Figure 2. Novel BRAF exon 15 mutations in BRAF V600E-negative MA.

(A,D,G,J) H&E, (B,E,H,K) BRAF V600E IHC, and (C,F,I,L) BRAF exon 15 genomic sequencing of four MA cases. All four cases show the typical histomorphologic features of MA but demonstrate negative to weak cytoplasmic and/or nuclear staining. Adjacent non-neoplastic kidney parenchyma shows negative to weak cytoplasmic staining and weak to moderate nuclear staining. For one case, Sanger sequencing of BRAF exon 15 revealed no mutations (wild type sequence at codons 600 and 601 shown in panel C). Two additional cases of V600E-negative MA showed novel BRAF exon 15 mutations by Sanger sequencing. For one case, compound substitution of adenine and thymidine at codon 600 for thymidine and guanine (arrowheads in F) results in the missense BRAF V600D mutation; for the other case, a complex compound substitution and/or indel at codons 600 and 601 (arrowheads in I) results in the compound missense BRAF mutations V600D and K601L. (These novel BRAF exon 15 mutations were subsequently confirmed by Sanger sequencing of the subcloned PCR product.) Finally, for one case, Sanger sequencing demonstrated a thymidine to adenine substitution at codon 600 (arrowhead in L), corresponding to a BRAF V600E missense mutation; this is the only instance of discordance between V600E mutation-specific immunohistochemistry and Sanger sequencing. Magnification: (A,D,G,J) 20X; and, (B,E,H,K) 40X.