Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5–10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2–46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0–7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.
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Chan JA, Zhang H, Roberts PS, Jozwiak S, Wieslawa G, Lewin-Kowalik J, et al. Pathogenesis of tuberous sclerosis subependymal giant cell astrocytomas: biallelic inactivation of TSC1 or TSC2 leads to mTOR activation. J Neuropathol Exp Neurol. 2004;63:1236–42.
Kotulska K, Borkowska J, Roszkowski M, Mandera M, Daszkiewicz P, Drabik K, et al. Surgical treatment of subependymal giant cell astrocytoma in tuberous sclerosis complex patients. Pediatr Neurol. 2014;50:307–12.
Henske EP, Jóźwiak S, Kingswood JC, Sampson JR, Thiele EA. Tuberous sclerosis complex. Nat Rev Dis Prim. 2016;2:16035.
Salussolia CL, Klonowska K, Kwiatkowski DJ, Sahin M. Genetic etiologies, diagnosis, and treatment of tuberous sclerosis complex. Annu Rev Genomics Hum Genet. 2019;20:217–40.
Bongaarts A, Giannikou K, Reinten RJ, Anink JJ, Mills JD, Jansen FE, et al. Subependymal giant cell astrocytomas in Tuberous sclerosis complex have consistent TSC1/TSC2 biallelic inactivation, and no BRAF mutations. Oncotarget. 2017;8:95516–29.
Martin KR, Zhou W, Bowman MJ, Shih J, Au KS, Dittenhafer-Reed KE, et al. The genomic landscape of tuberous sclerosis complex. Nat Commun 2017;8:15816. https://doi.org/10.1038/ncomms15816.
Lee D, Cho YH, Kang SY, Yoon N, Sung CO, Suh YL. BRAF V600E mutations are frequent in dysembryoplastic neuroepithelial tumors and subependymal giant cell astrocytomas. J Surg Oncol. 2015;111:359–64.
Debiec-Rychter M, Jesionek-Kupnicka D, Zakrzewski K, Liberski PP. Cytogenetic changes in two cases of subependymal giant-cell astrocytoma. Cancer Genet Cytogenet. 1999;109:29–33.
Franz DN, Belousova E, Sparagana S, Bebin EM, Frost M, Kuperman R, et al. Everolimus for subependymal giant cell astrocytoma in patients with tuberous sclerosis complex: 2-year open-label extension of the randomised EXIST-1 study. Lancet Oncol. 2014;15:1513–2150.
Franz DN, Agricola K, Mays M, Tudor C, Care MM, Holland-Bouley K, et al. Everolimus for subependymal giant cell astrocytoma: 5-year final analysis. Ann Neurol. 2015;78:929–38.
Fogarasi A, De Waele L, Bartalini G, Jozwiak S, Laforgia N, Verhelst H, et al. EFFECTS: an expanded access program of everolimus for patients with subependymal giant cell astrocytoma associated with tuberous sclerosis complex. BMC Neurol. 2016;16:126.
Tyburczy ME, Kotulska K, Pokarowski P, Mieczkowski J, Kucharska J, Grajkowska W, et al. Novel proteins regulated by mTOR in subependymal giant cell astrocytomas of patients with tuberous sclerosis complex and new therapeutic implications. Am J Pathol. 2010;176:1878–90.
Bongaarts A, van Scheppingen J, Korotkov A, Mijnsbergen C, Anink JJ, Jansen FE, et al. The coding and non-coding transcriptional landscape of subependymal giant cell astrocytomas. Brain. 2020;143:131–49.
Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol. 2016;131:803–20.
Wen PY, Huse JT. 2016 World Health Organization classification of central nervous system tumors. Contin (Minneap Minn). 2017;23:1531–47.
Diamandis P, Aldape K. World Health Organization 2016 classification of central nervous system tumors. Neurol Clin. 2018;36:439–47.
Northrup H, Krueger DA, International Tuberous Sclerosis Complex Consensus Group. Tuberous sclerosis complex diagnostic criteria update: recommendations of the 2012 International Tuberous Sclerosis Complex Consensus Conference. Pediatr Neurol. 2013;49:243–54.
Kim W, Giannikou K, Dreier JR, Lee S, Tyburczy ME, Silverman EK, et al. A genome-wide association study implicates NR2F2 in lymphangioleiomyomatosis pathogenesis. Eur Respir J. 2019;53(6):1900329. https://doi.org/10.1183/13993003.00329-2019. pii
Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol. 2009;27:182–9.
Fisher S, Barry A, Abreu J, Minie B, Nolan J, Delorey TM, et al. A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Genome Biol. 2011;12:R1.
Carter SL, Cibulskis K, Helman E, McKenna A, Shen H, Zack T, et al. Absolute quantification of somatic DNA alterations in human cancer. Nat Biotechnol. 2012;30:413–21.
Tyburczy ME, Dies KA, Glass J, Camposano S, Chekaluk Y, Thorner AR, et al. Mosaic and intronic mutations in TSC1/TSC2 explain the majority of TSC patients with no mutation identified by conventional testing. PLoS Genet. 2015;11:e1005637.
Giannikou K, Lasseter KD, Grevelink JM, Tyburczy ME, Dies KA, Zhu Z, et al. Low-level mosaicism in tuberous sclerosis complex: prevalence, clinical features, and risk of disease transmission. Genet Med. 2019;21:2639–43.
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.
Cornwell M, Vangala M, Taing L, Hebert Z, Köster J, Li B, et al. VIPER: visualization pipeline for RNA-seq, a snakemake workflow for efficient and complete RNA-seq analysis. BMC Bioinforma. 2018;19:135.
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc, 2012;7:562–78.
Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323. https://doi.org/10.1186/1471-2105-12-323.
