Taishi Nakamura

Revisiting TP53 Mutations and Immunohistochemistry—A Comparative Study in 157 Diffuse Gliomas

The association between p53 immunohistochemistry and TP53 mutation status has been controversial. The present study aims to re‐evaluate the efficacy of p53 immunohistochemistry to predict the mutational status of TP53. A total of 157 diffuse gliomas (World Health Organization grades II–IV) were assessed by exon‐by‐exon DNA sequencing from exon 4 through 10 of TP53 using frozen tissue samples. Immunohistochemistry with a p53 antibody (DO‐7) on paired formalin‐fixed paraffin‐embedded materials was assessed for the extent and intensity of reactivity in all cases. A total of 72 mutations were detected in 66 samples. They included 60 missense mutations, five nonsense mutations, four deletions and three alterations in the splicing sites. A receiver operating characteristic curve analysis revealed that strong p53 immunoreactivity in more than 10% of cells provided the most accurate prediction of mutation. Using this cutoff value, 52 of 55 immunopositive cases harbored a mutation, whereas only 14 of 102 immunonegative cases showed mutations, sensitivity and specificity being 78.8% and 96.7%. Tumors with frameshift mutations frequently showed negative immunostaining. Staining interpretation by an independent observer yielded comparable accuracy. We thus propose p53 immunohistochemistry as a moderately sensitive and highly specific marker to predict TP53 mutation.

Recurrent mutations of CD79B and MYD88 are the hallmark of primary central nervous system lymphomas

Primary central nervous system lymphoma (PCNSL) manifest aggressive clinical behaviour and have poor prognosis. Although constitutive activation of the nuclear factor‐κB (NF‐κB) pathway has been documented, knowledge about the genetic alterations leading to the impairment of the NF‐κB pathway in PCNSLs is still limited. This study was aimed to unravel the underlying genetic profiles of PCNSL.

A combination of TERT promoter mutation and MGMT methylation status predicts clinically relevant subgroups of newly diagnosed glioblastomas

The prognostic impact of TERT mutations has been controversial in IDH-wild tumors, particularly in glioblastomas (GBM). The controversy may be attributable to presence of potential confounding factors such as MGMT methylation status or patients’ treatment. This study aimed to evaluate the impact of TERT status on patient outcome in association with various factors in a large series of adult diffuse gliomas. We analyzed a total of 951 adult diffuse gliomas from two cohorts (Cohort 1, n = 758; Cohort 2, n = 193) for IDH1/2, 1p/19q, and TERT promoter status. The combined IDH/TERT classification divided Cohort 1 into four molecular groups with distinct outcomes. The overall survival (OS) was the shortest in IDH wild-type/TERT mutated groups, which mostly consisted of GBMs (P < 0.0001). To investigate the association between TERT mutations and MGMT methylation on survival of patients with GBM, samples from a combined cohort of 453 IDH-wild-type GBM cases treated with radiation and temozolomide were analyzed. A multivariate Cox regression model revealed that the interaction between TERT and MGMT was significant for OS (P = 0.0064). Compared with TERT mutant-MGMT unmethylated GBMs, the hazard ratio (HR) for OS incorporating the interaction was the lowest in the TERT mutant-MGMT methylated GBM (HR, 0.266), followed by the TERT wild-type-MGMT methylated (HR, 0.317) and the TERT wild-type-MGMT unmethylated GBMs (HR, 0.542). Thus, patients with TERT mutant-MGMT unmethylated GBM have the poorest prognosis. Our findings suggest that a combination of IDH, TERT, and MGMT refines the classification of grade II-IV diffuse gliomas.

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

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.

Genome-wide DNA methylation profiling identifies primary central nervous system lymphoma as a distinct entity different from systemic diffuse large B-cell lymphoma

Taishi Nakamura1,2 · Satoshi Yamashita3 · Kazutaka Fukumura4 · Jun Nakabayashi5 · Kazuhiro Tanaka6 · Kaoru Tamura7 · Kensuke Tateishi2 · Manabu Kinoshita8 · Shintaro Fukushima1 · Hirokazu Takami1 · Kohei Fukuoka1 · Kai Yamazaki1 · Yuko Matsushita9 · Makoto Ohno9 · Yasuji Miyakita9 · Soichiro Shibui10 · Atsuhiko Kubo11 · Takashi Shuto12 · Sylvia Kocialkowski13 · Shoji Yamanaka14 · Akitake Mukasa15 · Takashi Sasayama6 · Kazuhiko Mishima16 · Taketoshi Maehara7 · Nobutaka Kawahara2 · Motoo Nagane17 · Yoshitaka Narita9 · Hiroyuki Mano4 · Toshikazu Ushijima3 · Koichi Ichimura2

Encouraging option of multi-staged gross total resection for a C11orf - RelA fusion-positive supratentorial anaplastic ependymoma

Ependymomas are primary neuroepithelial malignancies that mainly occur during childhood, and arise from ependymal cells along the ventricular systems of the CNS. Recently, it was elucidated that two-thirds of supratentorial (ST) ependymomas harbor oncogenic fusions of RELA, whose protein product is the principal effector of canonical NF-κB signaling. RELA fusion proteins activate signaling for tumor proliferation and malignant progression, resulting in poorer prognoses in these patients compared to those in patients with other ST ependymomas. In this study, we encountered a case of C11orf–RelA fusion-positive ST anaplastic ependymoma that was diagnosed in first tumor resection surgery of multi-staged gross total resection with molecular evidence. In ependymomas, regardless of tumor location or pathological grade, subtotal resection is associated with higher rates of mortality compared with GTR. Molecular analysis based on the application of recent molecular knowledge for ST ependymomas performs a role in appropriate and individualized treatment strategies.

