The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

• DOI: 10.3791/50221
• Publication Date: 2013-01-22T22:19:08Z
• Authors: Fiach C. O'Mahony; Jyoti Nanda; Alexander Laird; Peter Mullen; Helen Caldwell; Ian M. Overton; Lel Eory; Marie O'Donnell; Dana Faratian; Thomas Powles; David J. Harrison; Grant D. Stewart
• Keywords: Targeted Therapy; Grading (engineering); Kidney cancer; Tumour heterogeneity

Currently there is no curative treatment for metastatic clear cell renal cancer, the commonest variant of disease. A key factor in this resistance thought to be molecular complexity Targeted therapy such as tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% patients will respond, with overwhelming majority these relapsing within 1 year. As question intrinsic and acquired cancer highly relevant. In order study TKIs, ultimate goal developing effective, personalized treatments, sequential tissue after a specific period targeted required, an approach which had proved successful chronic myeloid leukaemia. However application strategy carcinoma complicated by high level both inter- intratumoral heterogeneity, feature well other solid tumors. Intertumoral heterogeneity due transcriptomic genetic differences established even similar presentation, stage grade tumor. addition it that great morphological (intratumoral) RCC, likely represent greater heterogeneity. Detailed mapping categorization RCC tumors combined analysis Fuhrman grading allows selection representative areas proteomic analysis. Protein based attractive its widespread availability pathology laboratories; however, can problematic limited antibodies. Due dot blot nature Reverse Phase Arrays (RPPA), antibody specificity must pre-validated; strict quality control antibodies used paramount importance. Despite limitation format does allow assay miniaturization, allowing printing hundreds samples onto single nitrocellulose slide. Printed slides then analyzed fashion Western use target primary fluorescently labelled secondary antibodies, multiplexing. Differential protein expression across all on slide simultaneously comparing relative fluorescence more cost-effective high-throughput manner.