(P 43) UROKINASE PLASMINOGEN ACTIVATOR RECEPTOR-ASSOCIATED PROTEIN AS A POTENTIAL NEXT GENERATION MOLECULAR TARGET FOR TREATMENT OF GASTROINTESTINAL STROMAL TUMOR

Objective: Most gastrointestinal stromal tumors (GIST) are resistant to chemotherapy but highly sensitive to treatment with tyrosine kinase inhibitors. Primary and secondary mutations in KIT and PDGFRA genes, encoding for receptor tyrosine kinases, limit the use of these oral compounds in clinical routine. To overcome or delay the resistance and to develop curative strategies for treatment of GIST, molecular targets beyond traditional receptor kinases may be of interest.
Urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180) is a recycling endocytic receptor with a key role in collagen turnover and extracellular matrix homeostasis. In normal tissue, uPARAP is preferentially expressed in cells of mesodermal origin, and the protein is known to be overexpressed in a variety of soft tissue sarcomas (see abstract Wozniak et al. at this meeting).
In the current project we aim to explore the molecular epidemiology of uPARAP in GIST, using archival tumor material from GIST patients as well as cell line and patient-derived xenograft material.

Methods: Expression of uPARAP was assessed by immunohistochemistry in GIST specific tissue microarrays (TMA) readily available in our Laboratory. We selected one clinical TMA, and six TMAs from 13 patient-derived and three cell line-derived GIST xenografts from our XenoSarc platform. For these models at least two passages of each tumor were included in the TMAs, with 2–3 cores per passage (523 cores in total). All these models have KIT or PDGFRA driver mutation and five of them harbor secondary mutations. The clinical GIST TMA is constructed from 71 GIST patients (76 samples, 2 cores per each) with different molecular background: primary mutation in KIT exon 11 (44%), PDGFRA exon 18 (17%) and KIT exon 9 (13%), secondary mutation in KIT exon 13 (8%) and KIT exon 17 (8%), and wild type (10%).
Xenograft TMAs were manually immunostained with Goat Anti-Human Mrc2 Polyclonal Antibody (R&D systems, cat. No #AF5770). Primary antibody incubation with a 1:100 dilution was performed overnight at 4 °C. Histologic evaluation was performed by estimating the percentage of positive cells (0-100%) and the expression intensity (0-3+) of each core. High uPARAP expression was defined as intensity score of 2 or 3 in ≥ 50% of tumor cells in at least 2 cores. The clinical TMA will be stained with MRC2 Mouse Monoclonal Antibody (clone OTI9G4; OriGene Technologies, cat. No #TA811858).

Results: uPARAP was found to be highly expressed in 9/16 established xenograft models in at least one passage; in three of these models the high expression was preserved across several passages, all harboring a primary KIT exon 11 mutation. Positive models showed only moderate inter-passage variability in uPARAP expression, and positivity was not lost over subsequent passages.
Immunostaining and analysis of uPARAP in the clinical GIST TMA are currently ongoing and results will be disclosed at the meeting.

Conclusion: uPARAP is highly expressed in several GIST xenograft models across multiple passages of ex-mouse tumors. Considering its preferential expression in GIST and other mesenchymal tumors and the recycling character of this molecular target, uPARAP may serve as a powerful emerging target for systemic treatment of GIST beyond traditional kinase inhibitors or in combination with the latter. uPARAP positive xenograft models can be used for in vivo evaluation of such anti-uPARAP treatment.