Qaddoumi I, Orisme W, Wen J, Santiago T, Gupta K, Dalton JD, et al. Genetic alterations in uncommon low-grade neuroepithelial tumors: BRAF, FGFR1, and MYB mutations occur at high frequency and align with morphology. Acta Neuropathol. 2016;131:833–45.
Ceccarelli M, Barthel FP, Malta TM, Sabetot TS, Salama SR, Murray BA, et al. Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. Cell 2016;164:550–63.
Tsurubuchi T, Nakano Y, Hirato J, Yoshida A, Muroi A, Sakamoto N, et al. Subependymal giant cell astrocytoma harboring a PRRC2B-ALK fusion: a case report. Pediatr Blood Cancer. 2019;66:e27995. https://doi.org/10.1002/pbc.27995.
Giannikou K, Malinowska IA, Pugh TJ, Yan R, Tseng YY, Oh C, et al. Whole exome sequencing identifies TSC1/TSC2 biallelic loss as the primary and sufficient driver event for renal angiomyolipoma development. PLoS Genet. 2016;12:e1006242. 5.
Bailey MH, Tokheim C, Porta-Pardo E, Sengupta S, Bertrand D, Weerasinghe A, et al. Comprehensive characterization of cancer driver genes and mutations. Cell 2018;174:1034–5.
Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 2013;499:214–8.
Robertson AG, Kim J, Al-Ahmadie H, Bellmunt J, Guo G, Cherniack AD, et al. Comprehensive molecular characterization of muscle-invasive bladder. Cancer Cell. 2017;171:540–56.
Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.
Lonsdale J, Thomas J, Salvatore M, Phillips R, Lo E, Shad S, et al. The Genotype-Tissue Expression (GTEx) project. Nat Genet. 2013;45:580–5.
Zhang B, Guo D, Han L, Rensing N, Satoh A, Wong M. Hypothalamic orexin and mechanistic target of rapamycin activation mediate sleep dysfunction in a mouse model of Tuberous sclerosis complex. Neurobiol Dis 2020;134:104615.
Onda H, Crino PB, Zhang H, Murphey RD, Rastelli L, Gould BE, et al. Tsc2 null murine neuroepithelial cells are a model for human tuber giant cells, and show activation of an mTOR pathway. Mol Cell Neurosci. 2002;21:561–74.
Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9:559. https://doi.org/10.1186/1471-2105-9-559.
Bettegowda C, Agrawal N, Jiao Y, Wang Y, Wood LD, Rodriguez FJ, et al. Exomic sequencing of four rare central nervous system tumor types. Oncotarget. 2013;4:572–83.
Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature. 2008;455:1061–8.
Kline CN, Joseph NM, Grenert JP, van Ziffle J, Talevich E, Onodera C, et al. Targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy. Neuro Oncol. 2017;19:699–709.
Correa DD, Satagopan J, Martin A, Braun E, Kryza-Lacombe M, Cheung K, et al. Genetic variants and cognitive functions in patients with brain tumors. Neuro Oncol. 2019;21:1297–309.
Muskens IS, Zhang C, de Smith AJ, Biegel JA, Walsh KM, Wiemels JL. Germline genetic landscape of pediatric central nervous system tumors. Neuro Oncol. 2019;21:1376–88.
Feng Y, Xu Q. Pivotal role of hmx2 and hmx3 in zebrafish inner ear and lateral line development. Dev Biol. 2010;339:507–18.
Wang W, Grimmer JF, Van De Water TR, Lufkin T. Hmx2 and Hmx3 homeobox genes direct development of the murine inner ear and hypothalamus and can be functionally replaced by Drosophila Hmx. Dev Cell. 2004;7:439–53.
Yu Z, Sun Y, She X, Wang Z, Chen S, Deng Z, et al. SIX3, a tumor suppressor, inhibits astrocytoma tumorigenesis by transcriptional repression of AURKA/B. J Hematol Oncol. 2017;10:115. https://doi.org/10.1186/s13045-017-0483-2.
Xie Z, Ma X, Ji W, Zhou G, Lu Y, Xiang Z, et al. Zbtb20 is essential for the specification of CA1 field identity in the developing hippocampus. Proc Natl Acad Sci USA. 2010;10:6510–5.
Guo XM, Chen B, Lv JM, Lei Q, Pan YJ, Yang Q. Knockdown of IRF6 attenuates hydrogen dioxide-induced oxidative stress via inhibiting mitochondrial dysfunction in HT22 cells. Cell Mol Neurobiol. 2016;36:1077–86. https://doi.org/10.1007/s10571-015-0301-8.
Kozlenkov A, Li J, Apontes P, Hurd Y, Byne WM, Koonin EV, et al. A unique role for DNA (hydroxy)methylation in epigenetic regulation of human inhibitory neurons. Sci Adv. 2018;4:eaau6190. https://doi.org/10.1126/sciadv.aau6190. 26.
The authors thank all subjects who participated in this study; the clinicians who referred and evaluated the patients and the neurosurgeons who performed the surgical resections; as well as Karthik V. Karnik and Edward R. Kwiatkowski for their work on customized code for MPS analysis, as well as Yana Stackpole for assistance with Qlucore analysis; Stichting Kinderen Kankervrij; Stichting AMC Foundation; Stichting TSC Fonds (EA, AB).
This work was supported by the Engles Family Fund for Research in TSC and LAM.
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Giannikou, K., Zhu, Z., Kim, J. et al. Subependymal giant cell astrocytomas are characterized by mTORC1 hyperactivation, a very low somatic mutation rate, and a unique gene expression profile. Mod Pathol 34, 264–279 (2021). https://doi.org/10.1038/s41379-020-00659-9