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PURPOSE Fluorescence imaging is an important surgical adjunct in malignant glioma surgery. 5-aminolevulinic acid (5-ALA) has been proven effective for radical tumor resection and extended progression-free survival in a phase III randomized trial and therefore integrated into surgery for malignant glioma. Importantly, however, some limitations still exist in its use, which include false positivity and false negativity as well as inability of re-administration. In this study, we aimed to develop a novel, spray-type fluorescent probe using hydroxymethyl rhodamine green (HMRG) as a fluorescent scaffold. METHODS We have previously established a fluorescent probe library comprised of more than 320 kinds of HMRG probes. They have HMRG as a fluorescent scaffold with various types of dipeptides attached to it. Primary probe screening was performed using the homogenized tumor samples from patients with glioblastoma operated at our institution. Secondary screening followed using the selected probes and fresh tumor samples obtained from patients with glioblastoma operated from 2016 until 2018. Diced electrophoresis gel (DEG) assay, two-dimensional gel electrophoresis followed by a multi-well plate-based fluorometric assay, was performed to identify responsible enzymes for the selected probe. Further experiments with inhibitors, real-time PCR, immunohistochemistry, and western blotting were performed for confirmation. RESULTS Proline-arginine-HMRG (PR-HMRG) was selected as a candidate probe based upon the above two-step screenings. It achieved 79.4% accuracy in receiver operating characteristic curve analysis. Calpain-1 was found to be responsible to cleave PR-HMRG probe by DEG-proteome analysis. Calpain-1 protein was highly expressed in tumor tissues which reacted to PR-HMRG probe. CONCLUSIONS Our innovative screening method was able to find PR-HMRG as a novel fluorescent probe effective for rapid detection of glioblastoma. A preclinical study is planned to assess the efficacy and safety of the selected probe.

Background Central nervous system (CNS) germ cell tumors (GCTs) are neoplasms predominantly arising in pediatric and young adult populations. While germinomas generally respond to chemotherapy and radiation, non-germinomatous GCTs (NGGCTs) require more intensive treatment. This study aimed to determine whether 12p gain could predict the prognosis of CNS GCTs. Methods Eighty-two CNS GCTs were included in this study. The 12p gain was defined by an additional 12p in the background of potential polyploidy or polysomy. Cases were analyzed using an Illumina methylation 450K array for copy number investigations and validated by fluorescence in situ hybridization (FISH). Results A 12p gain was found in 25-out-of-82 cases (30%) and was more frequent in NGGCTs (12% of germinoma cases and 50% of NGGCT cases), particularly in cases with malignant components, such as immature teratoma, yolk sac tumor, choriocarcinoma, and embryonal carcinoma. 12p gain and KIT mutation were mutually exclusive events. The presence of 12p gain correlated with shorter progression-free (PFS) and overall survival (OS) (10-year OS: 59% vs. 94%, with and without 12p gain, respectively, P = 0.0002), even with histology and tumor markers incorporated in the multivariate analysis. Among NGGCTs, 12p gain still had prognostic significance for PFS and OS (10-year OS: 47% vs. 90%, respectively, P = 0.02). The 12p copy number status was shared among histological components in mixed GCTs. Conclusions 12p gain may predict the presence of malignant components of NGGCTs, and poor prognosis of the patients. It may be associated with early tumorigenesis of CNS GCT.

Background: Germ cell tumors (GCTs) preferentially occurs in pediatric and young adult age groups. Chemo- and radiation therapies cause long-term sequelae in their later lives. We searched for clinical and histopathological features to predict the prognosis and affect treatment response, with a future goal of treatment stratification.Methods: A total of 154 GCT cases were included in the analysis. Total of 114 germinoma cases underwent measurement of tumor cell content on H-E specimen, and 82 GCT cases underwent 450K methylation analysis. 12p gain was determined on methylation-based copy number computation and FISH. Association with progression-free and overall survival (PFS/OS) was investigated. Results: The tumor cell content was widely distributed from =50%) showed shorter PFS than those with a lower tumor cell content (

