SWI/SNF Alterations in Squamous Bladder Cancers

Abstract

Dysfunction of the SWI/SNF complex has been observed in various cancers including urothelial carcinomas. However, the clinical impact of the SWI/SNF complex in squamous-differentiated bladder cancers (sq-BLCA) remains unclear. Therefore, we aimed to analyze potential expression loss and genetic alterations of (putative) key components of the SWI/SNF complex considering the co-occurrence of genetic driver mutations and PD-L1 expression as indicators for therapeutic implications. Assessment of ARID1A, SMARCA2, SMARCA4, SMARCB1/INI1, SMARCC1, SMARCC2 and PBRM1 mutations in a TCGA data set of sq-BLCA (n = 45) revealed that ARID1A was the most frequently altered SWI/SNF gene (15%) while being associated with protein downregulation. Genetic alterations and loss of ARID1A were confirmed by Targeted Next Generation Sequencing (NGS) (3/6) and immunohistochemistry (6/116). Correlation with further mutational data and PD-L1 expression revealed co-occurrence of ARID1A loss and TP53 mutations, while positive correlations with other driver mutations such as PIK3CA were not observed. Finally, a rare number of sq-BLCA samples were characterized by both ARID1A protein loss and strong PD-L1 expression suggesting a putative benefit upon immune checkpoint inhibitor therapy. Hence, for the first time, our data revealed expression loss of SWI/SNF subunits in sq-BLCA, highlighting ARID1A as a putative target of a small subgroup of patients eligible for novel therapeutic strategies.

Keywords: ARID1A; SWI/SNF complex; immune checkpoint inhibitors; squamous bladder cancer.

Figure 1

SWI/SNF alterations and ARID1A protein expression in the sq-BLCA cohort of the TCGA network. (A) Genetic alterations of different subunits of the SWI/SNF complex (including missense mutations, truncating mutations, amplifications and deep deletions). (B) Mutation Mapper illustrating positions of identified ARID1A alterations (nonsense and missense) relative to the protein sequence and domains (green and red box). Protein domains are indicated according to PFAM: green box: ARID domain; red box: BAF250_C domain. (C) Mutational frequencies of analyzed subunits of the SWI/SNF complex. (D) Box plots illustrating ARID1A protein expression classified by ARID1A mutations. BAF (dark blue): BRG1/BRM-associated factor; PBAF (light blue): polybromo-associated BAF; MUT: mutated; WT: wildtype. * p < 0.05.

Figure 2

Protein expression of subunits of the SWI/SNF complexes BAF and PBAF in squamous-differentiated bladder cancers (sq-BLCA). (A) H&E and immunohistochemical staining of seven subunits of the SWI/SNF complex are shown for a representative tissue core with ARID1A mutation (c.4005-2A>G, p.(?)). Black scale bar: 100 µM. For further immunohistochemical ARID1A staining according to the range of Immune Reactive Scores (IRSs) see Supplementary Figure S3. (B) Box plot graphs show overall distribution of IRS staining results of subunits for urothelial cancers with squamous components (MIX SCC) and pure squamous cancers (SCC). The numeric values correspond to the median value. (C) Frequencies of expression loss (IRS ≤ 2) shown for all analyzed subunits. (D) Expression network illustrating the statistical correlation between analyzed subunits of BAF and PBAF. Only significant (p ≤ 0.05) spearman correlations (r-values as indicated) are shown. Dark blue: component of the BAF complex; light blue: component of the PBAF complex; * p < 0.05.

Figure 3

Expression loss of SWI/SNF subunits in sq-BLCA with known genetic driver mutations. (A) Upper lines: mutational spectrum of genes (TP53, FGFR3, PIK3CA, CDKN2A) potentially involved in bladder cancer development and progression (SCC: n = 30; MIX SCC: n = 36). TP53 was the most frequently mutated gene in pure SCC (9/29) as well as MIX-SCC (20/36), while FGFR3 (SCC: 4/29; MIX: 3/36) and CDKN2A (SCC: 4/29; MIX: 2/35) mutations were less abundant (SCC: 4/29; MIX: 3/36). PIK3CA driver mutations were observed in 6/29 (SCC) and in 6/36 (MIX) tumors. Lower lines: corresponding expression loss of subunits of the SWI/SNF complex. MUT: mutations; EXP: expression. (B) Overall mutational frequencies of analyzed driver genes.

Figure 4

ARID1A mutations in sq-BLCA. (A) Exemplary illustration of a nonsense ARID1A mutation with an allele frequency of 20% (c.1001C>T, p.Ser334Ter, estimated tumor cell content 40%). (B) Summarized ARID1A mutations identified in Sq-BLCA with clear expression loss (IRS < 2). Protein domains are indicated according to PFAM: green box: ARID domain; red box: BAF250_C domain.