Genome-wide methylation profiles in primary intracranial germ cell tumors indicate a primordial germ cell origin for germinomas

Acta Neuropathologica volume 133, pages445–462(2017)Cite this article

Abstract

Intracranial germ cell tumors (iGCTs) are the second most common brain tumors among children under 14 in Japan. The World Health Organization classification recognizes several subtypes of iGCTs, which are conventionally subclassified into pure germinoma or non-germinomatous GCTs. Recent exhaustive genomic studies showed that mutations of the genes involved in the MAPK and/or PI3K pathways are common in iGCTs; however, the mechanisms of how different subtypes develop, often as a mixed-GCT, are unknown. To elucidate the pathogenesis of iGCTs, we investigated 61 GCTs of various subtypes by genome-wide DNA methylation profiling. We showed that pure germinomas are characterized by global low DNA methylation, a unique epigenetic feature making them distinct from all other iGCTs subtypes. The patterns of methylation strongly resemble that of primordial germ cells (PGC) at the migration phase, possibly indicating the cell of origin for these tumors. Unlike PGC, however, hypomethylation extends to long interspersed nuclear element retrotransposons. Histologically and epigenetically distinct microdissected components of mixed-GCTs shared identical somatic mutations in the MAPK or PI3K pathways, indicating that they developed from a common ancestral cell.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. 1.

    Amatruda JF, Ross JA, Christensen B, Fustino NJ, Chen KS, Hooten AJ, Nelson H, Kuriger JK, Rakheja D, Frazier AL, Poynter JN (2013) DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors. BMC Cancer 13:313

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Brait M, Maldonado L, Begum S, Loyo M, Wehle D, Tavora FF, Looijenga LH, Kowalski J, Zhang Z, Rosenbaum E, Halachmi S, Netto GJ, Hoque MO (2012) DNA methylation profiles delineate epigenetic heterogeneity in seminoma and non-seminoma. Br J Cancer 106:414–423

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Dolecek TA, Propp JM, Stroup NE, Kruchko C (2012) CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2005–2009. Neuro Oncol 14(Suppl 5):v1–v49

    Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Fukushima S, Otsuka A, Suzuki T, Yanagisawa T, Mishima K, Mukasa A, Saito N, Kumabe T, Kanamori M, Tominaga T, Narita Y, Shibui S, Kato M, Shibata T, Matsutani M, Nishikawa R, Ichimura K (2014) Mutually exclusive mutations of KIT and RAS are associated with KIT mRNA expression and chromosomal instability in primary intracranial pure germinomas. Acta Neuropathol 127:911–925

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Gobel U, Schneider DT, Calaminus G, Haas RJ, Schmidt P, Harms D (2000) Germ-cell tumors in childhood and adolescence. GPOH MAKEI and the MAHO study groups. Ann Oncol 11:263–271

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Guo H, Zhu P, Yan L, Li R, Hu B, Lian Y, Yan J, Ren X, Lin S, Li J, Jin X, Shi X, Liu P, Wang X, Wang W, Wei Y, Li X, Guo F, Wu X, Fan X, Yong J, Wen L, Xie SX, Tang F, Qiao J (2014) The DNA methylation landscape of human early embryos. Nature 511:606–610

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Hovestadt V, Remke M, Kool M, Pietsch T, Northcott PA, Fischer R, Cavalli FM, Ramaswamy V, Zapatka M, Reifenberger G, Rutkowski S, Schick M, Bewerunge-Hudler M, Korshunov A, Lichter P, Taylor MD, Pfister SM, Jones DT (2013) Robust molecular subgrouping and copy-number profiling of medulloblastoma from small amounts of archival tumour material using high-density DNA methylation arrays. Acta Neuropathol 125:913–916

