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The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers
Published on: January 22, 2013
The use of reverse phase protein arrays (RPPA) to explore protein expression variation within individual renal cell
Fiach C O'Mahony1, Jyoti Nanda, Alexander Laird
1Edinburgh Urological Cancer Group, University of Edinburgh. fomahony@staffmail.ed.ac.uk
Abstract:
Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant. In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma as well as other solid tumors. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis. Protein based analysis of RCC is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.
Insights
Understanding resistance to tyrosine kinase inhibitors (TKIs) in clear cell renal cell cancer is crucial. This study explores proteomic analysis of tumor heterogeneity to develop personalized TKI treatments for kidney cancer patients.
Area of Science:
- Oncology
- Molecular Biology
- Proteomics
Background:
- Metastatic clear cell renal cell cancer (ccRCC) lacks curative treatments, with targeted therapies like sunitinib showing limited efficacy due to intrinsic and acquired resistance.
- Tumor heterogeneity, both inter- and intratumoral, presents a significant challenge in understanding and overcoming treatment resistance in ccRCC.
- Sequential tissue analysis is essential for studying TKI resistance, mirroring successful strategies in other cancers, but is complicated by ccRCC's heterogeneity.
Purpose of the Study:
- To investigate the molecular complexity of ccRCC and identify mechanisms of resistance to tyrosine kinase inhibitors (TKIs).
- To establish a method for analyzing proteomic differences in heterogeneous ccRCC tumors to inform personalized treatment strategies.
- To address the need for effective treatments for patients with metastatic clear cell renal cell cancer who do not respond to current therapies.
Main Methods:
- Utilized combined morphological analysis and Fuhrman grading to categorize ccRCC tumors and select representative areas for analysis.
- Employed Reverse Phase Protein Arrays (RPPA) for proteomic analysis, requiring strict antibody quality control due to the dot blot format.
- Analyzed differential protein expression across multiple samples on a single slide using validated antibodies and fluorescent detection for multiplexing and high-throughput analysis.
Main Results:
- Detailed mapping and categorization of RCC tumors enabled selection of representative areas for proteomic analysis.
- Reverse Phase Protein Arrays (RPPA) allowed for miniaturized, high-throughput proteomic analysis of hundreds of samples.
- Simultaneous differential protein expression analysis was achieved through comparative fluorescence levels, facilitating cost-effective investigation.
Conclusions:
- Proteomic analysis, despite challenges with antibody specificity, offers a viable approach to study ccRCC heterogeneity and TKI resistance.
- The RPPA platform enables miniaturized, high-throughput proteomic analysis crucial for understanding complex solid tumors like ccRCC.
- This approach supports the development of personalized treatments by providing insights into the molecular drivers of resistance in renal cell cancer.
