Carbonic Anhydrase 9 Expression Increases with Vascular Endothelial Growth Factor–Targeted Therapy and Is Predictive of Outcome in Metastatic Clear Cell Renal Cancer

Abstract

BackgroundThere 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.ObjectiveTo explore if dynamic, targeted-therapy-driven molecular changes correlate with mccRCC outcome.Design, setting, and participantsMultiple 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.InterventionThree cycles of sunitinib 50 mg (4 wk on, 2 wk off).Outcome measurements and statistical analysisReverse phase protein arrays (training set) and immunofluorescence automated quantitative analysis (validation set) assessed protein expression.Results and limitationsDifferential 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.ConclusionsCA9 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 summaryDrug 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.

Sunitinib Treatment Exacerbates Intratumoral Heterogeneity in Metastatic Renal Cancer

“…The final finding was that despite ITH, consistent chromosomal, RNA and protein changes to the targets of sunitinib were seen with treatment, in keeping with our previous findings regarding consistent changes in CAIX protein expression following sunitinib treatment (12). Notable CNVs identified were losses of the renal cancer driver genes SETD2, BAP1, VHL, and PBRM1 (latter two genes also had a significantly lower level of gene expression) following sunitinib therapy (20).…”

“…Reverse phase protein array (RPPA) samples protein levels were determined and slides spotted as described previously (7,12). Briefly, batch effects across the three slides per marker were mitigated using ComBat (13) and normalized (VSN; 14).…”

Angiogenesis- and Hypoxia-Associated Proteins as Early Indicators of the Outcome in Patients with Metastatic Breast Cancer Given First-Line Bevacizumab-Based Therapy

“…Of importance, a treatment-induced increase of CA9 in our patients was associated with improved PFS and OS. Although this may seem counterintuitive, increased intratumoral CA9 expression in patients with advanced renal cell carcinoma treated with sunitinib for 18 weeks has also been associated with better OS (43). An increased level in CA9 has also been observed in glioblastoma patients during treatment with cediranib and chemoradiation or chemoradiation alone (31).…”

Re: Grant D. Stewart, Fiach C. O’Mahony, Alexander Laird, et al. Carbonic Anhydrase 9 Expression Increases with Vascular Endothelial Growth Factor–Targeted Therapy and Is Predictive of Outcome in Metastatic Clear Cell Renal Cancer. Eur Urol. In press. http://dx.doi.org/10.1016/j.eururo.2014.04.007

“…Older data from our institution suggested that CAIX had a role as a predictor of treatment response to high-dose interleukin 2 (IL-2) therapy. This finding was externally validated and prospectively confirmed by our own institution [2,3]; however, a large, multicenter, prospective, randomized trial (SELEC trial) conducted by the Cytokine Working Group failed to confirm a predictive role of CAIX for treatment response to IL-2. More recently, retrospective studies had suggested that there may be a predictive role for CAIX expression in patients treated with targeted therapy with sorafenib [4]; however, this finding could not be confirmed in a larger cohort of the TARGET (Treatment Approaches in Renal Cancer Global Evaluation Trial) study.…”

“…We read with great interest the current study of Stewart et al, who described the independent association of carbonic anhydrase IX (CAIX) expression and survival outcomes in patients with clear cell renal cell carcinoma (ccRCC) treated with sunitinib [1]. Despite having been approved for >7 yr, effective predictive biomarkers for sunitinib treatment response are still lacking.…”