BACKGROUND In BIOMARK trial, patients with newly diagnosed glioblastoma were treated with standard chemoradiotherapy combined with first-line bevacizumab; a subset of patients continued bevacizumab beyond progression (BBP). Neurocognitive function (NCF), symptom burden, and health-related quality of life (HRQoL) were examined as secondary endpoints. PATIENTS AND METHODS In the primary protocol, newly diagnosed glioblastoma patients aged 20-75 received standard 6-week radiotherapy combined with temozolomide and bevacizumab followed by 4-week cycles of temozolomide plus bevacizumab, and then 2-3-week cycles of bevacizumab monotherapy. Upon recurrence, patients were subjected to the secondary protocol with 2-3-week cycles of bevacizumab monotherapy with or without other chemotherapeutic agents. NCF tests (Hopkins verbal learning test-revised, trail making test, and controlled oral word association), EORTC QLQ-C30/BN20, and MDASI-BT were completed by the patients. Time to deterioration (TTD) was defined as the time from randomization until a pre-specified change from baseline without further improvement or death. The Kaplan-Meier method and the log-rank test were used to assess TTD for each subscale of the above tests. RESULTS Overall, 94 patients were enrolled in the study. Analyses were based on the full analysis set cohort (N=90), excluding non-glioblastoma diagnosis by central review. The median overall survival (OS) and progression-free survival (PFS) were 25.0 and 14.9 months, respectively. Baseline HRQoL and symptom burden subscales (emotional functioning, symptom severity score, affective factor, and focal factor) were significantly associated with PFS. The median TTD was 8.7, 7.5, 8.1 months for global health status/QoL, symptom severity score, interference score, respectively. Among patients who experienced recurrence, disease progression was apparently preceded by deterioration in terms of symptom burden. CONCLUSIONS Detailed analysis of HRQoL and symptom burden may aid care of glioblastoma patients throughout the disease trajectory.

We previously reported that there was a subgroup of IDH-mutated astrocytomas harboring only 19q-loss showing oligodendroglioma-like morphology and significantly longer overall survival (OS) compared with 19q-intact astrocytomas. To further explore the biological characteristics of this possible subgroup and obtain insight into the mechanism of their clinical behavior, we compared gene expression pattern between five 19q-loss and five 19q-intact IDH-mutated astrocytomas by microarray analysis. Comparing expression level of each genes between 19q-loss and 19q-intact astrocytomas,136 up-regulated genes and 203 down-regulated genes were extracted. Gene expression patterns of 19q-loss astrocytomas were partially different from that of 19q-intact astrocytomas. More down-regulated genes distributed on 19q and 4p, and more up-regulated genes distributed on 4q. Multiple genes associated with stem cell maintenance were down-regulated in 19q-loss astrocytomas, and genes associated with glioma progression were differentially expressed. Comparing expression patterns among 19q-loss astrocytomas and other IDH-mutant glioma subgroups using TCGA datasets by t-SNE analysis revealed that expression pattern of 19q-loss astrocytomas did not shift to that of oligodendrogliomas with 1p/19q codeletion but were a subgroup in astrocytomas. These results indicated that 19q-loss in astrocytomas was an acquired event different from 1p/19q codeletion in oligodendrogliomas, and better prognosis morphological features in 19q-loss astrocytomas were derived from differentially expressed genes associated with stem cell maintenance and glioma progression.

