Table 1.
Patient characteristics.
Table 2.
Primer sequences.
Fig 1.
Screening for patient samples harboring the KIAA-BRAF fusion gene and breakpoint detection.
(A) RT-PCR was performed to detect samples harboring the KIAA-BRAF fusion gene. cDNA of U87 and water were the negative controls. Among the six patient samples, S1, S4, and S6 expressed the KIAA1549 exon15, BRAF exon 9 fusion gene (left). None of the six samples manifested the KIAA1549 exon16, BRAF exon 9 fusion gene (right). (B) Sanger sequencing of S1, S4, and S6 revealed the KIAA1549 exon15, BRAF exon 9 fusion junctions. (C) LA-PCR assay of genomic DNA performed to obtain long amplified products including breakpoints. The genome of U87 and water were the negative controls. S1, S4, and S6 contained amplified products around 7 kb in length. (D) Breakpoints in S1, S4, and S6 detected by Sanger sequencing. Although the fusion pattern was the same, the breakpoints were different. For details, see S8 Fig.
Fig 2.
Histopathological findings of MVP in PA.
H & E staining: MVP exhibiting a glomerular structure. CD34: Inner layer of MVP was stained against this marker. α-SMA: Cellular components of MVP were stained diffusely against this marker. GFAP and Olig2: No cellular components of MVP were stained against these markers. Original magnification: 20x. Scale bar: 100 μm.
Fig 3.
Evaluation of the KIAA-BRAF fusion gene in cellular components of MVP by genomic PCR.
(A) Cellular components of MVP were collected from FFPE slides with laser microdissection (arrows). (B) Special primers designed for around breakpoints. (C) PCR analysis with the special primers was performed for fusion gene detection. Samples S1 and S6 exhibited a positive band for tumor and for cellular components of MVP. (D) Sanger sequencing was performed to confirm the amplified products. In S1 and S6, tumor cells and cellular components of MVP exhibited the same base sequence, including breakpoints.
Fig 4.
Digital PCR for quantitative fusion gene evaluation.
(A) Raw digital PCR data for S1. RNase-P was employed as an internal control. Red dots showed droplets with RNase-P DNA and blue dots with the KIAA-BRAF fusion gene. Yellow dots showed droplets with no DNA. (B) The relative expression of the KIAA-BRAF fusion gene in tumor cells and cellular components of MVP is shown. The ratio of cells harboring the KIAA-BRAF fusion gene (cellular components of MVP vs. tumor cells) was 42% in S1 and 76% in S6. The results are expressed as the mean +- S.E.M., n = 3.
Fig 5.
FISH analysis for the detection of the KIAA-BRAF fusion gene.
(A) FISH probes at the duplication site of the BRAF gene (7q34) are labeled with Texas red. The centromere of chromosome 7q (7q11.21) is labeled with fluorescein isothiocyanate (FITC, green). Due to tandem duplication at 7q34 leading to the expression of the KIAA-BRAF fusion gene, cells with the fusion gene demonstrated gain of a red signal (total: three signals) in comparison with the green signal (two signals). (B) In both S1 and S6, tumor cells and cellular components of MVP were positive for the KIAA-BRAF fusion gene.