LAUSR

Spinal cord (7, 15, 16) and brainstem (1, 2, 12, 14) diffuse midline gliomas have been shown to have H3F3A (K27M) mutations, which are paralleled by presence of strong, diffuse nuclear immunoreactivity using a high fidelity immunohistochemistry (IHC) stain (17). Although a World Health Organization (WHO) grade of IV has been assigned to diffuse midline glioma, H3 K27Mmutant (8), it is becoming apparent that rarely low grade tumors, including pilocytic astrocytoma, may also show this mutation (9). However, in the latter case, despite the long patient survival, the tumor eventually progressed and caused demise (9). This suggests that the presence of mutation may portend adverse prognosis in any tumor type in which it occurs. H3 K27M mutation appears to be very rare in pilocytic astrocytomas in general. K27M was reported to be negative/absent in 15 of 15 midline pilocytic astrocytomas (by IHC) (16), 40 of 40 pilocytic astrocytomas (by IHC, from unspecified anatomical locations) (17), and 15 of 15 pilocytic and 5 of 5 pilomyxoid astrocytomas (by pyrosequencing, from unspecified anatomical locations) (6). However, one high grade glioma positive for K27M IHC has been reported that contained a prominent pilomyxoid component (16). Gangliogliomas (GGs) are another tumor type that usually lack H3 K27M mutation. Gielen et al in a large survey of 163 pediatric tumors of all types showed that the majority of tumor types from unspecified anatomical locations lack mutation (by pyrosequencing), including 10 of 10 GGs (6). Venneti et al demonstrated that 17 of 17 pediatric and 2 of 2 adult GGs were negative for mutation (IHC and sequencing), all WHO grade I (17). Anatomical location was not specified, however, and thus it is unclear how many midline examples were included. Nevertheless, similar to the situation with pilocytic astrocytoma, exceptional reports of GGs positive for H3 K27M IHC are starting to appear. These include two spinal cord anaplastic GGs (6), one malignant glioma with epithelioid and rhabdoid features as well as ganglionic differentiation (16), one thalamic GG initially grade I and then recurrent as anaplastic grade III 7 years later (11), and one cerebellar GG, initially WHO grade I followed by rapid progression and anaplastic GG 2 months later (11). Similar to the above-cited pilocytic astrocytoma with mutation (9), the two patients with progression suffered demise at 8 years, 2 months and 33 months after diagnosis (11). Given our previous experience with a cohort of pediatric patients with brainstem GGs on whom we had testing for BRAF V600E mutational status and clinical follow-up (3), as well as with two adult patients with spinal cord GGs who succumbed and had massive metastatic tumor dissemination at autopsy (13), we reinterrogated these cases for the H3K27M mutation, using the highfidelity antibody (Millipore, Temecula, CA) (17). We also assessed three additional unpublished midline pediatric GGs with autopsy documentation of metastatic disease, as well as several surviving adult patients with midline GGs. Given the known difficulty of diagnosis of GG on small biopsies, especially from brainstem and spinal cord locations, and the inability to utilize IDH1 R132H immunohistochemistry to distinguish diffuse infiltrating gliomas with entrapped non-neoplastic ganglion cells from true GGs in these sites (5, 10), only definitive GG examples were utilized for study. Previously published cases had been well-documented histologically (3, 13). Details of patient age, tumor anatomical location, and results for H3 K27M IHC, BRAF V600E mutation via Sanger sequencing, BRAF VE1 IHC and ATRX IHC are documented in the Table 1. Four of the five autopsied cases had disseminated metastatic disease, including the two previously published adult cases (13). The other three autopsied cases in our study were all from pediatric patients and had not been previously published. Patient 7 was a child who presented with an extensive cervical and upper thoracic cord tumor (Figure 1a) that intraoperatively manifested as a bulging, hyperemic intrinsic mass (Figure 1b). Extensive resection was diagnosed as GG, WHO grade I, based on the presence of numerous neoplastic ganglion cells (Figure 1b), binucleate tumor ganglion cells (Figure 1c), multifocal calcifications (Figure 1d, arrowheads), near-absence of mitotic activity and low MIB-1 cell cycle labeling, as performed on three different blocks. (See also Supporting Information File 1 for more images of this case, including prominent calcifications.) The patient had local tumor progression and, despite chemoradiation, developed multifocal metastases to bilateral frontal horns, septum pellucidum and left occipital horn 11 months after initial diagnosis. Biopsy from one of her ventricular metastases now showed anaplastic transformation of the GG. Death occurred 9 months later, at which time autopsy documented involvement of C6-T2, as well as the extensive cerebral intraventricular metastatic deposits. Both metastatic cerebral deposits and recurrent spinal cord disease showed anaplastic transformation of the GG, with palisading necrosis. Initial spinal cord resection specimen, biopsy sample from the intraventricular metastasis, and an autopsy sample from her central nervous system metastasis were all positive for H3 K27M (see Table 1) (Figure 1d, initial biopsy illustrated, arrowheads highlighting multifocal calcifications). Testing of tissue specimens taken at three different time points, all positive, indicates neither gain nor loss of mutation over the course of disease. While double labeling with immunostains for H3 K27M and neuronal markers was not performed, it was felt that both neoplastic ganglion cells and neoplastic glial cells were positive for the mutation. Patient 8 was a child who presented with a pontine mass (Figure 1e) that was diagnosed on stereotactic biopsy as a GG with numerous neoplastic ganglion cells (Figure 1f, arrowhead) and scattered basophilic calcifications (Figure 1f). The tumor showed no mitotic activity, only rare cells with MIB-1 cell cycle labeling, and was diagnosed as GG, WHO grade I (see also Supporting Information File 2 for more images of this case). Both neoplastic ganglion cells