Molecular characterization of low grade and high grade bladder cancer

Abstract

Background: Bladder cancer (BC) is the 9th most common cancer diagnosis worldwide. Low grade (LG) represents 70% of all BCs, characterized by recurrence and rare ability (10-15%) to progress to high grade (HG) and invade. The remaining 30% is high grade (HG), fast invasive BC, which is resistant to therapy. Identifying biomarkers for predicting those tumors able to progress is a key goal for patient outcome improvement. This study focuses on the most promising prognostic markers.

Materials and methods: TP53 and FGFR3 mutational status, Survivin, CK19, CK20, E-cadherin and CD44 gene expression analysis were performed on 66 BCs.

Results: Survivin was found associated to tumor grade (p<0.05). Moreover, Survivin correlated with CD44 in TP53 wild type (p = 0.0242) and FGFR3 wild type (p = 0.0036) tumors. In particular the Survivin-CD44 correlation was associated to HG FGFR3 wild type BCs (p = 0.0045). Unsupervised hierarchical clustering based on gene expression data identified four distinct molecular groups reflecting the patient histology (p = 0.038).

Conclusion: We suggest Survivin, both as a biomarker associated to G3 BCs but negatively related to TP53 mutational status, and as a potential novel therapeutic target.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Example of the histologic classification of G1 (A), G2 (B) and G3 (C) as well as LG (D) and HG (E) BCs among study cases (original magnification x20).

Fig 2. Differential gene expression analysis between HG and LG tumors.

Survivin (A), CK20 (B), E-Cadherin (C) and CD44 (D) gene expression levels in LG and HG BCs. LG: Low Grade; HG: High Grade. Standard error of the mean (SEM) is indicated by the bars. n indicates the number of analyzed samples. * indicates p-value

Fig 3. Differential gene expression of CK20, CD44, E-cadherin, and Survivin in relation to FGFR3 and TP53 mutational status of LG and HG BCs.

CK20, E-cadherin, CD44 and Survivin mRNA levels in TP53-FGFR3 wild type, FGFR3 mutated, TP53 mutated and FGFR3-TP53 mutated LG tumors (A). CK20, E-cadherin, CD44 and Survivin in TP53-FGFR3 wild type, FGFR3 mutated and TP53 mutated HG tumors (B). Only one HG tumor showed overlapped TP53-FGFR3 mutations. It is no sufficient for any statistical analysis. CK20: cytokeratin 20; E-CAD: E-cadherin: Low Grade; HG: High Grade. Standard error of the mean (SEM) is indicated by the bars. n indicates the number of analyzed samples. * indicates p-value

Fig 4. Intergene multivariable analysis.

Multivariable analysis of CD44, CK20, E-cadherin (ECAD) and Survivin in all BCs, HG and LG BCs taking in account also the mutational status of both TP53 and FGFR3 genes. To underline the statistically significant correlations, p-value is reported in red. (StatGraphics XVI software).

Fig 5. Differential gene expression analysis within tumor grading.

Survivin (A), CK20 (B), E-cadherin (C) and CD44 (D) gene expression levels in G1, G2 and G3 BCs. G1: Grade 1; G2: Grade 2; G3: Grade 3. Standard error of the mean (SEM) is indicated by the bars. n indicates the number of analyzed samples. * indicates p value

Fig 6. Differential gene expression analysis within BC morphology.

Survivin (A), CK20 (B), E-cadherin (C) and CD44 (D) gene expression levels in the BCs subtypes defined by the BC morphology. G1+G2 were LG with papillary morphology; papillary G3 were HG characterized as papillary protrusion; flat G3 were HG with non papillary morphology. Standard error of the mean (SEM) has indicated by the bars. n indicates the number of analyzed samples. * indicates p value

Fig 7. Clustering analysis.

Unsupervised clustering analysis combining gene expression levels of CK20, CD44, E-CAD and Survivin genes created 4 distinct clusters (A). Gene expression levels of CK20, CD44, E-CAD and Survivin genes in the clustering of BCs defined by their grading and morphology (B). It is possible to identify similar trends in gene expression profile between Cluster 2 and G2 group and Cluster 4 and G3 non papillary (G3 flat) group.

Fig 8. Histotype distribution in clusters.

Unsupervised clustering analysis performed on gene expression values of CD44, E-cadherin, Survivin and CK20 in BCs revealed specific BC histotype distribution within clusters. LG: low grade; HG: high grade.