Login to Access Video or Poster Abstract: MP39-10
Sources of Funding: NIH R21-CA169964

Introduction

Intra-tumor heterogeneity (ITH) is the presence of cell clones with different genetic or histologic phenotypes that occupy distinct spaces within a tumor. Whether genomic ITH translates into functional ITH on the level of protein expression and activity is less well characterized. We hypothesized that variation in expression and activation of critical RCC signaling proteins is less than genomic ITH, resulting in a limited number of functional phenotypes in a tumor. This framework would allow for representative sampling of RCC using renal mass biopsy and advanced proteomic techniques.

Methods

We profiled the expression and activation state of extracellular signal-regulated kinase (ERK), a critical effector of the MAPK signaling pathway, in 131 ex vivo biopsies from 39 clear cell RCC tumors and adjacent normal parenchyma after nephrectomy. We performed fine-needle aspirate (FNA) biopsies of grossly representative sections after bivalving the kidney. We performed nano-scale immunoassays (NIA) to quantify the absolute and relative abundances of the phospho-isoforms of ERK1/2 using the Peggy Sue instrument (Protein Simple). ERK1/2 was interrogated with a pan-ERK antibody. Expression levels of ERK levels were compared across samples by normalization to the levels of the ubiquitously expressed protein HSP70._x000D_

Results

ERK1 was infrequently activated, with most samples (69%) demonstrating no phosphorylation of ERK1. In contrast, ERK2 demonstrated phosphorylation in the majority of the samples. Two distinct mono-phosphorylated ERK2 isoforms were detected (pERK2a and pERK2b), with relative abundances ranging from 0-58% and 1-57%, respectively. The relative abundance of dual-phosphorylated ERK2 (ppERK2) ranged from 0-26%. The overall ITH of ERK2 activation was low (average standard deviation [SD] 6%) compared with the difference in ERK2 activation among the 39 tumors (SD 5% - 22%).

Conclusions

The abundance and activation of ERK1/2 can be measured from scant FNAs of RCC using novel proteomic methods. We found little ITH of ERK, which suggests that protein ITH is generally less than functional heterogeneity among tumors. These data suggest that a single renal mass biopsy may accurately measure the activity of relevant signaling pathways within a tumor, which may facilitate future precision medicine approaches.

Funding

NIH R21-CA169964