Beschreibung:
<jats:title>Abstract</jats:title>
<jats:p>Introduction: Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction in immune cells. While most analytical methods focus on the detection of these crucial enzymes at the RNA or protein level, a method was lacking to monitor multiple enzymatic activities in patient-derived materials like blood and (tumor) tissues. Such a substrate-specific assay is a new tool for biomarker discovery in immuno-oncology (I-O), because the I-O targets - checkpoint and immune receptors like PD1, CTLA4, LAG3, 4-1BB, CD40, CD20, OX40, TIGIT and GITR - are controlled by phosphatases. The aim of this study was to develop a multiplex PTP assay and investigate the phosphatase activity in metastatic renal cell carcinoma (mRCC) tissues with high and low levels of TILs (tumor-infiltrating lymphocytes).</jats:p>
<jats:p>Methods: On the basis of hematoxylin and eosin staining of mRCC tissues, 9 tissues with high TILS-scores were selected, as well as 10 tissues without TILS, matched on basis of the percentage of viable tumor cells. The technology used is a second generation peptide microarray, which is fully automated with short turnaround times and a high throughput of clinical samples in a 96-well setup. Arrays comprise 46 unique nitrophosphotyrosine containing peptides derived from known phosphosites. When a clinical sample is applied to this peptide microarray, this will lead to a pattern of peptide dephosphorylations, which is monitored in real time (kinetic readout).</jats:p>
<jats:p>Results: The PTP assay was first developed for PBMC profiling. 0.10 ug protein/array was used, corresponding to 10000 cells. The signals were concentration dependent and could be inhibited by PTP inhibitors. For tissue profiling 2.0 µg protein input (0.02 mm3 tissue) was used. A 2-group comparison analysis of the phosphatase activity in tissues with high scores of TILs (massive infiltration, N=9) vs. without (N=10), revealed that 29 of 46 peptide substrates showed significantly higher dephosphorylation (PTP) activities in the tissues with high levels of lymphocyte infiltrates. A PCA revealed a clear separation between the groups of high and low TILS scores. Discriminating phosphosites (p&lt;0.0002) were derived from the following 10 signaling proteins RET, ERBB2, PAXI, PGFRB, CBL, FRK, PDPK1, INSR, PECA1 and the T-cell kinase LCK.</jats:p>
<jats:p>Conclusions: Proof of concept was shown in mRCC samples that high PTP activity correlates with high levels of TILS. As a TILS-score itself is already regarded to be a candidate biomarker for I-O therapy response, the observed correlation is a basis for the development of more mechanistic biomarkers predicting therapy response. This method can be a starting point for the development of an enzymatic and thus sensitive and quantitative TILS test.</jats:p>
<jats:p>This study has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 259939.</jats:p>
<jats:p>Citation Format: Rob Ruijtenbeek, Liesbeth Houkes-van Kerkhoff, Jeannette Oosterwijk, Liesbeth Hovestad-Bijl1, Riet Hilhorst, Alejandro H. Gomez, Juan F. Rodriguez, Jesus Garcia-Donas, Bart Kiemeney, Egbert Oosterwijk. Correlation of phosphatase activity with lymphocyte infiltrates in metastatic renal cell carcinoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5608. doi:10.1158/1538-7445.AM2017-5608</jats:p>