Purpose: The aim of this study was to investigate the effect of VEGF targeted therapy (sunitinib) on molecular intratumoral heterogeneity (ITH) in metastatic clear cell renal cancer (mccRCC). Experimental design: Multiple tumor samples (n=187 samples) were taken from the primary renal tumors of mccRCC patients who were sunitinib treated (n=23, SuMR clinical trial) or untreated (n=23, SCOTRRCC study). ITH of pathological grade, DNA (aCGH), mRNA (Illumina Beadarray) and candidate proteins

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... phase protein array) were evaluated using unsupervised and supervised analyses (driver mutations, hypoxia and stromal related genes). ITH was analysed using intratumoral protein variance distributions and distribution of individual patient aCGH and gene expression clustering. Results: Tumor grade heterogeneity was greater in treated compared to untreated tumors (P=0.002). In unsupervised analysis, sunitinib therapy was not associated with increased ITH in DNA or mRNA. However, there was an increase in ITH for the driver mutation gene signature (DNA and mRNA) as well as increasing variability of protein expression with treatment (p<0.05). Despite this variability, significant chromosomal and transcript changes to key targets of sunitinib, such as VHL, PBRM1 and CAIX, occurred in the treated samples. Conclusions: These findings suggest that sunitinib treatment has significant effects on the expression and ITH of key tumor and treatment specific genes/proteins in mccRCC. The results, based on primary tumor analysis, do not support the hypothesis that resistant clones are selected and predominate following targeted therapy. Research. on June 5, 2015. RNA and protein level; and (ii) a period of sunitinib treatment (18 weeks) exacerbated molecular heterogeneity. Ideally, to answer the latter question one would undertake multi-region tumor sampling both before and after the sunitinib treatment, but before nephrectomy. Such an experiment would not be ethical or permissible. As such, we used a comparator group in the form of patients with well-matched sunitinib naïve tumors. Sunitinib has specific molecular Research. on June 5, 2015. reverse phase protein arrays (RPPA) to explore protein expression variation within individual renal cell cancers.