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Ichimura K, Fukushima S, Totoki Y, Matsushita Y, Otsuka A, Tomiyama A, Niwa T, Takami H, Nakamura T, Suzuki T, Fukuoka K, Yanagisawa T, Mishima K, Nakazato Y, Hosoda F, Narita Y, Shibui S, Yoshida A, Mukasa A, Saito N, Kumabe T, Kanamori M, Tominaga T, Kobayashi K, Shimizu S, Nagane M, Iuchi T, Mizoguchi M, Yoshimoto K, Tamura K, Maehara T, Sugiyama K, Nakada M, Sakai K, Kanemura Y, Nonaka M, Asai A, Yokogami K, Takeshima H, Kawahara N, Takayama T, Yao M, Kato M, Nakamura H, Hama N, Sakai R, Ushijima T, Matsutani M, Shibata T, Nishikawa R, Intracranial Germ Cell Tumor Genome Analysis C (2016) Recurrent neomorphic mutations of MTOR in central nervous system and testicular germ cell tumors may be targeted for therapy. Acta Neuropathol 131:889–901

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Igarashi S, Suzuki H, Niinuma T, Shimizu H, Nojima M, Iwaki H, Nobuoka T, Nishida T, Miyazaki Y, Takamaru H, Yamamoto E, Yamamoto H, Tokino T, Hasegawa T, Hirata K, Imai K, Toyota M, Shinomura Y (2010) A novel correlation between LINE-1 hypomethylation and the malignancy of gastrointestinal stromal tumors. Clin Cancer Res 16:5114–5123

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Jennings MT, Gelman R, Hochberg F (1985) Intracranial germ-cell tumors: natural history and pathogenesis. J Neurosurg 63:155–167

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Jeyapalan JN, Noor DA, Lee SH, Tan CL, Appleby VA, Kilday JP, Palmer RD, Schwalbe EC, Clifford SC, Walker DA, Murray MJ, Coleman N, Nicholson JC, Scotting PJ (2011) Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance. Br J Cancer 105:575–585

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Kim JG, Takeshima H, Niwa T, Rehnberg E, Shigematsu Y, Yoda Y, Yamashita S, Kushima R, Maekita T, Ichinose M, Katai H, Park WS, Hong YS, Park CH, Ushijima T (2013) Comprehensive DNA methylation and extensive mutation analyses reveal an association between the CpG island methylator phenotype and oncogenic mutations in gastric cancers. Cancer Lett 330:33–40

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Kobayashi H, Sakurai T, Miura F, Imai M, Mochiduki K, Yanagisawa E, Sakashita A, Wakai T, Suzuki Y, Ito T, Matsui Y, Kono T (2013) High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice. Genome Res 23:616–627

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Korshunov A, Ryzhova M, Hovestadt V, Bender S, Sturm D, Capper D, Meyer J, Schrimpf D, Kool M, Northcott PA, Zheludkova O, Milde T, Witt O, Kulozik AE, Reifenberger G, Jabado N, Perry A, Lichter P, von Deimling A, Pfister SM, Jones DT (2015) Integrated analysis of pediatric glioblastoma reveals a subset of biologically favorable tumors with associated molecular prognostic markers. Acta Neuropathol 129:669–678

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Lambert SR, Witt H, Hovestadt V, Zucknick M, Kool M, Pearson DM, Korshunov A, Ryzhova M, Ichimura K, Jabado N, Fontebasso AM, Lichter P, Pfister SM, Collins VP, Jones DT (2013) Differential expression and methylation of brain developmental genes define location-specific subsets of pilocytic astrocytoma. Acta Neuropathol 126:291–301

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Lee SH, Appleby V, Jeyapalan JN, Palmer RD, Nicholson JC, Sottile V, Gao E, Coleman N, Scotting PJ (2011) Variable methylation of the imprinted gene, SNRPN, supports a relationship between intracranial germ cell tumours and neural stem cells. J Neurooncol 101:419–428

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Looijenga LH, Van Agthoven T, Biermann K (2013) Development of malignant germ cells—the genvironmental hypothesis. Int J Dev Biol 57:241–253

