Congenital glioblastomas (cGBMs) are uncommon tumors presenting in early infancy, variably defined as diagnosed at birth or at age less than 3 months by strict criteria, or more loosely, as occurring in very young children less than 12 months of age. Previous studies have shown that cGBMs are histologically indistinguishable from GBMs in older children or adults, but may have a more favorable clinical outcome, suggesting biological differences between congenital versus other GBMs. Due to the infrequency of cGBMs, especially when employing strict inclusion criteria, molecular features have not been sufficiently explored.
Archer FusionPlex Solid Tumor Kit, Archer VariantPlex Solid Tumor Kit, Illumina RNAseq were utilized to study cGBMs seen at our institution since 2002. A strict definition for cGBM was utilized, with only infants less than age 3 months at clinical presentation sought for this study.
Of the 8 cGBM cases identified in our files, 7 had sufficient materials for molecular analyses, and 3 of 7 cases analyzed showed fusions of the ALK gene (involving MAP4, MZT2Bex2 and EML4 genes as fusion partners). One case showed ROS1 fusion. Somatic mutations in TSC22D1, BMG1 and DGCR6 were identified in 1 case. None of the cases showed alterations in IDH1/2, histone genes, or the TERT gene, alterations which can be associated with GBMs in older children or adults.
Our results show that cGBMs are genetically heterogeneous and biologically different from pediatric and adult GBMs. Identification of ALK and ROS1 raise the possibility of targeted therapy with FDA-approved targeted inhibitors.
Congenital glioblastomas (cGBMs), defined by presentation prenatally or in early infancy , are uncommon tumors with less than one hundred cases reported [2,3,4,5,6,7,8,9,10,11,12,13,14]. cGBMs differ from other pediatric brain tumors in several important respects. First, most cGBMs are supratentorial, in contrast to the infratentorial predilection of many other pediatric brain tumors such as ependymoma, medulloblastoma, diffuse midline glioma. Second, cGBMs respond favorably to surgery and chemotherapy and have a more favorable outcome as compared to pediatric and adult GBMs, leading to long-term survival (greater than 5 years) in a large number of patients [5, 7]. Third, cGBMs do not show the characteristic molecular alterations of either pediatric or adult gliomas in the limited studies published to date [5, 7, 15].
Previously, we reported clinicopathologic features of 5 patients with cGBM with favorable outcome following subtotal resection and moderate intensity chemotherapy, indicating biological differences compared to pediatric and adult glioblastoma . The wide availability of next generation sequencing (NGS) technologies encouraged us to further explore the molecular features of cGBM. We now present further molecular characterization and of our 5 previously-reported cases  with 3 additional cGBMs.
Cases were identified by electronic medical record search from 2002 to 2018 inclusive. Appropriate informed consent was obtained at the time of surgery and the study was approved by the University of Colorado Institutional Review Board (IRB protocol number 95-500). All cases were reviewed at the University of Colorado Children’s Hospital. Five out of 8 cases underwent surgical resection at our institution and 3 had received surgery at outside hospitals and patients and slides had been sent to us for consultation.
The 8 cases consisted of 4 males and 4 females. Given the known debate about the definition of congenital GBM, we used the definition of Solitaire and Krigman , who in 1964 provided one of the most stringent definitions of cGBM, defining three distinct categories of congenital brain tumor, namely: (1) definitely congenital with symptoms occurring at birth, (2) probably congenital with symptoms occurring within the first week of life, and (3) possibly congenital with symptoms occurring within the first few months of life. All of our cases presented within the first 3 months of life (range: 1–12 weeks) and hence corresponded to at least the “possibly congenital” category according to the Solitaire and Krigman classification . It is noteworthy that many researchers today use a less stringent cut off of presentation before 1 year of age for “congenital/infantile” gliomas [9, 15, 16].
Clinical features such as age at presentation, gender, location of tumor, tumor recurrence and survival duration were noted for each patient. All tumors occurred in the supratentorial compartment; 6 were hemispheric and 2 thalamic (Fig. 1a, b), neither of which showed H3 K27M mutation by immunohistochemical or molecular analysis. Clinical, radiologic and survival data are summarized in Table 1. Selected examples of imaging and histopathologic findings are shown in Fig. 1.