Background The Delphi consensus statements on the management of germ cell tumors (GCTs) failed to reach agreements on the statement that the cases with (i) pineal and neurohypophyseal bifocal lesion, (ii) with diabetes insipidus, and (iii) with negative tumor markers can be diagnosed as germinoma without histological verification. To answer this, multicenter retrospective analysis was performed. Methods A questionnaire on clinical findings, histological diagnosis, and details of surgical procedures was sent to 86 neurosurgical and 35 pediatrics departments in Japan. Results Fifty-one institutes reported 132 cases that fulfilled the 3 criteria. Tissue sampling was performed in 91 cases from pineal (n = 44), neurohypophyseal (n = 32), both (n = 6), and distant (n = 9) lesions. Histological diagnosis was established in 89 cases: pure germinoma or germinoma with syncytiotrophoblastic giant cells in 82 (92.1%) cases, germinoma and mature teratoma in 2 cases, and granulomatous inflammation in 2 cases. Histological diagnosis was not established in 2 cases. Although no tumors other than GCTs were identified, 3 (3.4%) patients had non-germinomatous GCTs (NGGCTs). None of the patients developed permanent complications after endoscopic or stereotactic biopsy. Thirty-nine patients underwent simultaneous procedure for acute hydrocephalus without permanent complications, and hydrocephalus was controlled in 94.9% of them. Conclusion All patients who fulfilled the 3 criteria had GCTs or granulomatous inflammation, but not other types of tumors. However, no fewer than 3.4% of the patients had NGGCTs. Considering the safety and the effects of simultaneous procedures for acute hydrocephalus, biopsy was recommended in such patients.

TERT promoter mutation is associated with 1p/19q codeletion and favorable prognosis in IDH-mutated gliomas. Prognostic and diagnostic significance of TERT promoter mutation is well-recognized in IDH-wildtype glioblastomas, but not in IDH-mutated gliomas. We investigated prognostic efficacy of TERT mutation in a cohort of 560 Japanese IDH-mutated adult gliomas. The molecular status of IDH, TERT and 1p/19q and patient clinical data including Karnofsky performance status (KPS) were collected in all cases. TERT mutations and 1p/19q codeletions were found in 303 and 285 cases, respectively. The patient cohort was divided into four groups by a combination of the 1p/19q and TERT status. The characteristics of 1p/19q intact-TERT mutated group (Astro-TERT group, n=24) were compared with those of 1p/19q intact-TERT wild (Astro-group, n=251) or 1p/19q codeleted-TERT mutated (Oligo-group, n=279) cases. Astro-TERT group with any grade showed intermediate overall survival between the Oligo-group and Astro-group although the survival differences were not statistically significant (median overall survival (OS) not reached (NR) versus NR, and 106 months, respectively. p >0.05). We further conducted subgroup analysis by adjusting KPS and WHO grade as Cox regression analysis for survival indicated the unfavorable survival impact of KPS < 90 and WHO grade IV. In the subgroup with favorable KPS (90–100) and grade II-III (n=438), The OS of Astro-TERT group (median NR) was significantly longer survival than that of Astro-group (median 120.2 months, p=0.032), and was comparable with that of the Oligo-group (median NR, p >0.05). On the other hand, OS of none of the molecular groups significantly differ in poorer KPS subgroups (p >0.05). In grade IV tumors, the OS of the Astro-TERT group (NR) was comparable with that of Astro-group (29 months, p=0.19) rather than Oligo-group (NR, p=0.051). Thus, TERT promoter status provides a valuable prognostic information for IDH-mutated grade II-III gliomas in the current molecular diagnostic system.

BACKGROUND The anti-programmed cell death protein 1 (PD-1) immune checkpoint antibody, nivolumab, may be beneficial in patients with recurrent glioblastoma. OBJECTIVE This open-label, non-comparative study assessed the efficacy and safety of nivolumab in Japanese patients with first recurrence of glioblastoma. METHODS Patients with a first recurrence of histologically confirmed World Health Organization (WHO) Grade IV glioma, after treatment with temozolomide plus radiotherapy, received nivolumab 3 mg/kg every 2 weeks until confirmed disease progression based on Response Assessment in Neuro-Oncology (RANO) criteria or development of toxicity. The primary endpoint was 1-year overall survival rate (1y-OS) assessed using a Bayesian approach. The prespecified efficacy criteria were that the Bayesian posterior mean 1y-OS with nivolumab would be 49%, and the Bayesian posterior probability threshold for exceeding the 1y-OS of bevacizumab (34.5%) from the Japanese Phase II study (JO22506) would be 93%. RESULTS Of the 50 patients enrolled, 44 (93.5%) had recurrent malignant glioma (glioblastoma and gliosarcoma), 26 (59.1%) had measurable disease, and 4 (9.1%) received corticosteroids at baseline. The posterior mean (90% Bayesian credible intervals) 1y-OS with nivolumab was 54.35% (42.27–66.21), and the observed posterior probability of exceeding the 1y-OS of bevacizumab (34.5%, JO22506) was 99.7%. The median (90% confidence interval) overall and progression-free survival (PFS) was 13.09 months (10.38, 17.68) and 1.45 months (1.41, 1.54), respectively. One partial response was observed (objective response rate [ORR] 1/26 evaluable patients [3.8%]). The grade 3–5 treatment-related adverse event rate was 16.0%; most adverse events resolved and were manageable. CONCLUSION The 1y-OS with nivolumab monotherapy in Japanese patients was higher than that in the bevacizumab group in the JO22506 study. However, PFS and ORR were not improved compared with JO22506. The safety profile of nivolumab was consistent with that observed in other tumor types.