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Mack SC, Witt H, Piro RM, Gu L, Zuyderduyn S, Stutz AM, Wang X, Gallo M, Garzia L, Zayne K, Zhang X, Ramaswamy V, Jager N, Jones DT, Sill M, Pugh TJ, Ryzhova M, Wani KM, Shih DJ, Head R, Remke M, Bailey SD, Zichner T, Faria CC, Barszczyk M, Stark S, Seker-Cin H, Hutter S, Johann P, Bender S, Hovestadt V, Tzaridis T, Dubuc AM, Northcott PA, Peacock J, Bertrand KC, Agnihotri S, Cavalli FM, Clarke I, Nethery-Brokx K, Creasy CL, Verma SK, Koster J, Wu X, Yao Y, Milde T, Sin-Chan P, Zuccaro J, Lau L, Pereira S, Castelo-Branco P, Hirst M, Marra MA, Roberts SS, Fults D, Massimi L, Cho YJ, Van Meter T, Grajkowska W, Lach B, Kulozik AE, von Deimling A, Witt O, Scherer SW, Fan X, Muraszko KM, Kool M, Pomeroy SL, Gupta N, Phillips J, Huang A, Tabori U, Hawkins C, Malkin D, Kongkham PN, Weiss WA, Jabado N, Rutka JT, Bouffet E, Korbel JO, Lupien M, Aldape KD, Bader GD, Eils R, Lichter P, Dirks PB, Pfister SM, Korshunov A, Taylor MD (2014) Epigenomic alterations define lethal CIMP-positive ependymomas of infancy. Nature 506:445–450

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Matsutani M, Sano K, Takakura K, Fujimaki T, Nakamura O, Funata N, Seto T (1997) Primary intracranial germ cell tumors: a clinical analysis of 153 histologically verified cases. J Neurosurg 86:446–455

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Naumov VA, Generozov EV, Zaharjevskaya NB, Matushkina DS, Larin AK, Chernyshov SV, Alekseev MV, Shelygin YA, Govorun VM (2013) Genome-scale analysis of DNA methylation in colorectal cancer using Infinium HumanMethylation450 BeadChips. Epigenetics 8:921–934

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Netto GJ, Nakai Y, Nakayama M, Jadallah S, Toubaji A, Nonomura N, Albadine R, Hicks JL, Epstein JI, Yegnasubramanian S, Nelson WG, De Marzo AM (2008) Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors. Mod Pathol 21:1337–1344

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Ohka F, Natsume A, Motomura K, Kishida Y, Kondo Y, Abe T, Nakasu Y, Namba H, Wakai K, Fukui T, Momota H, Iwami K, Kinjo S, Ito M, Fujii M, Wakabayashi T (2011) The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma. PLoS One 6:e23332

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Oosterhuis JW, Stoop H, Honecker F, Looijenga LH (2007) Why human extragonadal germ cell tumours occur in the midline of the body: old concepts, new perspectives. Int J Androl 30:256–263 (discussion 263–254)

    Article  PubMed  Google Scholar 

  24. 24.

    Pietsch T, Schmidt R, Remke M, Korshunov A, Hovestadt V, Jones DT, Felsberg J, Kaulich K, Goschzik T, Kool M, Northcott PA, von Hoff K, von Bueren AO, Friedrich C, Mynarek M, Skladny H, Fleischhack G, Taylor MD, Cremer F, Lichter P, Faldum A, Reifenberger G, Rutkowski S, Pfister SM (2014) Prognostic significance of clinical, histopathological, and molecular characteristics of medulloblastomas in the prospective HIT2000 multicenter clinical trial cohort. Acta Neuropathol 128:137–149

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Rajpert-De Meyts E (2006) Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects. Hum Reprod Update 12:303–323

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Rijlaarsdam MA, Tax DM, Gillis AJ, Dorssers LC, Koestler DC, de Ridder J, Looijenga LH (2015) Genome wide DNA methylation profiles provide clues to the origin and pathogenesis of germ cell tumors. PLoS One 10:e0122146

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Rodriguez J, Frigola J, Vendrell E, Risques RA, Fraga MF, Morales C, Moreno V, Esteller M, Capella G, Ribas M, Peinado MA (2006) Chromosomal instability correlates with genome-wide DNA demethylation in human primary colorectal cancers. Cancer Res 66:8462–9468

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Rosenblum MK, Nakazato Y, Matsutani M (2007) CNS germ cell tumours. In: Louis DN (ed) WHO Classification of Tumours of the Central Nervous System, 4th edn. International Agency for Research on Cancer, Lyon, pp 197–204

  29. 29.