Mutational and fusion analyses were undertaken for 7 of 8 cases with available tissue, using Archer FusionPlex Solid Tumor Kit, Archer VariantPlex Solid Tumor Kit and Illumina RNA-seq as previously detailed . In short, a total of 53 genes (listed in Table 2), known to be involved in tumor-associated fusion events were examined using Anchored Multiplex PCR technology from ArcherDx (Boulder, CO). This assay utilizes a proprietary Anchored Multiplex PCR (AMP TM) based enrichment to detect all fusions associated with the genes in a single sequencing assay, without prior knowledge of fusion partners or breakpoints. The VariantPlex assay tests for mutations in 69 genes (Table 3) with known involvement in oncogenesis using AMP technology.
In addition to these CLIA-certified clinically-available panels, all cases also received research based RNA sequencing. Methods are briefly described here. RNA libraries were synthesized from PolyA-selected total RNA using the Nugen Universal Plus mRNA-Seq kit and sequenced using either HiSeq 4000 or NovaSeq 6000 (Illumina). Reads were aligned to the human genome assembly GRCh38 using GSNAP (Genomic Short-read Nucleotide Alignment Program, version 2014-12-17). Fusion events in RNAseq data were identified using EricScript fusion finding software . Somatic variants were isolated by filtering out germline from tumor variants that were identified using FreeBayes (v1.0.1-2-g0cb2697).
Three cases of the 7 evaluable cases exhibited ALK fusions (involving MAP4, MZT2Bex2 and EML4 genes as fusion partners, see Table 1). Fusion breakpoints are shown in Fig. 2. One case demonstrated a ROS1 fusion (clinical findings for the ROS1 fusion positive case are described in detail in an upcoming publication ). One additional case (case 3) harbored somatic mutations in TSC22D1 (c.2711A>C; p.G;m904Pro), BMS1 (c.2572C>G; p.Gln858Glu) and DGCR6 (c.424G>A; p.Val142Ile) genes. Each of these variants is of unknown clinical significance; however TSC22D1, a transcription factor with suppressor activity, is relatively well studied in cancer . As reported previously by us , and others [16, 20, 21], none of the cases showed alterations in IDH1/2, ATRX, TERT, EGFR amplification or PTEN loss/chromosome 7 gain, alterations common in adult GBMs. In addition, there was an absence of mutations in histone 3 genes or TP53, which are frequently seen in pediatric and adolescent GBMs. Molecular features of low grade gliomas such as BRAF-KIAA1549 fusion were also not identified in cGBM.
As reported previously [7, 16], all 8 cases met WHO 2016 histological criteria for GBM, including presence of cytologically atypical astrocytic cells (Fig. 1c, d) with increased proliferative index and presence of necrosis or microvascular proliferation. These features are not significantly different from those seen in many adult and pediatric GBMs. Variably present were frequent fresh and chronic hemorrhage (Fig. 1d), vascular thromboses and microvascular proliferation. Histopathologic examination and immunohistochemical stains were utilized to exclude diagnosis of other common congenital/early neonatal brain tumors such as teratoma, choroid plexus tumors, craniopharyngioma, and embryonal (formerly primitive neuroectodermal) tumors . The histologic findings were not predictive of the specific molecular alterations consistent to our findings in pediatric glial and glioneuronal tumors .
Clinical outcome including recurrence and survival was variable but markedly more favorable as a whole than that for GBMs in many older pediatric patients [22, 23]. Long term follow-up shows that 6 out of 8 patients are alive and free of recurrence (range: 1.5–17 years) at the time of this report, following receipt of moderately-intense adjuvant chemotherapy treatment without radiation. Specific individual survival durations are 1.5 years, 4 years, 10 years, 10 years, 11 years and 17 years (Table 1). Two of 8 deceased patients died within 6 weeks of surgery with 1 expiring at the time of the operation due to massive intratumoral bleeding (see Table 1). The second patient died of progressive disease 6 weeks after diagnosis after the family declined surgical resection or any other therapeutic intervention. None of the 6 surviving patients have experienced a recurrence requiring second surgical resection, to date. Three patients have mild to moderate developmental delay and 3 have other neurologic abnormalities, but all have an otherwise normal quality of life (Table 1).
