We assessed EGFR phosphorylation on tyrosine 1173 in all patient samples for which residual frozen tumor was attainable and compared it to EGFR phosphorylation in 49 tumor samples from GBM patients who had not received any EGFR kinase inhibitor before surgical procedure . Given that EGFR levels in GBM assortment above two to 3 orders of magnitude , we chose an electrochemiluminescent detection kinase using a broad linear choice of detection . This platform made available the supplemental advantage that it allowed us to determine complete and phospho EGFR signal for every sample in a single nicely and run all clinical trial and control samples collectively in a 96 effectively format. When compared to control samples , the group of lapatinib treated tumors showed significantly less EGFR phosphorylation per total EGFR signal . Having said that, all lapatinib handled tumors showed residual EGFR phosphorylation over amounts observed in lapatinib na?e tumors not overexpressing EGFR.
For all tumors with adequate residual sample, we also performed immunoblot analysis . EGFR immunoblot analysis showed EGFR overexpression in 12 27 tumors; a 140 KDa band, constant with all the EGFRvIII deletion, was detected LY2940680 clinical trial in seven 27 of tumors, all inside the group of tumors overexpressing EGFR . Only one of these tumors harbored a missense mutation in the EGFR ectodomain . A comparison of EGFR phosphorylation concerning lapatinib handled tumors with EGFR overexpression and handle tumors showed that lapatinib taken care of GBMs showed reduce ranges of EGFR phosphorylation than controls with very similar levels of EGFR overexpression . All lapatinib handled tumors showed residual EGFR phosphorylation over ranges seen in GBM controls lacking EGFR overexpression, steady with our ELISA results.
Because all sufferers underwent surgical tumor resection, Amygdalin we could not assess the radiographic tumor responses to lapatinib. 5. Level of EGFR inhibition determines cell death response in EGFR mutant GBM cells Scientific studies in cancer cell lines have shown that cell death induction by lapatinib involves drug concentrations of two three M, drug concentrations over the IC50s for inhibition of EGFR phosphorylation and inhibition of cell proliferation . Comprehensive dose response experiments in EGFR mutant SF268 , SKMG3 and KNS 81 FD GBM cells similarly showed dose dependent cell death induction only over lapatinib concentrations of 1500 1750 nM . Whilst lapatinib ranks amongst the most selective ATP blog competitive kinase inhibitors , we sought to verify that this cell death threshold reflected a necessity for near finish EGFR inhibition instead of likely off target results of lapatinib.
We performed titration experiments with a retroviral EGFR shRNA construct in GBM cells with EGFR EC mutations. At a virus dilution of one:27, SF268 GBM cells showed clear reductions in EGFR protein levels and EGFR phosphorylation and greater than 50 growth inhibition, but no evidence for cell death .