Summary of mutations found in the <i>ARID1A</i> resequencing study.

<p>Non-synonymous mutations found at a frequency ≥10% that were confirmed by Sanger sequencing are shown here.</p>*<p>denotes a truncating mutation.</p

<i>ARID1A</i> mutations and expression in UBC.

<p><i>Panel A</i>. A G>C transversion identified through Solexa resequencing, confirmed by Sanger sequencing of independent PCR products, leading to a predicted Q2210H substitution in VMCUB-3 cells. <i>Panel B</i>. Western blotting analysis in a panel of UBC cell lines identifies a subset with undetectable expression, including VMCUB-3. mRNA expression was analyzed by RT-qPCR; results are shown as values normalized with respect to the housekeeping gene <i>HPRT</i>. <i>Panel C</i>. A C>T mutation in codon 403, leading to a premature stop codon, was identified in a primary T1G3 tumor. The mutation was absent from matched normal leukocyte DNA. Lack of protein expression in the corresponding tumor tissue was confirmed using immunohistochemistry. The red arrowhead points to a tumor cell lacking ARID1A staining, whereas the black arrowhead indicates a positive stromal cell. For comparison, a TaG1 tumor with wild type <i>ARID1A</i> sequence is shown.</p

Loss of ARID1A expression is associated with more aggressive UBC.

<p>UBC cases were classified in three categories: low grade NMI (TaG1 and TaG2 tumors), high grade NMI (TaG3 and T1G3 tumors), and MI (≥T2 tumors). <i>Panel A</i>. ARID1A immunohistochemical score is significantly lower in more aggressive, advanced tumors. FGFR3 immunohistochemical score, which is directly associated with <i>FGFR3</i> mutations, is also significantly lower in more aggressive tumors. By contrast, p53 score is higher in more aggressive tumors. <i>Panel B</i>. Differential expression of <i>ARID1A, FGFR3</i> and <i>TP53</i> at the mRNA level is observed in two different, independent UBC microarray series: the mRNA levels of all 3 genes are significantly lower in MIBC. *denotes an FDR adjusted <i>P</i>-value <0.5.</p

Loss of ARID1A expression is associated with more aggressive UBC and with patient outcome.

<p>ARID1A expression was assessed by IHC on tissue microarrays. Patients (n = 84) were followed-up as indicated in Methods and classified as having “recurred”, “progressed”, or being free of disease. Patients with high ARID1A-expresssing tumors display a lower risk of recurrence and a higher risk of progression indicating a more aggressive clinical course.</p

Characteristics of the patients and tumors included in series 2 (tissue microarray).

<p>Characteristics of the patients and tumors included in series 2 (tissue microarray).</p

Characteristics of the patients from whom fresh tumor was used for <i>ARID1A</i> sequence analysis.

<p>Characteristics of the patients from whom fresh tumor was used for <i>ARID1A</i> sequence analysis.</p

Relationship between ARID1A and cell differentiation markers, as detected using immunohistochemistry in tumor tissue microarrays.

<p>UBC cases were classified in three categories: LG-NMIBC (TaG1 and TaG2 tumors), HG-NMIBC (TaG3 and T1G3 tumors), and MI (≥T2 tumors). Non-hierarchical clustering of IHC scores for ARID1A, FGFR3, KRT5/6, KRT14, and KRT20 was performed. IHC scores are shown in a green-red color code. Color bars below the dendogram include information about tumor stage and grade (tones of blue) and <i>FGFR3</i> mutational status (grey/black) when known. White squares indicate that information for that parameter is not available.</p

Effects of <i>ARID1A</i> knockdown in UBC cell lines.

<p><i>Panel A</i>. <i>ARID1A</i> was knocked-down using three different shRNAs in the RT112 and VMCUB-3 cells. The knock-down was efficient at both the protein and mRNA levels. The bars represent the relative quantification of ARID1A mRNA levels taking non-targeting shRNA interfered cells as controls. <i>Panel B</i>. The quantification colony formation is shown, with error intervals of results from triplicate experiments denoting SEM. In RT112 cells, ARID1A knockdown was associated with reduced colony formation. By contrast, no major effects were observed in VMCUB-3 cells harboring an <i>ARID1A</i> mutation. Representative morphological changes in cells interfered with control shNT (scrambled shRNA) and with one of the shRNAs targeting <i>ARID1A</i> are shown.</p