Andrew M. Donson

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

Claudin 6 Is a Positive Marker for Atypical Teratoid/Rhabdoid Tumors

Atypical teratoid/rhabdoid tumors (AT/RTs) are highly aggressive pediatric brain tumors characterized by the presence of rhabdoid cells and negative immunostaining for INI1 (BAF47). Histogenesis is unknown and diagnosis can be challenging because of their extreme morphological and immunophenotypic heterogeneity. Currently no signature markers other than INI1 loss have been identified. To search for possible candidate proteins of interest in AT/RTs, Affymetrix GeneChip® microarrays were utilized to investigate nine AT/RTs vs. 124 other tumor samples. The most distinctive gene identified was claudin 6 (CLDN6), a key component of tight junctions. CLDN6 showed moderate or higher mRNA expression in eight of nine AT/RTs, with little to no expression in 114 of 115 other tumors. Average expression was 38‐fold higher in AT/RTs vs. other samples. Immunohistochemical (IHC) staining of 33 tumor specimens found positive membrane staining in seven of seven AT/RTs, and was negative in 26 of 27 other brain tumor samples. Notably, none of the 16 medulloblastomas/primitive neuroectodermal tumors showed IHC staining for CLDN6. IHC staining results closely matched the level of mRNA expression detected by microarray. CLDN6 may be a useful positive marker to help further identify AT/RTs for diagnostic and treatment purposes.

Pediatric brainstem gangliogliomas show BRAFV600E mutation in a high percentage of cases

Brainstem gangliogliomas (GGs), often cannot be resected, have a much poorer prognosis than those located in more common supratentorial sites and may benefit from novel therapeutic approaches. Therapeutically targetable BRAF c.1799T>A (p.V600E) (BRAFV600E) mutations are harbored in roughly 50% of collective GGs taken from all anatomical sites. Large numbers of pediatric brainstem GGs, however, have not been specifically assessed and anatomic—and age‐restricted assessment of genetic and biological factors are becoming increasingly important. Pediatric brainstem GGs (n = 13), non‐brainstem GGs (n = 11) and brainstem pilocytic astrocytomas (PAs) (n = 8) were screened by standard Sanger DNA sequencing of BRAF exon 15. Five of 13 (38%) pediatric GG harbored a definitive BRAFV600E mutation, with two others exhibiting an equivocal result by this method. BRAFV600E was also seen in five of 11 (45%) non‐brainstem GGs and one of eight (13%) brainstem PAs. VE1 immunostaining for BRAFV600E showed concordance with sequencing in nine of nine brainstem GGs including the two cases equivocal by Sanger. The equivocal brainstem GGs were subsequently shown to harbor BRAFV600E using a novel, more sensitive, RNA‐sequencing approach, yielding a final BRAFV600E mutation frequency of 54% (seven of 13) in brainstem GGs. BRAFV600E‐targeted therapeutics should be a consideration for the high percentage of pediatric brainstem GGs refractory to conventional therapies.

Unique Molecular Characteristics of Pediatric Myxopapillary Ependymoma

Myxopapillary ependymoma (MEPN) generally can be cured by gross total surgical resection and usually manifest a favorable prognosis. However, surgery is less curative in tumors that are large, multifocal or extend outside the thecal sac. Late recurrences may occur, particularly in pediatric patients. The role of adjuvant therapy is unclear in the clinical management of recurrent tumors. Clinical trial design requires a better understanding of tumor biology. Unique molecular features of MEPN were investigated by using microarray technology to compare the gene expression of five pediatric MEPN to 24 pediatric intracranial ependymoma (EPN). The upregulation of three genes of interest, homeobox B13 (HOXB13), neurofilament, light polypeptide (NEFL) and PDGFRα, was further studied by immunohistochemistry in a larger cohort that included adult MEPN and EPN specimens. Protein expression in MEPN was compared to subependymoma, spinal EPN, intracranial EPN and normal fetal and adult ependyma. Immunoreactivity for HOXB13, NEFL and PDGFRα was strongest in MEPN and virtually absent in subependymoma. Spinal and intracranial EPN generally expressed weak or focal staining. MEPN manifests unique gene and protein expression patterns compared to other EPNs. Aberrant expression of HOXB13 suggests possible recapitulation of developmental pathways in MEPN tumorigenesis. PDGFRα may be a potential therapeutic target in recurrent MEPN.

Pediatric brainstem gangliogliomas show overexpression of neuropeptide prepronociceptin (PNOC) by microarray and immunohistochemistry.

Gangliogliomas (GGs) primary to brainstem are rare, with the overwhelming majority of GGs occurring in supratentorial, especially temporal lobe, locations. A less favorable prognosis exists for brainstem GGs, despite their usually identical WHO grade I status. Few large clinical series, and limited biological information, exists on these tumors, especially gene expression.

H3 K27M mutation in gangliogliomas can be associated with poor prognosis

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

SOX10 Distinguishes Pilocytic and Pilomyxoid Astrocytomas From Ependymomas but Shows No Differences in Expression Level in Ependymomas From Infants Versus Older Children or Among Molecular Subgroups.

SOX10 is important in nonneoplastic oligodendroglial development, but mRNA transcripts and protein expression are identified in a wider variety of CNS glial neoplasms than oligodendrogliomas. We previously demonstrated high levels of SOX10 mRNA and protein in pilocytic astrocytomas (PAs) but not ependymomas (EPNs). We now extend these studies to investigate subsets of these 2 tumors that affect infants, pilomyxoid astrocytomas (PMAs) and infant (<1 year) ependymomas (iEPNs). By gene expression microarray analysis, we found that iEPNs and all EPNs in older children showed very low SOX10 expression levels, on average 7.1-fold below normal control tissues. EPN groups showed no significant difference in SOX10 expression between iEPN and EPN. PAs/PMAs had 24.1/29.4-fold higher transcript levels, respectively, than those in normal tissues. Using immunohistochemical analysis of adult, pediatric, and infantile EPNs and of PAs/PMAs, we found that EPNs from multiple anatomical locations and both age groups (n = 228) never showed 3+ diffuse nuclear immunostaining for SOX10; the majority were scored at 0 or 1+. Conversely, almost all pediatric and adult PAs and PMAs (n = 47) were scored as 3+. These results suggest that in select settings, SOX10 immunohistochemistry can supplement the diagnosis of PMA and PA and aid in distinguishing them from EPNs.

Desmoplastic infantile astrocytoma/ganglioglioma with rare BRAF V600D mutation

Desmoplastic infantile astrocytoma (DIA) and desmoplastic infantile gangliogliomas (DIGs) are rare, massive, cystic and solid tumors of infants usually found in superficial cerebral hemispheres. They manifest prominent desmoplastic stroma, admixed neoplastic astrocytes, primitive‐appearing small cells, and additional neoplastic ganglion cells in the case of DIGs. While v‐Raf murine sarcoma viral oncogene homolog B (BRAF) mutation is found in up to 50% of pediatric gangliogliomas, two recent studies found that it was rare in DIA/DIGs; we sought to assess BRAF status in DIA/DIGs from our institution.