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Genome analysis of intracranial germ cell tumors
In contrast to adults, brain tumors are very common among children, being the most frequent solid malignancy in pediatric patients. The spectrum of brain tumors in children also differs from that of adults. Recent extensive genetic studies in medulloblastomas, pilocytic astrocytomas, pediatric glioblastomas and ependymomas have identified a number of novel molecular features in these tumors. Based on those findings, medulloblastomas are now subdivided into four groups that have distinct molecular profiles and clinical courses. The results are now being translated into molecular classifications and may possibly lead to the development of individualized treatment. Intracranial germ cell tumors (iGCTs) are one of the few pediatric brain tumors that are yet to be explored. They are the second most common brain tumor in children under the age of 14 in Japan. iGCTs are histopathologically divided into 5 subtypes, i.e., germinomas, teratomas, embryonal carcinomas, choriocarcinomas and yolk sac tumors, as well as mixed tumors of any combination. How these diverse types of germ cell tumors develop is currently unknown. Mature teratomas may be surgically curable and germinomas generally respond well to combined radio-chemotherapeutic regimens. However, a subset of germinomas and other iGCTs show resistance to therapy and often signify a poor prognosis for the patients. Their molecular pathogenesis is largely unknown. These tumors require more attention which should be implemented with a full molecular investigation.
To comprehensively study the biology of iGCTs, we have established the Intracranial Germ Cell Tumors Genome Analysis Consortium of Japan, a nation-wide collaborative initiative to centrally collect patients’ materials and clinical information. Over 50 centers in Japan have joined the Consortium, through which more than 250 iGCT cases of various histological subtypes have been obtained so far. A whole exome sequencing in 41 cases of iGCTs and targeted sequencing in 83 iGCTs as well as 65 testicular GCTs and 8 metastatic GCTs showed that genes involved in the MAPK pathway (e.g., KIT, RAS) and/or the PI3K pathway (e.g. MTOR, PTEN) were frequently mutated in approximately 50% of all GCTs examined. We have demonstrated that mutated MTOR had elevated activity which may be inhibited by a dual MTOR inhibitor, suggesting that MTOR inhibition may be an effective targeted therapy against GCTs with PI3K alterations. A genome-wide DNA methylation analysis has revealed that germinomas were characterized by global hypomethylation. The pattern of hypomethylation in germinomas were similar to that of premature primordial germ cells (PGC) in mice, strongly suggesting that PGC is a cell of origin for germinomas.
Fig. 1 Mutations landscape of central nervous system germ cell tumors（modified from Ichimura et al., Acta Neuropathol 2016）
Molecular analysis of gliomas
As a number of molecular genetic alterations have been identified in gliomas over the last two decades, it has become clear that 1) each subtype has a distinct genotype 2) adult and pediatric gliomas develop through different molecular pathogenesis. In adults, astrocytomas and oligodendrogliomas share frequent mutations of IDH1/IDH2 (50-80%). While astrocytomas of grade II and III often harbor TP53 mutations in addition to IDH1 mutations, oligodendrogliomas rarely have TP53 mutations but frequently show concurrent total 1p/19q loss and IDH1 mutations. These facts suggest that astrocytomas and oligodendrogliomas share a common cell of origin. Glioblastomas on the other hand seldom harbor IDH mutations but are characterized by alterations of genes involved in the RB1 and p53 pathways. In addition, mutations or amplifications of the MAPK/PI3K pathway genes are also very common.
In 2016, the revised 4th edition of the WHO Classification of Tumours of the Central Nervous System (WHO2016) was published. In WHO2016, molecular classification was officially introduced as a part of diagnostic criteria for some tumor types. For example, diagnosis of oligodendroglioma now requires demonstration of 1p/19q codeletion and IDH mutation. Oligoastrocytoma will no longer be diagnosed after molecular diagnosis as they will be classified either as oligodendroglioma or astrocytoma depending on the IDH and 1p/19q status.
Recently, we found that mutations in the promoter region of the TERT gene, a reverse transciptase subunit of telomerase that elongates telomeres and prevent cells undergo senescence, are very frequent event in adult gliomas. The mutations mutually exclusively occur at either of two hotspots, C228T and C250T, and create a novel GABPA transcription factor binding site, thereby upregulating TERT expression. Thus, we established promoter mutations of TERT as one of the major mechanisms of telomerase activation in gliomas. Furthermore, we found that TERT mutations are almost always present in tumors with concurrent IDH1 mutations and total 1p/19q loss (mainly oligodendrogliomas) and also very frequently found in glioblastomas without IDH1 mutations or total 1p/19q loss, however very rare among astrocytomas with IDH1 mutations but not total 1p/19q loss (Fig. 2). These findings suggest that TERT mutations are involved in the development of subtypes of adult gliomas and that they may have a diagnostic significance. In addition, a combined analysis of TERT promoter mutation, methylation and expression showed that all tumors with TERT promoter mutation had upregulation of TERT mRNA regardless of the methylation status while an inverse association between TERT promoter methylation and mRNA expression was observed in tumors without promoter mutation, indicating that the promoter mutation but not methylation is the main driving force of TERT transactivation.
