Muscle-invasive bladder carcinoma (MIBC) constitutes a heterogeneous group of tumors with a poor outcome. Molecular stratification of MIBC may identify clinically relevant tumor subgroups and help to provide effective targeted therapies. From seven series of large-scale transcriptomic data (383 tumors), we identified an MIBC subgroup accounting for 23.5% of MIBC, associated with shorter survival and displaying a basal-like phenotype, as shown by the expression of epithelial basal cell markers. Basal-like tumors presented an activation of the epidermal growth factor receptor (EGFR) pathway linked to frequent EGFR gains and activation of an EGFR autocrine loop. We used a 40-gene expression classifier derived from human tumors to identify human bladder cancer cell lines and a chemically induced mouse model of bladder cancer corresponding to human basal-like bladder cancer. We showed, in both models, that tumor cells were sensitive to anti-EGFR therapy. Our findings provide preclinical proof of concept that anti-EGFR therapy can be used to target a subset of particularly aggressive MIBC tumors expressing basal cell markers and provide diagnostic tools for identifying these tumors.
Cancer cells are often characterized by epigenetic changes, which include aberrant histone modifications. In particular, local or regional epigenetic silencing is a common mechanism in cancer for silencing expression of tumor suppressor genes. Though several tools have been created to enable detection of histone marks in ChIP-seq data from normal samples, it is unclear whether these tools can be efficiently applied to ChIP-seq data generated from cancer samples. Indeed, cancer genomes are often characterized by frequent copy number alterations: gains and losses of large regions of chromosomal material. Copy number alterations may create a substantial statistical bias in the evaluation of histone mark signal enrichment and result in underdetection of the signal in the regions of loss and overdetection of the signal in the regions of gain.
The PI3K/AKT pathway is considered to play a major role in bladder carcinogenesis, but its relationships with other molecular alterations observed in bladder cancer remain unknown. We investigated PI3K/AKT pathway activation in a series of human bladder urothelial carcinomas (UC) according to PTEN expression, PTEN deletions and FGFR3, PIK3CA, KRAS, HRAS, NRAS and TP53 gene mutations. The series included 6 normal bladder urothelial samples and 129 UC (Ta n = 25, T1 n = 34, T2-T3-T4 n = 70). Expression of phospho-AKT (pAKT), phospho-S6-Ribosomal Protein (pS6) (one downstream effector of PI3K/AKT pathway) and PTEN was evaluated by reverse phase protein Array. Expression of miR-21, miR-19a and miR-222, known to regulate PTEN expression, was also evaluated. pAKT expression levels were higher in tumors than in normal urothelium (p < 0.01), regardless of stage and showed a weak and positive correlation with pS6 (Spearman coefficient RS = 0.26; p = 0.002). No association was observed between pAKT or pS6 expression and the gene mutations studied. PTEN expression was decreased in PTEN-deleted tumors, and in T1 (p = 0.0089) and T2-T3-T4 (p < 0.001) tumors compared to Ta tumors; it was also negatively correlated with miR-19a (RS = -0.50; p = 0.0088) and miR-222 (RS = -0.48; p = 0.0132), but not miR-21 (RS = -0.27; p = 0.18) expression. pAKT and PTEN expressions were not negatively correlated, and, on the opposite, a positive and moderate correlation was observed in Ta (RS = 0.54; p = 0.0056) and T1 (RS = 0.56; p = 0.0006) tumors. Our study suggests that PI3K/AKT pathway activation occurs in the entire spectrum of bladder UC regardless of stage or known most frequent molecular alterations, and independently of low PTEN expression.
Epidermal nevus (EN) is a congenital disorder characterised by hyperpigmented epidermal thickening following a Blaschkos line. It is due to somatic mutations in either FGFR3 or PIK3CA in half of the cases, and remains of unknown genetic origin in the other half. EN is also seen as part of complex developmental disorders or in association with bladder carcinomas, also related to FGFR3 and PIK3CA mutations. Mosaic mutations of these genes have been occasionally found in syndromic EN.
TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR?=?0.25 [0.18-0.37], p?=?0.0001) or for pT1 tumours alone (OR?=?0.47 [0.28-0.79], p?=?0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR?=?0.56 [0.23-1.36] (p?=?0.12) and OR?=?0.99 [0.37-2.7] (p?=?0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.
The gene cyclin-dependent kinase inhibitor 2A (CDKN2A) is frequently inactivated by deletion in bladder carcinoma. However, its role in bladder tumourigenesis remains unclear. We investigated the role of CDKN2A deletion in urothelial carcinogenesis, as a function of FGFR3 mutation status, a marker for one of the two pathways of bladder tumour progression, the Ta pathway. We studied 288 bladder carcinomas: 177 non-muscle-invasive (123 Ta, 54 T1) and 111 muscle-invasive (T2-4) tumours. CDKN2A copy number was determined by multiplex ligation-dependent probe amplification, and FGFR3 mutations by SNaPshot analysis. FGFR3 mutation was detected in 124 tumours (43.1%) and CDKN2A homozygous deletion in 56 tumours (19.4%). CDKN2A homozygous deletion was significantly more frequent in FGFR3-mutated tumours than in wild-type FGFR3 tumours (p = 0.0015). This event was associated with muscle-invasive tumours within the FGFR3-mutated subgroup (p < 0.0001) but not in wild-type FGFR3 tumours. Similar findings were obtained for an independent series of 101 bladder carcinomas. The impact of CDKN2A deletions on recurrence-free and progression-free survival was then analysed in 89 patients with non-muscle-invasive FGFR3-mutated tumours. Kaplan-Meier survival analysis showed that CDKN2A losses (hemizygous and homozygous) were associated with progression (p = 0.0002), but not with recurrence, in these tumours. Multivariate Cox regression analysis identified CDKN2A loss as a predictor of progression independent of stage and grade. These findings highlight the crucial role of CDKN2A loss in the progression of non-muscle-invasive FGFR3-mutated bladder carcinomas and provide a potentially useful clinical marker for adapting the treatment of such tumours, which account for about 50% of cases at initial clinical presentation.
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