BACKGROUND Chordoma and chondrosarcoma account for the majority of skull base tumors affecting the petroclival region. Chondrosarcoma has better prognosis than chordoma; heavy particle therapy is often indicated for residual/recurrent chordoma. Preoperative, precise diagnosis of the tumor would be desirable, as it can potentially impact on the choice of a surgical approach and the aggressiveness of surgery. METHODS We conducted a radiomics study to create a machine learning model distinguishing chondrosarcoma from chordoma. We collected DICOM T2-weighted images and T1-weighted images with gadolinium (GdT1) enhancement in the consective patients of chordoma or chondrosarcoma who underwent surgery at The University of Tokyo Hospital from September of 2012 to January of 2019. We selected patients with uniform MRI images. VOI (volume of interest) was set using Monaco (https://www.elekta.com/software-solutions/treatment-management/external-beam-planning/monaco.html). Not only sematic features but also agnostic features were calculated. The original images and 8 wavelet transformed images were calculated for texture agnostic features such as Gray-Level Co-occurrence Matrix (GLCM). Features were selected by recursive feature elimination (RFE). The final model evaluation was performed by average area under the curve (AUC). RESULTS The study population included 17 chordomas and 22 chondrosarcomas in a total of 39 patients. 476 features were obtained per image sequence. The number of features per case was 476 × 2 = 952 The most accurate machine learning model was created using the extracted three features from only T2. The best AUC was 0.77 ± 0.11 in logistic regression (dataset was divided randomly into halves, average value of AUC calculated six times). CONCLUSIONS This novel machine learning model can differentiate chordoma and chondrosarcoma reasonably well. A validation study with a larger number of patients is warranted.

BACKGROUND Clinical benefit of continuing bevacizumab beyond progression is unknown in glioblastomas. A biomarker analysis of the AVAglio trial suggested that proneural subtype of IDH-wildtype glioblastoma patients may receive an OS benefit from a first-line bevacizumab. This study explored biomarkers that may predict survival of glioblastoma patients treated with concurrent irradiation, temozolomide (TMZ) and bevacizumab (BEV) followed by BEV beyond progression (BBP). METHODS In the primary protocol, newly diagnosed GBM patients aged 20–75 received concurrent TMZ (75 mg/m2, D1-42), irradiation (2 Gy x 5 QW x6) and BEV (10 mg/kg Q2W x 3) followed by ≤12 4-week cycles of TMZ (150–200 mg/m2, D1-5) plus BEV (10 mg/kg, D1 and 15) and then 2 or 3-week cycles of BEV monotherapy (15 or 10 mg/kg). Upon PD/recurrence during the primary protocol, the patients were subjected to the secondary protocol with 2-3-week cycles of BEV monotherapy with or without other chemotherapeutic agents (BBP). The primary endpoint was 2-year survival rate in patients receiving BBP. Fresh-frozen tumor specimen were subjected to genome-wide methylation, copy number, expression and mutation analysis. A subset of the control cohort of the AVAglio study was used as BEV-negative control. RESULTS A total of 94 GBM patients were enrolled at 39 centers between June 2015 and January 2017. Efficacy analyses were based on the full analysis set (FAS) cohort (N=90), excluding non-GBM diagnosis by central review. The median time to PD/recurrence was 453 days in the FAS and 348 days in patients receiving BBP (N=27). The 2-year survival rate (90% CI) was 52.4% (43.3–60.8%) and 28.8% (15.4–43.7%), respectively. The 2-year survival rate in GBM patients receiving BBP was lower than expected (50%). That of proneural and mesenchymal IDH-wt GBM in the biomarker cohort (n=83) were 52.9% (31.7–70.3%) and 56.6% (41.6–69.1%), respectively. A full biomarker analysis will be presented.

