Lps 019 a

To validate MAP3K8 gene rearrangements, we performed MAP3K8 break-apart assay in all FFPE tissue samples found by RNA-Seq to carry the gene fusion. A subset of samples with alternative alterations were also included as negative controls. In addition, we performed break-apart PRKCA assay in samples found by RNA-Seq to have PRKCA gene fusion, and break-apart TERT promoter assay in both the primary biopsy and the metastatic tumor of the index case. Custom probes were generated, as previously described ⁴¹ , using bacterial artificial chromosomes (BAC) clones, obtained from BACPAC Resources of Children’s Hospital of Oakland Research Institute (Oakland, CA, USA), flanking MAP3K8 (CH17–364M4; CH17–471D1), PRKCA (CH17–384F08 and CH17–209D17), and the TERT promoter (CH17–75N21 and CH17–410B01). Break-apart FISH for ALK was performed on samples found by RNA-Seq to have ALK fusion, using commercially available probes (CytoCell, Cat# LPS 019-A). Dual-color FISH was performed on 4-μm FFPE tissue sections, as previously described ^{1 (link)}. FISH images were captured using CytoVision 7.5 software.

BAC clones (BACPAC Resources) were used to develop break-apart probes for the following genes: BRAF (RP11-837G3, RP11-948O19), NTRK1 (CH17-67O18, RP11-1038N13), PTPRZ1 (CH17-132B19, RP11-99L10), IL6R (CH17-169C19, RP11-627K14), TPM3 (CH17-317C21, CH17-169C19), EML4 (CH17-315G08, RP11-885P15), ARID1B (RP11-230C9, CH17-280H05), and BAIAP2L1 (RP11-958G24, CH17-112O19). Break-apart FISH for ALK was performed by using a commercially available probe set (CytoCell, Cat# LPS 019-A). In addition, BAC clones (CH17-132B19, RP11-99L10 and CH17-57M15, CH17-240N01) were used to develop a fusion probe set for PTPRZ1–NFAM1. Dual-color FISH was performed on 4-µm FFPE sections, as previously described.^{20 (link)}