Abstract 4264: Whole exome and targeted sequencing identified the MAPK and PI3K pathways as the main targets in intracranial and testicular germ cell tumors

Intracranial germ cell tumors (iGCTs) are the second most common central nervous system tumors in patients under 14 in Japan. The majority of germinomas respond well to combined chemo- and radiotherapy, however non-germinoma germ cell tumors (NGGCTs) may show resistance to therapy and have a poor clinical outcome. Despite their clinical significance, the biology of iGCTs is mostly unknown. The aim of this study is to elucidate the molecular pathogenesis of iGCTs through a comprehensive genomic analysis. A total of 198 germ cell tumors (GCTs) including 133 iGCTs (69 pure germinomas, 56 NGGCTs and 8 metastatic tumors) as well as 65 testicular germ cell tumors (tGCTs) (39 seminomas and 26 non-seminoma GCTs) were collected from 13 centers participating in the Intracranial Germ Cell Tumor Consortium in Japan. Matched normal DNA was available for 70 iGCTs and 4 tGCTs. Somatic mutations in all coding exons were investigated by whole exome sequencing (WES) using SureSelectXT Human All Exon v4 and a GAIIx or HiSeq 2000 system in 41 tumors and the matched normal DNAs. Targeted sequencing with a set of custom made PCR primers was performed using either an IonTorrent PGM or Proton System. The results were integrated with the patients9 clinical information that was available for 124 iGCT patients. Mutations in selected candidate genes were further evaluated in an independent tumor cohort using the IonTorrent PGM system. On average, 15.4 non-synonymous somatic mutations were observed in each tumor, ranging from 1 to 140 by WES in 41 iGCTs. Based on the WES data, 41 candidate genes were selected according to the frequency and/or significance of the mutations found. All coding exons of these 41 genes spanning over 160kb were PCR-amplified in a further 157 GCTs using the IonTorrent system. The combined WES and IonTorrent screenings showed that KIT was the most frequently mutated gene in both iGCTs (27%) and tGCTs (18%). MTOR was the second most frequently mutated also in both iGCTs (7%) and tGCTs (6%). RAS mutations (KRAS, HRAS, NRAS) were altogether found in 13% of iGCTs and 12% of tGCTs. These mutations were mutually exclusive to each other and also to KIT mutations. Collectively, the genes involved in the MAPK pathway (e.g., KIT, RAS, NF1) and the PI3K/MTOR pathway (e.g., MTOR, PTEN) were mutated in 44% and 13% of all GCTs. These alterations were significantly more common among pure germinomas than NGGCTs. The mutated MTOR protein was shown to have increased kinase activity, which was suppressed by a specific MTOR inhibitor. Our comprehensive mutational genomic analysis of GCTs revealed that alterations of the MAPK and/or PI3K/MTOR pathways play a critical role in the pathogenesis of both iGCTs and tGCTs, although the extent of their involvement depends on the histopathological subtypes. Our findings will hopefully lead to the development of a targeted therapy for treatment-resistant iGCTs. Citation Format: Koichi Ichimura, Shintaro Fukushima, Yasushi Totoki, Yuko Matsushita, Ayaka Otsuka, Arata Tomiyama, Tohru Niwa, Ryuichi Sakai, Toshikazu Ushijima, Taishi Nakamura, Tomonari Suzuki, Kouhei Fukuoka, Takaaki Yanagisawa, Kazuhiko Mishima, Yoichi Nakazato, Fumie Hosoda, Yoshitaka Narita, Soichiro Shibui, Akihiko Yoshida, Hirokazu Takami, Akitake Mukasa, Koki Aihara, Nobuhito Saito, Toshihiro Kumabe, Masayuki Kanamori, Teiji Tominaga, Keiichi Kobayashi, Saki Shimizu, Motoo Nagane, Toshihiko Iuchi, Masahiro Mizoguchi, Koji Yoshimoto, Kaoru Tamura, Taketoshi Maehara, Kazuhiko Sugiyama, Mitsutoshi Nakada, Keiichi Sakai, Yonehiro Kanemura, Masahiro Nonaka, Kiyotaka Yokogami, Hideo Takeshima, Nobutaka Kawahara, Tatsuya Takayama, Masahiro Yao, Hiromi Nakamura, Natsuko Hama, Masao Matsutani, Tatsuhiro Shibata, Ryo Nishikawa. Whole exome and targeted sequencing identified the MAPK and PI3K pathways as the main targets in intracranial and testicular germ cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4264. doi:10.1158/1538-7445.AM2014-4264