    Saito T, Tanaka R, Kouno M, Washiyama K, Abe S, Kumanishi T (1989) Tumor-infiltrating lymphocytes and histocompatibility antigens in primary intracranial germinomas. J Neurosurg 70:81–85

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Saitou M, Yamaji M (2012) Primordial germ cells in mice. Cold Spring Harb Perspect Biol 4:a008375

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Sano K, Matsutani M, Seto T (1989) So-called intracranial germ cell tumours: personal experiences and a theory of their pathogenesis. Neurol Res 11:118–126

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Smiraglia DJ, Szymanska J, Kraggerud SM, Lothe RA, Peltomaki P, Plass C (2002) Distinct epigenetic phenotypes in seminomatous and nonseminomatous testicular germ cell tumors. Oncogene 21:3909–3916

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Smith ZD, Chan MM, Humm KC, Karnik R, Mekhoubad S, Regev A, Eggan K, Meissner A (2014) DNA methylation dynamics of the human preimplantation embryo. Nature 511:611–615

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Takeshima H, Niwa T, Takahashi T, Wakabayashi M, Yamashita S, Ando T, Inagawa Y, Taniguchi H, Katai H, Sugiyama T, Kiyono T, Ushijima T (2015) Frequent involvement of chromatin remodeler alterations in gastric field cancerization. Cancer Lett 357:328–338

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Tan C, Scotting PJ (2012) Stem cell research points the way to the cell of origin for intracranial germ cell tumours. An alternative cell of origin for intracranial germ cell tumours. J Pathol 229:4–11

    Article  PubMed  Google Scholar 

  36. 36.

    Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC, Eberhart CG, Parsons DW, Rutkowski S, Gajjar A, Ellison DW, Lichter P, Gilbertson RJ, Pomeroy SL, Kool M, Pfister SM (2012) Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol 123:465–472

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Teilum G (1965) Classification of endodermal sinus tumour (mesoblatoma vitellinum) and so-called “embryonal carcinoma” of the ovary. Acta Pathol Microbiol Scand 64:407–429

    CAS  PubMed  Google Scholar 

  38. 38.

    The committee of brain tumor registry of Japan (2009) Report of Brain Tumor Registry of Japan (1984–2000), 12th edn. Neurol Med Chir (Tokyo) 49(Suppl):1–101

  39. 39.

    Turcan S, Rohle D, Goenka A, Walsh LA, Fang F, Yilmaz E, Campos C, Fabius AW, Lu C, Ward PS, Thompson CB, Kaufman A, Guryanova O, Levine R, Heguy A, Viale A, Morris LG, Huse JT, Mellinghoff IK, Chan TA (2012) IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 483:479–483

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    van Meir EG, Oosterhuis JW, Looijenga LHJ (1998) Genesis and genetics of intracranial germ cell tumors. In: Sawamura Y (ed) Intracranial germ cell tumors, 1st edn. Springer, New York, pp 45–76

    Google Scholar 

  41. 41.

    Wang L, Yamaguchi S, Burstein MD, Terashima K, Chang K, Ng HK, Nakamura H, He Z, Doddapaneni H, Lewis L, Wang M, Suzuki T, Nishikawa R, Natsume A, Terasaka S, Dauser R, Whitehead W, Adekunle A, Sun J, Qiao Y, Marth G, Muzny DM, Gibbs RA, Leal SM, Wheeler DA, Lau CC (2014) Novel somatic and germline mutations in intracranial germ cell tumours. Nature 511:241–245

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Wermann H, Stoop H, Gillis AJ, Honecker F, van Gurp RJ, Ammerpohl O, Richter J, Oosterhuis JW, Bokemeyer C, Looijenga LH (2010) Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance. J Pathol 221:433–442

    CAS  PubMed  Google Scholar 

  43. 43.

    Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP (2004) A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 32:e38

    Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Yoshida T, Yamashita S, Takamura-Enya T, Niwa T, Ando T, Enomoto S, Maekita T, Nakazawa K, Tatematsu M, Ichinose M, Ushijima T (2011) Alu and Satalpha hypomethylation in Helicobacter pylori-infected gastric mucosae. Int J Cancer 128:33–39

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Zong L, Hattori N, Yoda Y, Yamashita S, Takeshima H, Takahashi T, Maeda M, Katai H, Nanjo S, Ando T, Seto Y, Ushijima T (2016) Establishment of a DNA methylation marker to evaluate cancer cell fraction in gastric cancer. Gastric Cancer 19:361–369

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank all the participating institutions of the iGCT Genome Analysis Consortium for their valuable support and contributions. This work was carried out as a research program of ‘The Project for Development of Innovative Research on Cancer Therapeutics’ (P-Direct) Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (No. 15cm0106066h0005), and supported by Grant-in-Aid for Young Scientists (B), KAKENHI No. 30529459 from the Japan Society for the Promotion of Science (JSPS). Data analysis was supported by National Cancer Center Research and Development Funds (26-A-5) (NCCRI; H.N., Y.T., M.K., and T.S.). S.F. is an awardee of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research in Japan for the 3rd Term Comprehensive 10-year Strategy for Cancer Control. This work is dedicated to the memory of Prof. Nobutaka Kawahara.

Author information

Affiliations

  1. Division of Brain Tumor Translational Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Shintaro Fukushima, Hirokazu Takami, Kohei Fukuoka, Taishi Nakamura, Kai Yamasaki, Yuko Matsushita & Koichi Ichimura

  2. Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Satoshi Yamashita & Toshikazu Ushijima

  3. Division of Cancer Genomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Hiromi Nakamura, Yasushi Totoki & Tatsuhiro Shibata

  4. Department of Bioinformatics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Mamoru Kato

  5. Pathology Division, Department of Pathology and Clinical Laboratories, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Shintaro Fukushima

  6. Department of Neurosurgery and Neurooncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Yuko Matsushita & Yoshitaka Narita

  7. NODAI Genome Research Center, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502, Japan

    Hisato Kobayashi

  8. Department of Neuro-Oncology/Neurosurgery, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka, Saitama, 350-1298, Japan

    Tomonari Suzuki, Kazuhiko Mishima, Takaaki Yanagisawa, Masao Matsutani & Ryo Nishikawa

  9. Division of Paediatric Neuro-Oncology, Department of Neurosurgery, Jikei University School of Medicine, Nishi-shinbashi 3-25-8, Minato-ku, Tokyo, 105-8461, Japan

    Takaaki Yanagisawa

  10. Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan

    Hirokazu Takami, Akitake Mukasa & Nobuhito Saito

  11. Department of Neurosurgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8574, Japan

    Masayuki Kanamori, Toshihiro Kumabe & Teiji Tominaga

  12. Department of Neurosurgery, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo, 181-8611, Japan

    Motoo Nagane

  13. Department of Neurosurgery, Chiba Cancer Center, 666-2 Nitona-cho, Chuo-ku, Chiba, Japan

    Toshihiko Iuchi

  14. Department of Neurosurgery, Graduate School of Medical Sciences Kyusyu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan

    Koji Yoshimoto & Masahiro Mizoguchi

  15. Department of Neurosurgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan

    Kaoru Tamura

  16. Department of Neurosurgery, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, 390-8621, Japan

    Keiichi Sakai

  17. Department of Neurosurgery, Shinshu Ueda Medical Center, Asahi 3-1-1, Matsumoto, 390-8621, Japan

    Keiichi Sakai

  18. Department of Clinical Oncology and Neuro-oncology Program, Cancer Treatment Center, Hiroshima University Hospital, 1-2-3 Kasumi, Minamiku, Hiroshima, 734-8551, Japan

