Protein lysate microarray analysis of uPA and PAI-1 from formalin-fixed breast cancers

Coordinator:    Prof. Dr. Karl-Friedrich Becker
Institution: Institut für Pathologie der Technischen Universität München

Subproject 1 of the Innovation Alliance “Biomarkers for breast cancer” addressed the quantification of the proteins uPA and PAI-1 in formalin-fixed, paraffin-embedded (FFPE) tissues to support therapy decisions in non-metastasised breast cancers.
The clinically approved ELISA (Enzyme Linked Immunosorbent Assay) test to measure uPA and PAI-1 is only established for cryo preserved samples. This hinders the propagation of the method as there often is only FFPE tissue available for this kind of analyses. Here we established techniques to extract uPA and PAI-1 from FFPE-tissues and analyzed them by standard methods e.g. Western blot analysis. Additionally, we successfully applied so called Reverse Phase Protein Arrays (RPPA) for the quantification of both proteins. The reproducibility of the assay was assessed. We assembled a set of protein markers comprising uPA, PAI-1 and 22 downstream signalling molecules and performed a signalling analysis utilizing RPPAs. We demonstrated that expression of PAI-1 correlates positively with the expression of Integrin-subunit ?V, uPAR, p-Akt, ER?, EGFR, HER3, HER4 and p-HER2. For uPA a positive correlation with the expression of ER?, p-Erk and Stat3 was found.
The translation of the clinically established ELISA to measure uPA and PAI-1 in FFPE tissues proved to be extremely challenging. None of the efforts to apply the assay to FFPE tissues, including elimination of inhibiting substances, precipitation and dilution of the proteins in an ELSIA-compatible buffer or use of alternative extraction buffers, did not result in a reliable and reproducible assay protocol. Alternatively we compared results achieved by the quantitative RPPA to those obtained by ELISA in a cohort of 156 patients with non-metastasised breast cancers. For both proteins discrepancies between the methods were observed. While results for uPA did not correlate significantly (rs = 0.051; p = 0.54) PAI-1 results correlated significantly between both methods (p < 0.001) but only with a low correlation coefficient rs = 0.573. Thus, no sufficient concordance of the two methods was achieved. Although uPA and PAI-1 can be analyzed in FFPE tissues by RPPA the method is currently not feasible for clinical use as it does not correlate with the gold standard ELISA.


A tissue sample embedded in formalin wax (© Institut für Pathologie, TU München).

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