Anaplastic pleomorphic xanthoastrocytoma (A-PXA, WHO grade III) is a newly defined entity with high-grade histopathologic features and a propensity for recurrence [6]. While PXA with low-grade histology (WHO grade II) can harbor a recurrent valine-to-glutamic acid (p.V600E) point mutation in BRAF in up to 78% of cases [6], the genomic drivers of A-PXA are poorly understood as the V600E mutation is absent in over half of A-PXAs [4]. Alterations reported to date in V600E-negative cases have included novel BRAF fusions and copy number alterations (Table 1).

Table 1 Genomic alterations in BRAF V600E-negative anaplastic pleomorphic xanthoastrocytoma
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The efficacy of therapeutic targeting oncogenically activated kinases in BRAF-mutant cancers depends on structural variations in the kinase domain. For example, the BRAF V600E mutation is often sensitive to kinase inhibitors such as vemurafenib, while β3-αC deletions and non-canonical BRAF mutations are often resistant to this small molecular inhibitor [2]. Therefore, from a therapeutic aspect, it is imperative to define the spectrum of BRAF alterations in these aggressive tumors. Here, we report two newly identified A-PXAs with activating mutations in the β3-αC loop of the BRAF kinase domain discovered through whole-exome, whole-genome, and transcriptome sequencing (Michigan Oncology Sequencing Project [MI-ONCOSEQ]) [8].

The first case is a 5-year-old male presenting with a large (11.7 × 7.3 cm) temporoparietal mass with subfalcine and uncal herniation (Fig. 1a). Molecular profiling revealed an oncogenic BRAF in-frame deletion (p.L485_P490delinsF) located adjacent to the β3-αC loop that results in a helix-constraining conformational change in the kinase domain. The second case is a 23-year-old male with a parietal ring-enhancing cystic mass. Sequencing revealed a novel 9 bp tandem duplication (p.V504_R506dup) in the β3-αC loop that results in a three codon in-frame insertion in the open reading frame (ORF) of BRAF [see Fig. 1a-d and Online Resource for details and representative images from both cases (Additional file 1)]. Consistently, both cases demonstrated MAPK activation with strong expression of phospho-ERK1/2 in tumor cells (Fig. 1g).

Fig. 1
figure 1

A-PXA with non-V600E activating mutations affecting the β3-αC loop in BRAF. Post-contrast T1-weighted coronal MR sequence showing a large space-occupying lesion with significant midline shift (a). Histologic sections from case #1 demonstrated pleomorphic giant cells (b), as well as pseudopalisading necrosis (c) and increased mitotic activity (d). Illustration of conformational changes of the β3 strand and αC helix in the kinase domain. The canonical BRAF V600E mutation results in monomeric activity and can accommodate the oncogenic BRAF inhibitor vemurafenib, which only binds when helix αC is “out” (e). In β3-αC deletion mutations, the β3-αC loop is shortened, effectively locking helix αC in the “in” position and conferring resistance to vemurafenib (f) (modified with permission from Trends in Cancer, 2 (12), Foster SA, Klijn C, Malek S, Tissue-Specific Mutations in BRAF and EGFR Necessitate Unique Therapeutic Approaches, p. 699–701, 2016, Supplemental Reference [2] [Online Resource]). MAPK signaling pathway activation was confirmed with immunohistochemistry with anti-phospho-p44/42 MAPK [Erk1/2] [Thr202/Tyr204] (g)

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Both of the mutations reported here affect the β3-αC loop in the kinase domain. To function properly, protein kinases must maintain a level of structural flexibility in order to switch between inactive and active states. This conformational change involves two regulatory regions in the catalytic domain: the activation segment and the αC-helix [2]. During this process, the αC-helix undergoes an “out” to “in” shift that facilitates interaction with the β3 strand and initiates catalysis [2] (Fig. 1e). Case #1 demonstrated a deletion mutation in the BRAF β3-αC loop that results in a shortened αC-helix that constrains the loop conformation to a constitutively kinase active “in” state. Similar “in” state activating alterations have been reported in other major signaling pathway kinases including HER2 and EGFR [2]. β3-αC deletion mutations render tumors resistant to small molecule inhibitors, such as vemurafenib, that bind to and inhibit kinases with an “out” conformation, but are ineffective against the “in” state [1, 2] (Fig. 1e, f). Case #2 contained a mutation in a structural element (R-spine) of the αC-helix [9]. Mutations in the R-spine have been shown to stabilize the active state and result in constitutive kinase activation [3]. However, the effect of this mutation on the conformational state of the kinase domain remains to be determined. Because RAF dimers are often formed in tumors with β3-αC kinase loop alterations, RAF dimer inhibition has been proposed as an alternative therapy for these genetic alterations [10].

Recent reports of clinical responses in V600E-mutated A-PXAs with BRAF “out” inhibitors [5, 7] have been encouraging. However, selection of effective targeted therapies requires a mechanistic understanding of oncogenic kinase activation in tumors. We present two A-PXAs that contain BRAF β3-αC loop alterations that may not be sensitive to traditional BRAF inhibitors. Therefore, treatment approaches for A-PXAs with or without V600E mutations may differ depending on the specific type of BRAF genetic alteration.