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. Author manuscript; available in PMC: 2022 Nov 18.
Published in final edited form as: Nat Med. 2019 Mar 4;25(4):597–602. doi: 10.1038/s41591-019-0373-y

Figure 3. Recurrent MAP3K8 fusions/truncations in a spitzoid melanoma cohort.

Figure 3.

A) Tile plot showing fusions, truncations and hotspot mutations in 49 spitzoid melanomas/atypical Spitz tumors. Each patient is a single column of the grid. Fusions are shown in purple, truncations in black, hotspot mutations in green, and no mutation detected in grey. MAP3K8 fusions and truncations were the most common event in the cohort, found in 33% of cases. B) Schematic representation of MAP3K8 fusions and truncations detected by RNA-Seq. The RNA-Seq fusion point and partner genes are shown as a black/white lollypop plot. All fusions joined exon 8 of MAP3K8 to a partner locus and removed autoinhibitory exon 9. C) Representative FISH and IHC images from MAP3K8 wildtype, fused and truncated samples. Break apart FISH (column 1) confirms the translocation in two samples with the 5’ of MAP3K8 shown in green and the 3’ shown in red. All FISH images are at 100X magnification; at least 100 nuclei were analyzed across multiple fields in each tumor section; MAP3K8 rearrangements predicted by RNA-Seq were confirmed in 13/17 samples; no MAP3K8 rearrangements were observed in 11 control samples. Immunohistochemistry shows MAP3K8 and Phospho-MEK1/2 expression in the tumors. IHC staining for phospho MEK1/2 was done in 48 tumor specimens: the majority of tumor cells expressed pMEK1/2. The intensity of expression was weak in 13 tumors and moderate in 35 tumors. IHC staining for MAP3K8 was done in 45 tumor specimens. In the majority of tissue specimens (42 of 45), the antibody stained the cytoplasm of tumor cells with moderate to strong intensity compared to the adjacent squamous cells that were stained weakly or negatively. All IHC images at 40X magnification. D) Results from colony forming assays in soft agar with NIH 3T3 cells transfected, with empty vector, truncated MAP3K8 and mutant NRAS. Bar height shows the number of colonies normalized to the empty vector control for two replicates (blue and orange). Underlying colony counts for replicate one (blue) are vector=46, truncated MAP3K8=66, NRAS=126 and replicate two (orange) are 146, 226 and 424, respectively.