To investigate the efficacy of TERT as an additional diagnostic marker, we conducted a multi-center collaborative study to collect 951 adult newly diagnosed gliomas and examined the statuses of IDH1/2, 1p/19q, TERT promoter and MGMT methylation. We found that a combination of IDH and TERT status predict diagnosis and prognosis of diffuse gliomas, suggesting that TERT promoter status can serve as an addition/alternative diagnostic marker.
Fig. 2. A model of glioma development.
Optimization of molecular marker tests
Established molecular markers for diagnosis, prognosis or chemosensitivity prediction include MGMT CpG island methylation, total 1p/19q loss, IDH1/IDH2 mutations and the KIAA1549-BRAF gene fusion. MGMT methylation predicts longer survival in glioblastoma patients (prognostic factor). It also anticipates better sensitivity to temozolomide among elderly patients with glioblastomas (predictive factor) according to the recent Phase III studies. Total 1p/19q loss, as a result of an unbalanced translocation t(1;19), is considered to be a hallmark of oligodendroglial tumors. Evidence of total 1p/19q loss predicts longer survival and better sensitivity to the PCV chemotherapy among anaplastic oligodendroglial tumors. As such, testing these molecular markers has practically become mandatory in any clinical trials involving gliomas. Utilization of molecular data in clinical decision making is on the horizon.
MGMT methylation is one of the most important molecular markers in glioblastomas. Methylation testing of MGMT is employed in most major clinical trials involving gliomas using various methods such as methylation-specific PCR. However an optimal method is yet to be agreed upon. The quantitative MSP-based method used in Phase III trials in Europe and the USA is not available in Japan. We have developed a robust pyrosequencing-based MGMT methylation assay (Fig. 3). To validate the efficacy of this assay for correctly predicting the prognosis of patients, the results are being compared with the outcome of approximately 140 primary glioblastomas which were treated with radiation and temozolomide in a multi-center collaboration. Specimens from all clinical trials involving brain tumors will be analyzed for MGMT methylation using our own optimized pyrosequencing protocol in the laboratory.
We are also developing a new FISH protocol to accurately detect 1p/19q codeletion with the aim to standardize molecular diagnosis of gliomas.
Fig. 3. A representative pyrosequencing result. Methylation of 10 consecutive CpG sites is simultaneously quantified in a single assay.
Molecular diagnosis of pediatric brain tumors
Our understanding on the molecular pathogenesis of pediatric brain tumors has considerably advanced in recent years through the extensive whole genome molecular analysis using the next generation sequencing technologies. Among those genetic/epigenetic changes, we aim to identify novel molecular markers or therapeutic targets that could be used for clinical decision making or development of more effective treatment.
Pediatric gliomas rarely show these changes but display an otherwise unique genetic profile. More than 80% of pilocytic astrocytomas (WHO grade I), which typically arise in children, have alterations of the MAPK pathway predominantly involving the KIAA1549-BRAF fusion gene or BRAF mutations. The BRAF fusion gene is so specific to pilocytic astrocytomas that it is considered as a diagnostic marker. Pediatric glioblastomas, unlike their adult counterparts, often have mutations in histone H3.3 (H3F3A). These facts strongly suggest that adult and pediatric gliomas develop through a distinct molecular pathogenesis despite the fact that they are morphologically indistinguishable. Based on these observations, we have proposed a hypothetical model of glioma development (Fig. 2). Ongoing comprehensive genome analysis will undoubtedly identify more genes involved in the pathogenesis of gliomas.
An international consensus has been made to molecularly classify medulloblastomas into 4 subtypes, WNT, SHH, Group 3 and Group 4 (Taylor, Acta Neuropathol 2012). A new molecular classification for ependymomas that recognizes 2 subtypes of supratentorial ependymomas (ST), one with C11orf95-RELA fusion and the other without, as well as 2 subtypes of posterior fossa ependymomas into PFA and PFB according to their methylation profiles (Mack, Nature 2014; Parker, Nature 2014). It is inevitable that future patient management will be based on these molecular classifications. In order to coordinate a central molecular diagnostics for pediatric brain tumors in Japan, we have established a Japan Pediatric Molecular Neuro-oncology Group (JPMNG), which is now accepting medulloblastomas and ependymomas for molecular classification according to the international consensus. Our lab is in charge of a genome-wide methylation profiling using a HumanMethylation450 BeadChip (Illumina) for ependymomas as well as other tumors. We are now in charge of central molecular diagnosis of pediatric brain tumors in collaboration with Japan Children’s Cancer Group (JCCG) and offering molecular diagnosis for the pediatric brain tumor patients nationwide.