    Kazuhiko Sugiyama

  19. Department of Neurosurgery, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8641, Japan

    Mitsutoshi Nakada

  20. Department of Neurosurgery, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, 889-1692, Japan

    Kiyotaka Yokogami & Hideo Takeshima

  21. Division of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka, 540-0006, Japan

    Yonehiro Kanemura

  22. Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan

    Masahide Matsuda & Akira Matsumura

  23. Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8558, Japan

    Kazuhiko Kurozumi

  24. Department of Neurosurgery, Dokkyo University School of Medicine, Kitakobayashi 880, Mibu, Tochighi, 321-0293, Japan

    Keisuke Ueki

  25. Department of Neurosurgery, Kansai Medical University Hirakata Hospital, 2-3-1 Shinmachi, Hirakata, Osaka, 573-1191, Japan

    Taishi Nakamura, Masahiro Nonaka & Akio Asai

  26. Department of Neurosurgery, Graduate School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan

    Nobutaka Kawahara

  27. Department of Neurosurgery, School of Medicine, Fujita Health University, 1-98, Kutsugake Dengakugakubo, Toyoake, Aichi, 470-1192, Japan

    Yuichi Hirose

  28. Department of Neurosurgery, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0374, Japan

    Toshihiro Kumabe

  29. Department of Urology, Hamamatsu University School of Medicine, 1-20-1 Higashi-ku, Handayama, Hamamatsu, Shizuoka, 431-3192, Japan

    Tatusya Takayama

  30. Department of Pathology, Hidaka Hospital, 886 Nakao-machi, Takasaki, Gunma, 370-0001, Japan

    Yoichi Nakazato

  31. Laboratory of Molecular Medicine, Human Genome Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Tatsuhiro Shibata

Authors
  1. Shintaro Fukushima
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Satoshi Yamashita
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hisato Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hirokazu Takami
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kohei Fukuoka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Taishi Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kai Yamasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yuko Matsushita
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hiromi Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yasushi Totoki
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Mamoru Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Tomonari Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Kazuhiko Mishima
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Takaaki Yanagisawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Akitake Mukasa
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Nobuhito Saito
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Masayuki Kanamori
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Toshihiro Kumabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Teiji Tominaga
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Motoo Nagane
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Toshihiko Iuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Koji Yoshimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Masahiro Mizoguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Kaoru Tamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Keiichi Sakai
    View author publications

    You can also search for this author in PubMed Google Scholar

  26. Kazuhiko Sugiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  27. Mitsutoshi Nakada
    View author publications

    You can also search for this author in PubMed Google Scholar

  28. Kiyotaka Yokogami
    View author publications

    You can also search for this author in PubMed Google Scholar

  29. Hideo Takeshima
    View author publications

    You can also search for this author in PubMed Google Scholar

  30. Yonehiro Kanemura
    View author publications

    You can also search for this author in PubMed Google Scholar

  31. Masahide Matsuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  32. Akira Matsumura
    View author publications

    You can also search for this author in PubMed Google Scholar

  33. Kazuhiko Kurozumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  34. Keisuke Ueki
    View author publications

    You can also search for this author in PubMed Google Scholar

  35. Masahiro Nonaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  36. Akio Asai
    View author publications

    You can also search for this author in PubMed Google Scholar

  37. Nobutaka Kawahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  38. Yuichi Hirose
    View author publications

    You can also search for this author in PubMed Google Scholar

  39. Tatusya Takayama
    View author publications

    You can also search for this author in PubMed Google Scholar

  40. Yoichi Nakazato
    View author publications

    You can also search for this author in PubMed Google Scholar

  41. Yoshitaka Narita
    View author publications

    You can also search for this author in PubMed Google Scholar

  42. Tatsuhiro Shibata
    View author publications

    You can also search for this author in PubMed Google Scholar

  43. Masao Matsutani
    View author publications

    You can also search for this author in PubMed Google Scholar

  44. Toshikazu Ushijima
    View author publications

    You can also search for this author in PubMed Google Scholar

  45. Ryo Nishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  46. Koichi Ichimura
    View author publications

    You can also search for this author in PubMed Google Scholar

Consortia

On behalf of The Intracranial Germ Cell Tumor Genome Analysis Consortium (The iGCTConsortium)

Corresponding author

Correspondence to Koichi Ichimura.

