Fig. 3

Immunohistochemical findings and molecular analyses of the salivary gland tumor. a C-WT1 shows nuclear positivity (scale bar: 50 μm). b N-WT1 (WT49) shows nuclear and cytoplasmic negativity. c N-WT1(6F-H2) shows cytoplasmic positivity. d FISH analysis using a break-apart probe for the EWSR1 gene region demonstrates the rearrangement in most of the cells. e The 3′/5′ expression imbalance assay based on RT-PCR reveals that the tumor lacked 5′-regional expression of the WT1 gene. The primers were designed to measure the expressions at two regions for each gene transcript: a 5′ probe pair located far upstream of the exons and a second pair located within the exons located further 3′ in the WT1 gene. PCR analysis was performed using these 5′ and 3′ primers, respectively. The Ct data were normalized to wild-type control tissue, and the normalized data was expressed as the relative gene expression level. WT cont; wild-type control (ovarian serous carcinoma), MT cont; mutant type control (typical DSRCT). f RT-PCR analysis showed that the EWS-WT1 fusion gene was present in the sample. M; marker, WT cont and MT cont; same as above