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11.01.2017 | Original Paper | Ausgabe 3/2017

Acta Neuropathologica 3/2017

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

Acta Neuropathologica > Ausgabe 3/2017
Shintaro Fukushima, Satoshi Yamashita, Hisato Kobayashi, Hirokazu Takami, Kohei Fukuoka, Taishi Nakamura, Kai Yamasaki, Yuko Matsushita, Hiromi Nakamura, Yasushi Totoki, Mamoru Kato, Tomonari Suzuki, Kazuhiko Mishima, Takaaki Yanagisawa, Akitake Mukasa, Nobuhito Saito, Masayuki Kanamori, Toshihiro Kumabe, Teiji Tominaga, Motoo Nagane, Toshihiko Iuchi, Koji Yoshimoto, Masahiro Mizoguchi, Kaoru Tamura, Keiichi Sakai, Kazuhiko Sugiyama, Mitsutoshi Nakada, Kiyotaka Yokogami, Hideo Takeshima, Yonehiro Kanemura, Masahide Matsuda, Akira Matsumura, Kazuhiko Kurozumi, Keisuke Ueki, Masahiro Nonaka, Akio Asai, Nobutaka Kawahara, Yuichi Hirose, Tatusya Takayama, Yoichi Nakazato, Yoshitaka Narita, Tatsuhiro Shibata, Masao Matsutani, Toshikazu Ushijima, Ryo Nishikawa, Koichi Ichimura, On behalf of The Intracranial Germ Cell Tumor Genome Analysis Consortium (The iGCTConsortium)
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s00401-017-1673-2) contains supplementary material, which is available to authorized users.


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.

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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
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