Lack of Ephrin Receptor A1 Is a Favorable Independent Prognostic Factor in Clear Cell Renal Cell Carcinoma

<div><p>The EPH receptor tyrosine kinases and their cell-bound ligands, the ephrins, have been shown to be associated with cancer development and progression. In this study, mRNA and protein expression of the receptors EPHA1 and EPHA2 as well as of their ligand EFNA1 and their prognostic relevance in clear cell renal cell carcinoma was evaluated. Gene expression was measured in 75 cryo-preserved primary tumors and matched non-malignant renal specimens by quantitative PCR. Protein expression was analyzed by immunohistochemistry on tissue microarrays comprising non-malignant, primary tumors and metastatic renal tissues of 241 patients. Gene and protein expression of all three factors was altered in tumor specimens with EPHA1 and EPHA2 being generally diminished in tumors compared to normal renal tissue, whereas EFNA1 was commonly elevated. A positive EPHA1 and EPHA2 protein staining as well as a low EFNA1 protein level were significantly linked to more aggressive tumor features, but only a positive EPHA1 immunoreactivity was significantly associated with poor survival. In subgroup analyses, EPHA1 and EPHA2 protein levels were significantly higher in metastatic than in primary lesions. Patients with EPHA1/EPHA2-positive tumors or with tumors with positive EPHA1 and low EFNA1 immunoreactivity had the shortest survival rates compared to the respective other combinations. In a multivariate model, EPHA1 was an independent prognostic marker for different survival endpoints. In conclusion, an impaired EPH-ephrin signaling could contribute to the pathogenesis and progression of clear cell renal cell carcinoma.</p></div

Univariate Cox regression analyses for PFS, TSS and OS dependent on clinicopathological parameters and molecular markers.

<p>P values highlighted in bold indicate statistically significant prognostic markers following correction for multiple comparisons (n = 20) by using the method of Benjamini and Hochberg.</p><p>Abbreviations: CI: confidence interval; HR: hazard ratio; n.d.: not determined.</p

Analyses of Potential Predictive Markers and Survival Data for a Response to Sunitinib in Patients with Metastatic Renal Cell Carcinoma

<div><p>Background</p><p>Patients with metastatic clear cell renal cell carcinoma (ccRCC) are frequently treated with tyrosine kinase inhibitors (TKI) such as sunitinib. It inhibits angiogenic pathways by mainly targeting the receptors of VEGF and PDGF. In ccRCC, angiogenesis is characterized by the inactivation of the <i>von Hippel-Lindau</i> gene (VHL) which in turn leads to the induction of HIF1α target genes such as CA9 and VEGF. Furthermore, the angiogenic phenotype of ccRCC is also reflected by endothelial markers (CD31, CD34) or other tumor-promoting factors like Ki67 or survivin.</p><p>Methods</p><p>Tissue microarrays from primary tumor specimens of 42 patients with metastatic ccRCC under sunitinib therapy were immunohistochemically stained for selected markers related to angiogenesis. The prognostic and predictive potential of theses markers was assessed on the basis of the objective response rate which was evaluated according to the RECIST criteria after 3, 6, 9 months and after last report (12–54 months) of sunitinib treatment. Additionally, VHL copy number and mutation analyses were performed on DNA from cryo-preserved tumor tissues of 20 ccRCC patients.</p><p>Results</p><p>Immunostaining of HIF-1α, CA9, Ki67, CD31, pVEGFR1, VEGFR1 and -2, pPDGFRα and -β was significantly associated with the sunitinib response after 6 and 9 months as well as last report under therapy. Furthermore, HIF-1α, CA9, CD34, VEGFR1 and -3 and PDGRFα showed significant associations with progression-free survival (PFS) and overall survival (OS). In multivariate Cox proportional hazards regression analyses high CA9 membrane staining and a response after 9 months were independent prognostic factors for longer OS. Frequently observed copy number loss and mutation of VHL gene lead to altered expression of VHL, HIF-1α, CA9, and VEGF.</p><p>Conclusions</p><p>Immunoexpression of HIF-1α, CA9, Ki67, CD31, pVEGFR1, VEGFR1 and -2, pPDGFRα and -β in the primary tumors of metastatic ccRCC patients might support the prediction of a good response to sunitinib treatment.</p></div

Effect of VHL gene alterations on protein expression of VHL and its target genes.

<p>Boxplots show the VHL score (A), HIF-1α score (B), CA9 score (C) and VEGFA intensity (D) of patients with normal VHL status, with VHL mutations and copy number losses.</p

Kaplan-Meier plots for IHC-markers with significant associations to PFS and OS.

<p>Significant differences in PFS (log-rank test) were observed for HIF-1α score (A), CD34 MVD (B) and VEGFR3 vessel staining (C). CA9 score (D), CA9 intensity (E) and CA9 membrane staining (F), VEGFR1 vessel staining (G), VEGFR3 vessel staining (H) and PDGFRα score (I) were significantly associated with OS. The data at each curve represent the number of patients per subgroup; the number of events for each subgroup is shown in brackets. The table under each Kaplan-Meier plot contains the median PFS or OS as well as the one-, two- or five-year PFS or OS for this marker.</p

Follow up data of patients.

1<p>two patients were excluded due to missing follow up data.</p

Univariate and multivariate analysis of response to sunitinib-treatment with regard to PFS and OS¹.

1<p>table contains trends and significant results (in bold) of univariate and multivariate analysis of OS and PFS.</p>2<p>non-responder is reference category and set as HR = 1.</p>3<p>only significant parameters from the univariate analyses of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076386#pone-0076386-t007" target="_blank">table 7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076386#pone-0076386-t008" target="_blank">8</a> were included in the multivariate analyses.</p

Primer for VHL mutation analyses.

<p>Primer for VHL mutation analyses.</p