Risk Prediction Tool for Aggressive Tumors in Clinical T1 Stage Clear Cell Renal Cell Carcinoma Using Molecular Biomarkers

Abstract

Some early-stage clear cell renal cell carcinomas (ccRCCs) of ≤7 cm are associated with a poor clinical outcome. In this study, we investigated molecular biomarkers associated with aggressive clinical T1 stage ccRCCs of ≤7 cm, which were used to develop a risk prediction tool toward guiding the decision of treatment. Among 1069 nephrectomies performed for ccRCC of ≤7 cm conducted between January 2008 and December 2014, 177 cases with available formalin-fixed paraffin-embedded tissue were evaluated. An aggressive tumor was defined as a tumor exhibiting synchronous metastasis, recurrence, or leading to cancer-specific death. Expression levels of six genes (FOXC2, CLIP4, PBRM1, BAP1, SETD2, and KDM5C) were measured by reverse-transcription polymerase chain reaction (qRT-PCR) and their relation to clinical outcomes was investigated. Immunohistochemistry was performed to validate the expression profiles of selected genes significantly associated with clinical outcomes in multivariate analysis. Using these genes, we developed a prediction model of aggressive ccRCC based on logistic regression and deep-learning methods. FOXC2, PBRM1, and BAP1 expression levels were significantly lower in aggressive ccRCC than non-aggressive ccRCC both in univariate and multivariate analysis. The immunohistochemistry result demonstrated the significant downregulation of FOXC2, PBRM1, and BAP1 expression in aggressive ccRCC. Adding immunohistochemical staining results to qRT-PCR, the aggressive ccRCC prediction models had the area under the curve (AUC) of 0.760 and 0.796 and accuracy of 0.759 and 0.852 using the logistic regression method and deep-learning method, respectively. Use of these biomarkers and the developed prediction model can help stratify patients with clinical T1 stage ccRCC.

Keywords: BAP1, BRCA1 associated protein-1; BMI, Body mass index; Biomarker; CLIP4, CAP-Gly, cytoskeleton-associated protein-glycine rich domain-containing linker protein family member 4; DNN, Deep neural network; EDTA, Ethylenediaminetetraacetic acid; FFPE, Formalin-fixed paraffin-embedded; FOXC2, Forkhead box protein C2; KDM5C, Lysine-specific demethylase 5C; MSKCC, Memorial Sloan Kettering Cancer Center; PBRM1, Polybromo 1; PBS, Phosphate-buffered saline; Prediction model; Renal cell cancer; SETD2, SET domain-containing 2; TNM, Tumor-node-metastasis; ccRCC, Clear cell renal cell carcinoma; qRT-PCR, Quantitative reverse transcription-polymerase chain reaction.

Graphical abstract

Fig. 1

Receiver operating characteristic curve from the logistic regression model and deep neural network model for predicting aggressive clear cell carcinoma.

Fig. 2

Immunohistochemical findings of FOXC2, PBRM1, and BAP1 (A) Positive expression of FOXC2 (B) Negative expression of FOXC2 (C) Positive expression of PBRM1 (D) Negative expression of PBRM1 (E) Positive expression of BAP1 (F) Negative expression of BAP1, Scale bars indicate 100 μM (original magnification x100; inset x400).