NGAL, L-FABP, and KIM-1 in comparison to established markers of renal dysfunction.

BACKGROUND: New urinary biomarkers like neutrophil gelatinase-associated lipocalin (NGAL), liver-type fatty acid binding protein (L-FABP), and kidney injury molecule-1 (KIM-1) open the opportunity to detect kidney injuries in early stages. Our study aimed at evaluating NGAL, L-FABP, and KIM-1 in comparison to established markers of urine protein differentiation for detection of renal dysfunction. METHODS: On the basis of the PROTIS expert system (for differentiation of glomerulo-/tubulopathy) urine and plasma samples of 263 randomly selected patients were routinely examined (urine: total protein, albumin, IgG, α1-microglobulin, creatinine, and dip stick results for leukocytes, blood, protein, glucose, pH, and nitrite; plasma: creatinine and cystatin C) followed by the analysis of the new urine biomarkers NGAL (CMIA), L-FABP (ECLIA), and KIM-1 (ELISA). RESULTS: Of the three new markers L-FABP showed the highest correlation with α1-microglobulin (r=0.76, p<0.01) and was closest associated with the degree of tubular proteinuria assessed by the PROTIS system. NGAL distinguished the PROTIS proteinuria groups with distinctive tubular proteinurias from the controls as well, but revealed a marked diagnostic influence by leukocyturia. Urinary KIM-1 revealed only a weak diagnostic value for the detection of renal injury. CONCLUSIONS: Urinary NGAL and L-FABP proved to be promising candidates for detecting injuries of the renal tubular system over a broad range of clinical conditions. L-FABP showed a better diagnostic performance and a lower interference by leukocyturia and hematuria than NGAL. Both markers may serve as sensitive tissue injury markers in addition to the established markers of renal functional impairment.

Quantitative RT-PCR assays for the determination of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 mRNA in primary tumor tissue of breast cancer patients: comparison to antigen quantification by ELISA.

Urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) play a key role in tumor-associated processes such as the degradation of extracellular matrix proteins, tissue remodeling, cell adhesion, migration, and invasion. High antigen levels of uPA and PAI-1 in tumor tissue of various solid malignant tumors, including breast cancer, are associated with poor patient prognosis. In the present study, we examined whether analysis of uPA and PAI-1 mRNA expression represents an alternative to the measurement of the respective antigen levels in breast cancer. Highly sensitive quantitative real-time PCR (QPCR) assays, based on the LightCycler technology, were established to quantify uPA and PAI-1 mRNA expression in breast cancer cell lines as well as in tumor tissue of breast cancer patients. mRNA concentrations were normalized to the expression level of the housekeeping gene h-G6PDH. The respective uPA and PAI-1 antigen concentrations were determined by established ELISA formats. QPCR mean interassay variation coefficients were 11% (uPA) and 8% (PAI-1). In breast cancer cell lines, mRNA and antigen values were highly correlated for both uPA and PAI-1 (each: rs=0.95; p<0.001). In contrast, correlations between uPA/PAI-1 mRNA and protein in the breast cancer samples were found to be distinctly weaker or not significant. Thus, quantitative determination of mRNA expression for both factors does not mirror antigen levels in breast cancer tissue, possibly due to posttranscriptional regulation. Except for nodal status being inversely correlated with uPA mRNA levels, no significant interrelations were observed between uPA or PAI-1 mRNA expression and clinicopathological parameters. On the protein level, elevated uPA and PAI-1 values were associated with a negative steroid hormone receptor status. In conclusion, the implementation of mRNA quantification of uPA and PAI-1 in breast tumors is unable to serve as a one-to-one substitution for antigen determination by ELISA.

Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells.

The human tissue kallikrein family of serine proteases (hK1-hK15 encoded by the genes KLK1-KLK15) is involved in several cancer-related processes. Accumulating evidence suggests that certain tissue kallikreins are part of an enzymatic cascade pathway that is activated in ovarian cancer and other malignant diseases. In the present study, OV-MZ-6 ovarian cancer cells were stably co-transfected with plasmids expressing hK4, hK5, hK6, and hK7. These cells displayed similar proliferative capacity as the vector-transfected control cells (which do not express any of the four tissue kallikreins), but showed significantly increased invasive behavior in an in vitro Matrigel invasion assay (p<0.01; Mann-Whitney U-test). For in vivo analysis, the cancer cells were inoculated into the peritoneum of nude mice. Simultaneous expression of hK4, hK5, hK6, and hK7 resulted in a remarkable 92% mean increase in tumor burden compared to the vector-control cell line. Five out of 14 mice in the 'tissue kallikrein overexpressing' group displayed a tumor/situs ratio greater than 0.198, while this weight limit was not exceeded at all in the vector control group consisting of 13 mice (p=0.017; chi2 test). Our results strongly support the view that tumor-associated overexpression of tissue kallikreins contributes to ovarian cancer progression.

Quantitative reverse transcription-PCR assay for detection of mRNA encoding full-length human tissue kallikrein 7: prognostic relevance of KLK7 mRNA expression in breast cancer.

BACKGROUND: The human tissue kallikrein gene family (KLK1 to KLK15) encodes a group of 15 serine proteases (hK1 to hK15), several of which have been implicated in cancer-related processes. METHODS: We established a specific quantitative reverse transcription-PCR assay for full-length KLK7 mRNA that excluded amplification of the exon 2 deletion splice variant (the latter does not encode a functional protease), and evaluated full-length KLK7 mRNA expression [normalized to human glucose-6-phosphate dehydrogenase (h-G6PDH)] in tumor tissue specimens from 155 breast cancer patients. RESULTS: High KLK7 mRNA expression (continuous) was significantly associated with a better patient outcome according to both univariate (P = 0.005) and multivariate (P = 0.046) Cox survival analysis. Separation of patients by optimized dichotomization revealed a significantly better prognosis for patients with high KLK7 mRNA status (n = 89) compared with patients with low KLK7 mRNA status (n = 66) [univariate hazard ratio (HR) = 0.45 (P = 0.001); multivariate HR = 0.50 (P = 0.005)]. In the subgroup of patients not receiving adjuvant treatment (n = 69), KLK7 mRNA status was a significant prognosticator [univariate HR = 0.29 (P = 0.002); multivariate HR = 0.40 (P = 0.034)]. This subgroup was least influenced by postoperative treatment and thus best showed the impact of KLK7 expression on the natural course of breast cancer disease. CONCLUSION: Expression of full-length KLK7 mRNA may represent a new prognostic marker in breast cancer disease.