Ethics declarations

Conflict of interest

Koichi Ichimura is a recipient of a research grant from Chugai Pharmaceuticals/EPS Co., Ltd.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 22 kb)

Supplementary material 2 (XLSX 20 kb)

Supplementary material 3 (DOCX 20 kb)

Supplementary material 4 (XLSX 28 kb)

Supplementary material 5 (DOCX 18 kb)

Supplementary material 6 (PDF 697 kb) Supplementary Figure S1. Three clusters classification for GCT by methylation profiles. (a) Heatmap for unsupervised hierarchical clustering of 61 GCTs, 20 gliomas, 20 colon cancers, and 6 normal samples into three groups; global low methylation (GLM), partial low methylation (PLM), and high methylation (HM), using a randomly selected probe set on the HumanMethylation 450 BeadChip (450k). The color keys from blue to red indicate low to high methylation level. The tumor histology is shown below the heatmap. S, seminoma; N, neural stem cell; C, cerebral cortex; B, blood; P, pineal gland; O, ovary; T, testis. (b) Box plots showing the differences of mean β-values of all probes on the 450k between pure germinomas versus NGGCTs, gliomas, colon cancers, and normal samples. Significant differences are displayed (***P < 0.001). (c) Heatmap for unsupervised hierarchical clustering of the chromosomal status examined by array comparative genomic hybridization in all 61 GCTs. The color keys from red to blue indicate copy-number loss to gain. The number following the histological diagnosis indicates the GCT-ID. GCTs with crosses on the chromosome Y are cases from female patients. The chromosomal instability was divided into two groups, a severe type (mosaic pattern), or a mild/negative type. (d) Illustrative copy-number profiles showing a mild type of chromosomal instability (top panel), and a severe type (bottom panel). (e) β-value histograms of the entire probe set on the 450k in pure germinomas. A histogram peak at the highest β-value indicates the contamination rate of non-neoplastic tissues. See also Fig. 2

Supplementary material 7 (PDF 1064 kb) Supplementary Figure S2. Global low DNA methylation profile in pure germinomas. (a, b, c, and d) β-value histograms of the normal tissue-unmethylated probes on the 450k (a, pure germinomas; b, NGGCTs; c, gliomas; d, colon cancers), in which the number n indicates the probe number and its proportion of β-value over 0.2. The number following the histological diagnosis indicates the GCT-ID. In pure germinomas, no histogram peak was seen above the β-value of 0.2 (red dotted-line), whereas one or more peaks were observed in the majority of NGGCTs, most gliomas, and all colon cancers. See also Fig. 3

Supplementary material 8 (PDF 535 kb) Supplementary Figure S3. Different methylation profile and shared mutation profile between germinoma and the other GCT component in Mixed-GCT. (a) Box plots showing the difference of mean β-values by 450k between the germinoma component and the non-germinoma component in 4 mixed-GCTs. Significant differences are observed (***P < 0.001). (b) Box plots showing the proportion of the probes with a β-value over 0.2 in each component. A significant difference was shown between the germinoma component (n = 6) and the non-germinoma component (n = 5) (***P < 0.001). (c and d) Photomicrographs of hematoxylin and eosin (H&E) staining slide in a mixed-GCT (GCT-ID 51). The area surrounded by a dotted line indicates a well-circumscribed germinoma component, while the area outside displays an immature teratoma and a yolk sac tumor component (top left panel, bar = 2mm). The germinoma component shows a sheeting proliferation of large round to polygonal cells with a clear cytoplasm (top center panel, bar = 50µm). The non-germinoma component includes an immature teratoma harboring primitive-like neuroepithelial structures, and a yolk sac tumor forming a reticular architecture of relatively small hyperchromatic cells (top right panel, bars = 50µm). β-value histograms of the normal tissue-ummethylated (tumor-methylated) probe set are shown below the photomicrographs. An enlarged image of the original histogram is shown in the inset for each figure, and the number of probes showing a β-value over 0.2 and their proportion are indicated. In the germinoma component, no histogram peak was seen among the probes with a β-value above 0.2 (middle center panel), whereas a modest peak was observed in mixed (middle left panel) and non-germinoma component (middle right panel) (red arrow). Somatic mutations in intron 8 of the CBL gene (IVS8-2A>G) detected in GCT51 by Sanger sequencing (arrow head, bottom left panel) in both the germinoma component (bottom center panel) and the other GCT component (bottom right panel), but not in the patient’s blood DNA (D, right panel). See also Fig. 4

