Prevalence and co-occurrence of actionable genomic alterations in high-grade bladder cancer

Abstract

Purpose: We sought to define the prevalence and co-occurrence of actionable genomic alterations in patients with high-grade bladder cancer to serve as a platform for therapeutic drug discovery.

Patients and methods: An integrative analysis of 97 high-grade bladder tumors was conducted to identify actionable drug targets, which are defined as genomic alterations that have been clinically validated in another cancer type (eg, BRAF mutation) or alterations for which a selective inhibitor of the target or pathway is under clinical investigation. DNA copy number alterations (CNAs) were defined by using array comparative genomic hybridization. Mutation profiling was performed by using both mass spectroscopy-based genotyping and Sanger sequencing.

Results: Sixty-one percent of tumors harbored potentially actionable genomic alterations. A core pathway analysis of the integrated data set revealed a nonoverlapping pattern of mutations in the RTK-RAS-RAF and phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathways and regulators of G1-S cell cycle progression. Unsupervised clustering of CNAs defined two distinct classes of bladder tumors that differed in the degree of their CNA burden. Integration of mutation and copy number analyses revealed that mutations in TP53 and RB1 were significantly more common in tumors with a high CNA burden (P < .001 and P < .003, respectively).

Conclusion: High-grade bladder cancer possesses substantial genomic heterogeneity. The majority of tumors harbor potentially tractable genomic alterations that may predict for response to target-selective agents. Given the genomic diversity of bladder cancers, optimal development of target-specific agents will require pretreatment genomic characterization.

Conflict of interest statement

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

Figures

Fig 1.

Landscape of DNA copy number alterations (CNAs) in high-grade bladder cancer. (A) Unsupervised hierarchical clustering of array comparative genomic hybridization data identified two distinct classes of bladder cancers. TP53 and RB1 alterations were significantly more common in the high copy number aberrant subset. (B) Fraction of the genome altered in the subsets of bladder tumors with high and low copy number aberrations and in additional select human cancers. The cohort of copy number data used for comparison was derived from multiple large tumor collections analyzed by array comparative genomic hybridization (both published and unpublished data) and includes The Cancer Genome Atlas tumor types with more than 100 available samples. It represents a composite, unbiased data set for comparing global copy number changes across myriad tumor subtypes (see Data Supplement for list of references from which these data were derived). (C) Statistically significant genomic amplifications (red) and deletions (blue) inferred from RAE analysis are indicated across the autosomes. Select genes located within recurrently amplified or deleted regions are highlighted.

Fig 2.

Co-occurrence of alterations within the RTK/RAS/RAF signaling pathway in high-grade bladder cancer. (A) Incidence (%) of amplifications, deletions, and mutations of select receptor tyrosine kinases and downstream targets. The heatmap compares the distribution of each alteration across tumor samples (bottom). (B) ERBB2-amplified samples are highlighted (left), and ERBB2 transcript expression levels as a function of ERBB2 gene copy number are indicated (right). ERBB2-amplified samples also exhibited significantly increased messenger RNA (mRNA) expression levels as compared with diploid samples. (C) Immunohistochemistry analysis of a representative ERBB2-amplified tumor sample exhibiting 3+ human epidermal growth factor receptor 2 (HER2) overexpression and an ERBB2-nonamplified tumor sample shown for comparison (right). Ampl., amplified; H&E, hematoxylin and eosin; Hetloss, heterozygous loss.

Fig 3.

Alterations within the TP53 and RB1/E2F3 pathways in high-grade bladder cancer. (A) Heatmap showing the distribution of TP53 and MDM2 alterations. (B) The RB1 cell cycle regulatory pathway with incidence of mutations and copy number alterations displayed for each gene along with a heatmap showing the co-occurrence of genetic alterations in this pathway. The boxplot (right) depicts the association between an E2F3 expression signature score and alterations within genes involved in the RB1 pathway (“multiple” refers to more than one gene in this pathway altered in the same tumor sample). Those samples harboring E2F3 amplification or amplification of multiple genes within the RB1 pathway display significantly increased E2F3 activity scores compared with tumors lacking amplification of any component of the pathway (labeled as “none”). P values were calculated by t test.

Fig 4.

Alterations within the phosphoinositide 3-kinase (PI3K)/AKT pathway in high-grade bladder cancer. (A) Key components of the PI3K/AKT signaling pathway displayed with their incidence of mutations and copy number abnormalities. The corresponding heatmap shows the distribution of pathway alterations across the tumor cohort. The mRNA expression score for PTEN-deleted samples is shown to the right of the heatmap. P value comparing expression scores between samples harboring homozygous deletion (homdel) versus diploid samples calculated by t test. (B) IC50 (concentration that inhibits 50%) values for a panel of urothelial cell lines with distinct PI3K/AKT pathway alterations (as annotated) as well as one cell line with an HRAS mutation. Each cell line was exposed to increasing concentrations of MK2206 and harvested 5 days after addition of drug. Cell viability was measured using trypan blue exclusion. (C) Immunoblot analysis of PI3K/AKT pathway downstream targets after exposure to MK2206 in the MGH-U4 (PIK3CA H1047R) and the HCV-29 (TSC1 Q55*) cell lines. Cell were harvested at 0, 1, 6, and 24 hours after addition of drug. Hetloss, heterozygous loss.