Carbonic anhydrase 9 expression increases with vascular endothelial growth factor-targeted therapy and is predictive of outcome in metastatic clear cell renal cancer

Abstract

Background: There is a lack of biomarkers to predict outcome with targeted therapy in metastatic clear cell renal cancer (mccRCC). This may be because dynamic molecular changes occur with therapy.

Objective: To explore if dynamic, targeted-therapy-driven molecular changes correlate with mccRCC outcome.

Design, setting, and participants: Multiple frozen samples from primary tumours were taken from sunitinib-naïve (n=22) and sunitinib-treated mccRCC patients (n=23) for protein analysis. A cohort (n=86) of paired, untreated and sunitinib/pazopanib-treated mccRCC samples was used for validation. Array comparative genomic hybridisation (CGH) analysis and RNA interference (RNAi) was used to support the findings.

Intervention: Three cycles of sunitinib 50mg (4 wk on, 2 wk off).

Outcome measurements and statistical analysis: Reverse phase protein arrays (training set) and immunofluorescence automated quantitative analysis (validation set) assessed protein expression.

Results and limitations: Differential expression between sunitinib-naïve and treated samples was seen in 30 of 55 proteins (p<0.05 for each). The proteins B-cell CLL/lymphoma 2 (BCL2), mutL homolog 1 (MLH1), carbonic anhydrase 9 (CA9), and mechanistic target of rapamycin (mTOR) (serine/threonine kinase) had both increased intratumoural variance and significant differential expression with therapy. The validation cohort confirmed increased CA9 expression with therapy. Multivariate analysis showed high CA9 expression after treatment was associated with longer survival (hazard ratio: 0.48; 95% confidence interval, 0.26-0.87; p=0.02). Array CGH profiles revealed sunitinib was associated with significant CA9 region loss. RNAi CA9 silencing in two cell lines inhibited the antiproliferative effects of sunitinib. Shortcomings of the study include selection of a specific protein for analysis, and the specific time points at which the treated tissue was analysed.

Conclusions: CA9 levels increase with targeted therapy in mccRCC. Lower CA9 levels are associated with a poor prognosis and possible resistance, as indicated by the validation cohort.

Patient summary: Drug treatment of advanced kidney cancer alters molecular markers of treatment resistance. Measuring carbonic anhydrase 9 levels may be helpful in determining which patients benefit from therapy.

Trial registration: ClinicalTrials.gov NCT01024205.

Keywords: Biomarker; CA9; Renal cancer; VEGF TKI.

Copyright © 2014 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1

Reverse phase protein array (RPPA) differential protein expression results. Box-and-whisker plot showing differential expression analysis of 55 proteins evaluated by RPPA in sunitinib-naïve (light orange) and treated (dark orange) metastatic clear cell renal cell carcinoma samples. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

Results for significantly variable and differentially expressed proteins in test and validation samples from sunitinib-naïve and treated patients. (a) Box-and-whisker plot showing test set reverse phase protein array differential expression results of four key proteins. Medians and interquartile ranges are shown. (b) Box-and-whisker plot showing automated quantitative analysis (AQUA) evaluated protein expression of four key proteins using a validation cohort of 61 sunitinib-treated and 25 pazopanib-treated and untreated paired metastatic clear cell renal cell carcinoma samples. Of the four proteins, carbonic anhydrase 9 (CA9) was the only one with significant differential protein expression (p = 0.01). Medians and interquartile ranges are shown. (c) Kaplan-Meier curve showing relationship of CA9 protein expression determined by AQUA in situ analysis (low vs high, as determined using X-tile [15]) in sunitinib/pazopanib-treated patients to overall survival (hazard ratio: 0.260; 95% confidence interval, 0.111–0.608; p = 0.001).

Fig. 3

Array comparative genomic hybridization and RNA interference carbonic anhydrase 9 (CA9) results. (a) Heat map plotting gains (red) and losses (blue) of CA9. The right-hand bar represents the logarithm of the odds score (−log10) of the adjusted p value (Fisher test), the dashed line represents p = 0.05. There were significantly more losses in the treated samples relative to the untreated patient samples (p = 0.002). Supplemental Figure 1 provides further description of regional chromosomal changes and Supplemental Figure 2 provides details of the genome-wide changes in gains and losses following sunitinib therapy. (b) RCC11 and (c) CAKI-2 human renal cell carcinoma transfected with either control or CA9 short interfering RNA (siRNA), followed by sunitinib treatment and cell viability analysis 5 d later. Error bars represent standard errors of the mean. To confirm silencing, cell lysates from RCC11 and CAKI-2 siRNA transfected cells were analysed by western blotting using CA9 and β-actin-specific antibodies, as indicated. VHL = von Hippel-Lindau; Del = deletion; mut = mutant; NC = no change; wt = wild-type.