PURPOSE: ALA is commonly used as an intraoperative tool in malignant glioma surgery, which has been proven effective for radical tumor resection and extended progression-free survival. However, there are some limitations in its use, such as false positivity, false negativity, and inability of re-administration. We aim to develop a novel fluorescent labeling system which can be repeatedly administered by spray during surgery, using hydroxymethyl rhodamine green (HMRG) as fluorescent scaffold originally designed at our university for cancer detection. [Methods]Primary probe screening was performed using the homogenized glioblastoma (GBM) samples with the fluorescent probe library comprised of more than 320 kinds of HMRG fluorescent scaffold combined with various types of dipeptides. Second probe screening was performed using fresh GBM specimens and the selected probes in primary screening. To identify the responsible enzymes, diced electrophoresis gel (DEG) assay was performed. This method utilizes the combination of two dimensional electrophoresis (isoelectric point and molecular weight) and a multiwell-plate-based fluorometric assay to find protein spots with the specified activities. [Results] The prominent probes were selected based upon the above two-step screenings. We identified two enzymes by proteome analysis and experiments using inhibitors, which was further confirmed with real-time PCR and western blotting. [Discussion] This screening methodology is innovative in that it is based on selecting probes from the probe library that respond to clinical samples rather than creating probes from the responsible enzymes. Practical fluorescent probes can be established even for low-grade gliomas, which would be a breakthrough for rapid intraoperative diagnosis in glioma surgery. [Conclusion] HMRG-based aminopeptidase fluorescent probes may be effective for GBM detection.

BACKGROUND Todai OncoPanel (TOP) is a targeted sequencing panel newly invented by the University of Tokyo, which allows us to prospectively investigate on genomic abnormalities of multiple cancer-related genes. The objective of this study was to assess its role in molecular diagnosis as well as in patient treatment. METHODS Thirteen cases of brain tumors were analyzed: 10 gliomas and glioneural tumors, 2 meningiomas, and 1 intracranial sarcoma. DNA and RNA were obtained from tumor samples; DNA was obtained from peripheral blood as well. The TOP DNA panel analyzed point mutations, insertions, deletions, and copy number variations of 464 cancer-related genes. The TOP RNA panel detected fusions, exon skipping, and expression abnormalities of 463 genes. RESULTS In all cases of gliomas, the tumor content was abundantly high and the analysis was performed with sufficient depth. The IDH1/2 mutation status and chromosome 1p/19q status were completely consistent with those obtained by Sanger sequencing and microsatellite analysis. In benign brain tumors, the tumor content tended to be low. The number of gene mutations identified also tended to be low. In a case of glioneuronal tumor, FGFR2 mutation was recognized, but there was no clinical trials of FGFR inhibitors open in Japan yet. DISCUSSION TOP is useful as an aid for molecular diagnosis of gliomas. In spite of some targetable genetic abnormalities detected in the tested tumors, its efficacy may be limited by the scant availability of respective molecularly targeted agents.

