tumor size, nodal status and advanced stage (20
). Using ELISA, no significant relationship was found between u-PA and overall survival (21
). These latter studies also investigated the role of other components of the u-PA system in subgroups of patients with NSCLC using ELISA, and showed a significant relationship between u-PAR and poor prognosis in squamous-cell carcinoma. In large-cell carcinoma of the lung, none of the components of the u-PA system were significantly associated with prognosis (10
). The differences in results between the studies on the prognostic value of the u-PA system in NSCLC may be explained by the small sizes of the histological subgroups and by the differences in patient groups and methods used.
Our present study results showed that both serum u-PA and u-PAR levels in patients with lung cancer were significantly higher than those in healthy controls. However, the levels of u-PA did not correlate with any of the clinicopathological parameters. By contrast, the levels of serum u-PAR were higher in patients with lymph node metastases than in those without metastases. This observation is well-correlated with earlier studies whose findings showed that the u-PA-u-PAR system played a crucial role in the invasion and metastasis of lung cancer cells (22
Of note is whether the elevations of serum u-PA and/or u-PAR concentrations are associated with the poor prognosis of lung cancer patients. In concordance with previous studies (10
), we found that the overall survival rate of lung cancer patients with simple serum u-PAR elevation, or both serum u-PA and u-PAR elevation, was significantly lower than that of patients with normal levels of u-PA and u-PAR, but serum u-PA was not a marker of independent prognosis. In this series, a significant correlation has also been found between u-PA and u-PAR. These findings indicate that a combination measurement of serum u-PA and u-PAR may provide additional information in determining prognosis.
We aimed to further determine whether the combination of variables would increase the prognostic stratification of the patients, using median values as a cut-off value for these parameters. We found that patients with high levels of cath B and cystatin C experienced significantly lower survival probability as compared to other combinations or with prognostic values of individual variables. Similarly, the patients with high levels of u-PA and u-PAR experienced significantly shorter overall survival. By contrast, combinations of other parameters exhibited no marked prognostic significance as compared to each parameter individually. Our results support recent data showing that cystatin C and cath B or u-PA and u-PAR interaction may participate in the modulation of the invasive phenotype of human tumors (8
In the multivariate Cox analysis, we statistically weighed cath B, cath L, cystatin C, u-PA and u-PAR activity with these established factors to assess their relative prognostic impact. Lymph node metastasis remained the most powerful predictor of survival for NSCLC patients; cath B and u-PAR activity also proved to be independent prognostic factors. However, clinical stage, age, histological classification, gender, malignancy grade, cath L, cystatin C and u-PA as constant parameters were not independent markers of poor outcome.
In conclusion, our preliminary study showed an association of higher serum levels of cath B, cath L, cystatin C, u-PA and u-PAR with the lung cancer patient group. Moreover, the prognostic significance of cath B and u-PAR has been defined, revealing a strong correlation of higher levels of the two enzymes, with a shorter survival rate of lung cancer patients. In addition, the combination measurement of five parameters may provide additional information in determining prognosis.