Sunitinib Treatment Exacerbates Intratumoral Heterogeneity in Metastatic Renal Cancer

Abstract

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 ¼ 187 samples) were taken from the primary renal tumors of patients with mccRCC who were sunitinib treated (n ¼ 23, SuMR clinical trial) or untreated (n ¼ 23, SCOTRRCC study). ITH of pathologic grade, DNA (aCGH), mRNA (Illumina Beadarray) and candidate proteins (reverse phase protein array) were evaluated using unsupervised and supervised analyses (driver mutations, hypoxia, and stromal-related genes). ITH was analyzed using intratumoral protein variance distributions and distribution of individual patient aCGH and gene-expression clustering.Results: Tumor grade heterogeneity was greater in treated compared with untreated tumors (P ¼ 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.

Overcoming intratumoural heterogeneity for reproducible molecular risk stratification: a case study in advanced kidney cancer

“…Each tumour sample analysed by RPPA reflected 50–75 mg of lysed tissue taken from a 1 cm 3 spatially mapped region [24]. Protein extraction, RPPA slide spotting, immunofluorescence data acquisition, data processing and identification of four markers that had increased variance associated with sunitinib treatment ( p  < 0.05) were described previously [20, 25]. Briefly, 1 mg/ml lysates were spotted onto nitrocellulose slides using a robotic spotter, and immunofluorescence imaging was performed with an Odyssey scanner (Li-Cor Biosciences, Lincoln, NB, USA).…”

The Efficacy of Sunitinib Treatment of Renal Cancer Cells Is Associated with the Protein PHAX In Vitro

“…Fresh-frozen primary ccRCC tissue for analysis was obtained from cytoreductive nephrectomy samples of 23 sunitinib-naive patients with metastatic ccRCC (mccRCC) as part of the Scottish Collaboration On Translational Research into Renal Cell Cancer (SCOTRRCC) study (UK CRN ID: 12229) [21]. Fresh-frozen primary tumour tissue was also obtained from 27 patients with mccRCC treated with three cycles of sunitinib (18 weeks) followed by a cytoreductive nephrectomy after 2 weeks off sunitinib as part of the Upfront Sunitinib (SU011248) Therapy Followed by Surgery in Patients with Metastatic Renal Cancer: A Pilot Phase II Study (SuMR; ClinicalTrials.gov identifier: NCT01024205); 23 of these patients had adequate tissue for analysis (see Table 1 in [22] for summary of patient characteristics). Investigations were approved by institutional review boards, and written informed consent was obtained from each patient.…”

“…As previously described [22], mRNA was amplified using the WT-Ovation FFPE System Version 2 (NuGEN, Redwood City, CA, USA), purified using the Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany), biotinylated using the Encore BiotinIL ModuleIL (NuGEN), purified using the minElute Reaction Cleanup Kit (Qiagen), and quantified using a Bioanalyser 2100 with the RNA 6000 Nano Kit (Agilent, Santa Clara, CA, USA). cRNA was then hybridized to Human HT-12v3 expression Beadarrays (Illumina, San Diego, CA, USA) according to the standard protocol for NuGEN-amplified samples.…”

3D-printed moulds for image-guided surgical biopsies: an open source computational platform

“…Often, however, tumours display such a high degree of heterogeneity that a single tissue sample is insu cient to capture the full molecular landscape of the disease 2 . A prime example of such spatial heterogeneity is renal cell carcinoma (RCC), which has been shown to be radiologically, genetically, and metabolically heterogeneous [3][4][5] . Macroscopic regions with distinct genotypes can be identified within a single tumour through multi-regional sampling 3,6 .…”