PURPOSE: Diffusion tensor tractography (DTT) is a method to estimate the direction of white matter fibers, but it is not easy to verify the relationship with brain functions accurately. We developed mixed reality computer graphics (MRCG) which integrates real space (brain surface image) and preoperative 3DCG (3-dimentional computer graphics). In our previous study, this method allowed high precision registration even after brain shift caused by craniotomy, and target registration error was 0.5 ± 0.04mm (mean±SE). We devised a method to investigate the spatial error of DTT and direct cortical stimulation (DCS) using MRCG. We examine its accuracy and clinical application. METHODS: We covered 4 patients with glioma who were underwent awake surgery. The sex of all the patients was male, and the average age was 40. MRCG was constructed by fusing 3DCG prepared before surgery and brain surface photograph (JPEG format) just after craniotomy using our original method, which is called thin plate spline method with some landmarks. Brain function mapping results by DCS were plotted on MRCG. In the surface photograph, we provided grids of 1cm square, then we jagged the site where positive symptoms appeared by DCS was true, and the negative site was false. The spatial error between the cortical cortex and brain function mapping estimated by arcuate fiber of DTT was measured. RESULTS: In 4 cases, DCS was performed in 36 places (average 9 places / case). The sensitivity and specificity of DTT were 0.21 and 0.68, false positive rate and false negative rate were 0.31 and 0.79, respectively. In surgery, the tumor resection was performed with reference to MRCG reflecting the brain function mapping result. CONCLUSION: The proposed method investigated at real space and virtual space suggests DTT does not necessarily match the results of DCS.

PURPOSE: 5-Amino Levulinic Acid (5-ALA) is commonly used as an intraoperative aid in malignant glioma surgery, which has been proved to be effective for more radical tumor resection and better patient prognosis. However, there are some limitations in its use, such as false positivity, false negativity, and inability of re-administration. We aim to develop a novel fluorescent labeling system, which can be repeatedly administered by spray during surgery, using hydroxymethyl rhodamine green (HMRG) and 2 methyl siliconrhodamine (2MeSiR600) as host fluorescent nuclei originally designed at our university for cancer detection, complementing 5-ALA. METHODS: Four groups of homogenized samples were prepared from frozen tissues which consisted of 10 peritumoral specimens, 5 glioblastomas, 5 astrocytomas, and 5 oligodendrogliomas. Probe screening was performed using the fluorescent probe library comprised of HMRG and 2MeSiR600 host fluorescent nuclei combined with various types of dipeptides. More than 720 kinds of fluorescent probes were applied to homogenized lysates. According to the fluorescence intensity measured over time after application, probes exhibiting strong focal fluorescent marks with large difference between peritumor and tumor tissues were selected as valid probes. The selected probes were then validated by the experiment using fresh specimens. RESULTS & DISCUSSION: The top probes were selected based upon the experiments using homogenized lysates as well as fresh specimens. They were validated prospectively with more surgical cases. With the combination of the two types of fluorescent host nuclei, glioblastoma can be identified in multi-color. CONCLUSION: Fluorescent probes with HMRG and 2MeSiR 600 host nuclei can be effective for intraoperative detection of glioma.

Limited success of immune-based therapies in glioma might be in part due to the lack of enough neoantigens (neoAgs) that can be targeted by immune effector cells. In addition, gliomas also show dynamic clonal evolution over time, possibly leading to immunoediting during progression. In this study, we evaluated neoAg expression change and its relation to immune microenvironment using matched primary and recurrent tumor samples from 25 glioma patients [8 IDH-wildtype glioblastoma (GBM), 9 IDH-mutant astrocytoma (LGG-A), and 8 oligodendroglioma (LGG-O)]. Predicted neoAgs (p-neoAgs) deriving from missense mutations were identified by whole-exome sequencing (WES) analysis and the MHC class I binding prediction algorism NetMHCpan2.8. Expressed neoAgs (e-neoAgs) among the p-neoAgs were determined by incorporating RNA sequencing (RNA-seq) data. The ratio of e-neoAgs to p-neoAgs (“neoAg expression ratio”) on each sample significantly decreased at recurrence (p = 0.003) and was particularly strong in neoAg with a higher affinity for MHC class I. Similar results were obtained for GBM, LGG-A, and LGG-O, when separately analyzed. RNA-seq-based differential gene expression analyses including gene ontology analysis and pair-designed gene set enrichment analysis illustrated that the cases with strongly reduced neoAg expression ratio compared to primary counterpart (top 8), but not those without (bottom 8), retained gene expression related to antigen presentation machinery and gained immune effector cells at recurrence. These in silicofindings were consistent with immunohistochemistry for CD8 and MHC class I on tumor samples. These data may suggest that the tumor cells with reduced neoAg expression survived at recurrence as a result of persistent anti-tumor immune responses in some gliomas under the standard treatment. Reduced neoAg expression and impaired APM during progression may play complementary roles in the immune evasion of gliomas.