These indicators of clinical behavior (response to treatment, recurrence and survival) in our cohort are consistent with many studies that generally report a relatively favorable prognosis for cGBMs [5, 12] with only a minority resulting in rapid deterioration and death [2, 10, 13]. In rare instances, spontaneous regression  or recurrence of cGBM as a ganglioglioma has been reported [9, 25]. This is in contrast to pediatric and adult glioblastoma that are characterized by rapid progression and recurrence and are usually fatal in 15–18 months, with less than 5% of adult GBM patients and less than 20% of pediatric GBM patients surviving more than 5 years [22, 23].
Genomic fusions were the most frequent molecular finding in our cohort, including 4 of 7 assessable cases with ALK fusion and 1 with ROS1 fusion. These findings are similar to recent publications that reported frequent alterations in ALK, ROS1 , NTRK, MET , as well as RAS/MAPK pathway in congenital and infant gliomas . Unlike the study by Guerriero Stucklin et al. , our study did not include low grade congenital/infantile gliomas and utilized a stringent cut off for congenital GBMs of < age 3 months as opposed to 1 year. Although our cohort is small, we observed that survival for our ALK/ROS1-altered congenital GBMs was markedly better at 100% versus 42% reported for high grade tumors with ALK/ROS1, as reported in the literature . Whether this difference is due to differences in patient age or simply secondary to small case numbers is uncertain.
It is important to emphasize that while ROS1, ALK, NTRK and MET alterations are enriched in cGBMs, none of these alterations is exclusive to this age group [6, 26]. Thus, a full panel of mutational/fusion testing is optimal for any pediatric high grade glioma when seeking possible targets for therapy.
Genetic alterations are frequently observed in tumors of the CNS  as well as in systemic solid organ tumors [27, 28]. Genetic rearrangements that create ROS1 fusion proteins in which the kinase domain of ROS1 becomes constitutively activated and drive cellular proliferation have been found in a variety of tumors including glioblastoma, non-small cell lung cancer, cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastic tumor, angiosarcoma, and epithelioid hemangioendothelioma . The transforming potential of ROS1 fusion proteins is well established. Expression of ROS1 fusion variants in fibroblasts was shown to result in anchorage-independent growth, foci formation, and tumorigenicity (for review see ). Similarly, ALK gene rearrangements initially discovered in a subset of anaplastic large-cell lymphomas have subsequently been found in a variety of tumors including non–small cell lung cancers  and CNS tumors . The fusion partner in ALK fused genes provides a dimerization domain that induces constitutive oligomerization and thus activation of the kinase . Targeting of ROS1 and ALK fusion proteins with the small-molecule inhibitor crizotinib has shown promise as an effective therapy in patients. In non-small cell lung cancer, crizotinib induces remissions and extends the lives of patients .
Our cohort of cGBM patients from a single institution shows that a majority of these infants enjoy a long term, recurrence-free survival when treated with resection followed by chemotherapy, favoring this approach as opposed to surgical resection alone . In addition, a substantial proportion of cGBM harbor ALK or ROS1 alterations, allowing for potential therapy with Food and Drug Administration (FDA)-approved drugs such as entrectinib , larotrectinib or Crizotinib . Although patients in our cohort were treated with tumor excision followed by carboplatin/etoposide therapy, and none have yet been treated with or have required ALK—targeted therapy, this remains a possibility in case of future tumor recurrence.
In summary, our molecular studies further extend our original work with cGBM  and confirm that these tumors are genetically heterogeneous and different from pediatric and adult HGG. We present extended follow-up of patients with cGBM and show that cGBM is compatible with long-term recurrence-free survival. Furthermore, actionable fusions, particularly in the ALK gene, can be frequently identified, which provides an additional potential target for therapy .
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This work was supported, in part, by the UCD Molecular Pathology Shared Resource (MPSR) and the UCD Genomics and Microarray Core (National Cancer Institute Cancer Center Support Grant No. P30-CA046934). Additional financial assistance was received from the Morgan Adams Foundation, and the Olivia Caldwell Foundation.
Conflict of interest
Kurt D. Davis has received sponsored travel from ArcherDx. Other authors report no conflicts of interest related to this study.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Gilani, A., Donson, A., Davies, K.D. et al. Targetable molecular alterations in congenital glioblastoma. J Neurooncol 146, 247–252 (2020). https://doi.org/10.1007/s11060-019-03377-8