Practicability of clinical application of bladder cancer molecular classification and additional value of epithelial-to-mesenchymal transition: prognostic value of vimentin expression

Abstract

Background: Bladder cancer (BlCa) taxonomy has proved its impact in patient outcome and selection for targeted therapies, but such transcriptomic-based classification has not yet translated to routine practice. Moreover, epithelial-to-mesenchymal transition (EMT) has shown relevance in acquisition of more aggressive BlCa phenotype. We aimed to test the usefulness of the molecular classification, as defined by immunohistochemistry (a routinely performed and easy-to-implement technique), in a well-defined BlCa cohort of both non-muscle invasive (NMIBC) and muscle invasive (MIBC) disease. Also, we aimed to assess the additional prognostic value of the mesenchymal marker vimentin to the stratification strategy.

Methods: A total of 186 samples were available. Immunohistochemistry/RT-qPCR for luminal markers GATA3/FOXA1, basal markers KRT5/KRT6A and vimentin were performed.

Results: mRNA expression levels of the markers positively correlated with immunoexpression scores. We found substantial overlapping in immunoexpression of luminal and basal markers, evidencing tumor heterogeneity. In MIBC, basal tumors developed recurrence more frequently. NMIBC patients with higher vimentin immunoexpression endured poorer disease-free survival, and increased expression was observed from normal bladder-NMIBC-MIBC-metastases.

Conclusions: The classification has the potential to be implemented in routine, but further adjustments in practical scoring should be defined; focusing on additional markers, including those related to EMT, may further refine BlCa molecular taxonomy.

Keywords: Basal; Bladder cancer; EMT; Luminal; Molecular classification; Pathology; Vimentin.

Fig. 1

Immunoexpression of luminal and basal markers in the bladder cancer cohort. a, b FOXA1 strong and diffuse immunoexpression in two bladder cancer specimens, one NMIBC (a) and one MIBC (b); c, d: GATA3 strong and diffuse immunoexpression in two bladder cancer specimens, one NMIBC (c) and one MIBC (d); e, f: CK5/6 strong multifocal immunoexpression in two bladder cancer specimens, one NMIBC (e) and one MIBC (f)

Fig. 2

Correlation between mRNA and protein expression of the several luminal and basal markers in the bladder cancer cohort (both MIBC and NMIBC included). FOXA1 (a and b), GATA3 (c and d), KRT5 (e and f) and KRT6A (g and h) analyses. mRNA expression levels are plotted as relative expression levels, normalized to GUSB. Red dash and bars represent median and interquartile range. The immunoexpression score (intensity × percentage) is plotted in the xx-axis. The graphs include n = 83 matched samples (*p < 0.05; ****p < 0.0001)

Fig. 3

Vimentin transcript and protein levels within the bladder cancer cohort. a differential mRNA expression of vimentin between non-muscle (NMIBC) and muscle-invasive (MIBC) bladder cancer. mRNA expression levels are plotted as relative expression levels, normalized to GUSB and HPRT1; b differential protein (immuno)expression of vimentin between NMIBC and MIBC. The immunoexpression score (intensity × percentage) is plotted; c immunoexpression of vimentin among normal bladder, NMIBC, MIBC and bladder cancer metastases. The immunoexpression score (intensity × percentage) is plotted. Red dash and bars represent median and interquartile range. Correction for multiple comparisons was employed and adjusted p-values are represented (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)

Fig. 4

Disease-free survival in non-muscle invasive bladder cancer (NMIBC) patients according to vimentin protein expression

Fig. 5

Immunoexpression of vimentin in the bladder cancer cohort. a, b: immunoexpression of vimentin in primary bladder cancer specimens, one NMIBC (a) and one MIBC (b); c and d: immunoexpression of vimentin in bladder cancer metastases