OBJECTIVE: A fluorescent probe such as 5-aminolevulinic acid achieves greater extent of resection in glioma surgery and thus serves as a valuable surgical adjunct. However, its sensitivity and specificity are yet to be perfectly satisfactory. Our novel spray-type fluorescence system using green hydroxymethyl rhodamine green (HMRG) probes has been tested for brain tumors. In this study, we investigated on red dimethyl silicone rhodamine 600 (2MeSiR600) probes, which have a higher sensitivity for peptidase activity than HMRG probes. MATERIALS AND METHODS: Fresh tumor tissues as well as fresh frozen tissues harvested from gliomas resected at our institutions were used in this study. The probe library included over 400 types with different combinations of amino acids attached to 2MeSiR600. Fluorescence intensity was measured over time after probes were applied to homogenized lysates made from fresh frozen tissues. Fluorescent probes which clearly discerned the tumor area from the peripheral area were selected, and then applied to fresh tumor tissues to confirm their selectivity. Tumor tissues were fixed with formalin and stained with hematoxylin and eosin. Immunohistochemistry staining was also performed. The correlation between fluorescence intensity and histopathology including cell density and MIB-1 labeling index was investigated. RESULTS: Probe screening demonstrated some 2MeSiR600 probes exhibited significantly higher fluorescence intensity in the peripheral area compared to the tumor area, which was confirmed when they were applied to fresh tumor tissues. CONCLUSION: 2MeSiR600 probes which selectively fluoresce the peripheral area may enable highly selective tumor recognition in conjunction with 5-aminolevulinic acid or our HMRG probes.

Gain of short arm of chromosome 12 (12p) is commonly observed in testicular germ cell tumors (tGCTs) and also seen in intracranial GCTs (iGCTs). However, little is known about the clinical significance of 12p gain in iGCTs. We have collected over 200 fresh frozen tissue samples of iGCTs through the Intracranial Germ Cell Tumor Genome Analysis Consortium in Japan. Firstly, we analyzed DNA methylation profile in 83 iGCTs, 3 tGCTs (seminomas) and 6 normal control samples using Infinium Human Methylation 450K BeadChip array (Illumina, CA, USA) in order to determine 12p gain status. Then, fluorescence in situ hybridization (FISH) study was carried out on 3 mixed iGCT cases using 12p/CEP12 probe (Abbott Molecular, Abbott park, IL, USA). Lastly, 58 iGCTs with clinicopathological information were analyzed for progression-free survival (PFS) and overall survival (OS). Gain of 12p was observed in 100% (3/3) of seminoma, 14% (3/22) of germinoma, 17% (1/6) of mature teratoma, 25% (1/4) of immature teratoma, 55% (11/20) of mixed germ cell tumor, 100% (4/4) of yolk sac tumor, 100% (1/1) of embryonal carcinoma, and 100% (1/1) of choriocarcinoma. In total, 45% (37/83) of iGCT showed 12p gain. Different histological components in each mixed GCT shared the same 12p copy number status within each mixed GCT case. Both PFS and OS were significantly worse in iGCTs with 12p gain (PFS: P=0.027, OS: P=0.0012). Gain of 12p can be a molecular marker to predict prognosis and represents an early event in tumorigenesis prior to histological differentiation in iGCTs.