Supplementary material 9 (PDF 1425 kb) Supplementary Figure S4. Comparison of methylation profiles between iGCTs and mouse PGCs. (a) Heatmaps for unsupervised clustering of the mean TSS200 methylation levels of 9,703 matched genes in mouse PGCs (E10.5, E13.5 and E16.5) (Kobayashi et al., Genome Res 2013, Ref.13), 18 iGCTs and 6 normal samples presented in a sex-specific manner. The color keys from blue to red indicate low to high methylation levels, respectively. The number following the histological diagnosis indicates the GCT-ID. (b-e) Bar charts showing the CpG methylation level of 35 germ cell-associated genes in 7 pure germinomas (b), 6 normal samples (c), and 16 NGGCTs (d) compared to those in mouse PGCs at E10.5, E13.5, and E16.5 (e). mPGC, male mouse PGC; fPGC, female mouse PGC. See also Fig. 5

Supplementary material 10 (PDF 385 kb) Supplementary Figure S5. Complete demethylation of the long interspersed nuclear element (LINE) 1 promoter region in pure germinomas. The LINE1 promoter methylation level examined by pyrosequencing in 2 representative pure germinoma cases (GCT-ID 29, 37), which are completely demethylated in contrast to a yolk sac tumor (GCT-ID 65) and normal cerebral cortex. See also Fig. 6

Supplementary material 11 (PDF 340 kb) Supplementary Figure S6. Identification of differentially highly-methylated genes in NGGCTs. (a) Heatmap by unsupervised hierarchical clustering into four groups (I, II, III, and IV) using transcription start site (TSS)200 probes of CpG islands in all 61 GCTs and 7 normal samples. Most pure germinomas were categorized in group III, while NGGCTs belong to group I, II or IV. The color keys from blue to red indicate low to high methylation level. The methylation cluster from Fig. 2 and the details for the tumor histology are shown below and above the heatmap, respectively. S, seminoma; Ge, germinoma; MT, mature teratoma; IT, immature teratoma; YT, yolk sac tumor; EC, embryonal carcinoma; CC, choriocarcinoma. (b) Volcano plots showing TSS200 probes significantly differentially methylated between pure germinoma and NGGCTs displaying a mean β-value difference of ≥0.2 and an adjusted p value <0.01. (in the area with transparent red; n = 523). See also Table 1 and Supplementary Table S4

Supplementary material 12 (PDF 182 kb) Supplementary Figure S7. Patients’ prognosis by histology and methylation status. (a, b, c and d) Kaplan-Meier survival curves of the overall survival (OS) and the progression-free survival (PFS) for all 58 iGCT patients compared by histology (pure germinoma, solid blue; mature teratoma, dotted red; NGGCT without mature teratoma, solid red) or methylation cluster (GLM, solid blue; PLM, dotted red; HM, solid red). A tendency of shorter OS was observed in NGGCTs without mature teratoma (a), although no significant differences were demonstrated in either analysis. (e and f) Kaplan-Meier survival curves of the OS and the PFS for 22 pure germinoma patients compared by methylation cluster (GLM, solid blue; PLM/HM, solid red). A tendency of shorter PFS was observed in pure germinomas classified to GLM (f), although no significant differences were demonstrated in either analysis. See also Supplementary Tables S1, S2, and S3

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fukushima, S., Yamashita, S., Kobayashi, H. et al. Genome-wide methylation profiles in primary intracranial germ cell tumors indicate a primordial germ cell origin for germinomas. Acta Neuropathol 133, 445–462 (2017). https://doi.org/10.1007/s00401-017-1673-2

Download citation

Keywords

  • iGCT
  • Germinoma
  • Global low DNA methylation
  • Primordial germ cell
  • LINE1 hypomethylation