Elucidation of the mechanism of the ALK-inhibitor resistance of ALK-positive tumors to the ALK and its application for cancer therapy
ALK encodes a type I transmembrane tyrosine kinase receptor that is normally expressed only in certain neuronal cells. The ALK gene was originally identified by cloning the t(2;5)(p23;q35) translocation identified in a subset of anaplastic large cell lymphomas (ALCLs). Although ALK-involved chromosomal translocations were originally identified in ALCLs, similar genetic abnormalities have been detected in diffuse large B-cell lymphomas (DLBCLs) and non-hematopoietic neoplasms, including inflammatory myofibroblastic tumors, renal adenocarcinoma and non-small-cell lung cancers (NSCLC). The ALK gene has been found together with many genes by chromosomal translocation in various cancers. Gene amplification and point mutations in the ALK gene have also been found in neuroblastomas. It has been revealed that crizotinib, an ALK inhibitor, has a marked effect on ALK-positive NSCLC, and this ALK inhibitor was recently approved for advanced ALK-positive NSCLC. However, the acquisition of crizotinib tolerance by secondary mutations, called gatekeeper mutations, in the ALK gene, amplification in the ALK gene locus and activation of pathways that bypass ALK-mediated signaling have also been reported. There have, so far, been reports that second-generation ALK inhibitors including CH5424802 and LDK378 (a derivative from TAE684) are being developed as drugs for ALK-positive tumors that occurred gatekeeper mutations in the ALK gene for crizotinib resistance. However, although ALK inhibitors against acquired crizotinib tolerance by secondary mutations and amplification of the ALK gene will be developed, activation of alternative pathways that bypass ALK-mediated signals has also been existed in some populations of ALK-positive tumors. Thus, in addition to the development of novel ALK inhibitors, further alternative therapeutic approaches for ALK-positive tumors are demanded to conquer the ALK inhibitor-resistance. In this project, we obtained intriguing findings that p53 is tyrosine phosphorylated by ALK-fusion proteins to inhibit the p53-mediated pathway through enhanced interaction of p53 with MDM2. We currently investigate the effect of combination treatment of the ALK inhibition with the p53 activation on ALK-positive tumors such as lung adenocarcinoma and neuroblastoma, and elucidate the mechanism of the acquisition of the ALK-inhibitor resistance to develop novel anti-cancer agents.
Development of cancer therapy targeting to tetraspanin family
Lung adenocarcinoma (ADC) has poor prognosis for lack of effective therapy. A part of lung ADC is believed to be derived from small airway epithelial cells and developed by accumulation of various genetic alternations, such as KRAS activation, CDKN2A and p53 inactivation. The mutation of p53 is more often observed in invasive lung ADC than noninvasive, indicating that tumor suppressor p53 regulates malignant transformation including invasiveness, metastasis and drug resistance. In this project, we established the system mimic the genetic alternation of lung ADC in vitro and identified TSPAN2, four transmembrane-spanning protein, as a candidate factor responsible for p53 inactivation-induced motility and invasiveness. TSPAN2 interacted with CD44, a cancer stem cell marker, and enhanced motility and invasiveness by reduction of intracellular reactive oxygen species (Otsubo et al., Cell Reports, 7, 527-538, 2014). We are establishing a system monitoring TSPAN2-CD44 interaction.
Elucidation of the mechanism of p53-mediated transcriptional selectivity and its application for overcoming resistance to anti-cancer drug
The p53 protein regulates cell-cycle progression, apoptosis and senescence for the prevention of tumorigenesis and primarily functions as a transcription factor that activates various genes responsible for anti-tumorigenesis. Although many factors that contribute to the regulation of p53 transcriptional activity have been reported, the detailed mechanisms concerning p53 selectivity of downstream target genes to determine cell fates following DNA damage are poorly understood. In this project, we found that NuMA bound to p53 and Cdk8 to modulate p53-mediated transcriptional selectivity (Ohata et al., Molecular & Cellular Biology, 33, 2447-2457, 2013). An analysis of the molecular mechanisms of p53-mediated transcriptional selectivity is important for the elucidation of carcinogenesis and the development of new anti-cancer drugs for drug resistance, and we currently try to identify small compounds that inhibit p53-NuMA interaction and specific inhibitors of Cdk8.