BACKGROUND: Limited success of immune-based therapies in glioma might be in part due to the lack of enough neoantigens (neoAgs) that can be targeted by immune effector cells. In addition, gliomas also show dynamic clonal evolution over time, possibly leading to immunoediting during progression. Here we conducted multiomics analysis on glioma primary and recurrent pair samples to evaluate temporal changes of glioma-immune system interactions and clarify the immune evasion mechanism characteristic of gliomas. MATERIALS AND METHODS: In total, 25 WHO grade II-IV glioma cases with primary and recurrent pair samples were enrolled [8 IDH-wildtype glioblastoma (GBM), 9 IDH-mutant astrocytoma (LGG-A), and 8 oligodendroglioma (LGG-O)]. All cases received only standard therapies, but not immunotheapies. Somatic missense mutations were identified by whole-exome sequencing. Predicted neoAgs (p-neoAgs) derived from missense mutations were identified based on the MHC class I binding prediction algorism NetMHCpan2.8. In addition, “expressed” neoAgs at mRNA levels (e-neoAgs) were specified according to RNA-seq data; the neoAg expression ratio of each sample was calculated by dividing e-neoAg count by p-neoAg. Differential gene expression analyses were performed using Gene Ontology (GO) analysis or pair-designed gene set enrichment analysis (GSEA) for the tumor-infiltrating leukocytes (TILs) and antigen presentation machinery (APM). Finally, immunohistochemistry (IHC) for CD8 and MHC class I on tumor samples was assessed for validation. RESULTS: Mutational load, p- and e-neoAg count did not significantly differ between primary and recurrent tumors. However, the neoAg expression ratio significantly decreased in recurrent tumors (p = 0.003) and was particularly strong in neoAgs with a higher affinity for MHC class I. Similar trends were demonstrated for GBM, LGG-A, and LGG-O, when analyzed separately. GO analysis revealed that a number of GO terms related to active immune response such as including “CXCR chemokine receptor binding” and “leukocyte migration” were significantly upregulated at recurrence in the cases with strongly reduced neoAg expression ratio (top 8 cases). In the same cases, GSEA for TILs demonstrated a few subpopulations including activated CD8+ T cells significantly increased at recurrence. On the other hand, several TIL subpopulations as well as many APM-related gene sets significantly depleted in those with less significantly reduced neoAg expression ratio (bottom 8). These findings were consistent with those of IHC for CD8 and MHC class I. CONCLUSION: These data may suggest that the tumor cells with reduced neoAg expression survived at recurrence as a result of persistent anti-tumor immune responses in some cases under the standard treatment. Reduced neoAg expression and impaired APM during progression may play complementary roles in the immune evasion of gliomas.

INTRODUCTION Recently, the cancer genome medical system has been rapidly promoted in Japan, such as cancer related gene panel test as NCC oncopanel and Fonduation One being covered by insurance. At the same time, there is a great deal of attention now on hereditary tumor diseases caused by abnormalities in cancer-related genes and their medical treatment. Von Hippel-Lindau (VHL) disease and tuberous sclerosis (TSC) are one such hereditary tumor disease, and develop tumors in multiple organs such as kidney tumors and lung tumors as well as brain tumors. The University of Tokyo Hospital has been developing an in-hospital medical care system for VHL disease and TSC. [VHL disease] If VHL disease is diagnosed at an early stage and strives for early detection and treatment of tumor, it is said that the prognosis is not largely different from normal people. Therefore, our hospital opened a specialized outpatient for VHL disease from 2012, and has carried out genetic diagnosis of VHL disease and medical treatment based on it. [TSC] Recently, the use of Afinitor for SEGA was covered by insurance in Japan, and a system in which multiple departments involved in our hospital cooperated was established, and regular meetings were held to determine treatment policies. DISCUSSION It was the development of genetic counseling system and multidisciplinary cooperation system that was important in any disease. Genetic counseling helps patients with illness and their families to genetically understand and adapt to illness. In the case of hereditary diseases, the practice of genetic counseling was very useful. In addition, since it is a disease involving multiple organs, close cooperation with other in-hospital clinics has been extremely useful for early detection and treatment of patient’s lesions.

BACKGROUND Most cases of glioblastoma recur within one year even under the standard treatment of surgical resection, radiation therapy and chemotherapy. 60–70% of recurrences are local, and in rare cases of metastasis, most are within the CNS. Extradural metastasis is considered exceedingly rare. CASE REPORT We present a 21-year-old man post total resection of right parietal lobe glioblastoma, diagnosed with lumber metastasis. He originally presented with impaired consciousness and left hemiplegia at the age of 20 and underwent gross total resection of the tumor. Pathology was IDH wild type, H3F3A K34R/V wild-type glioblastoma. Radiotherapy and adjuvant temozolomide per the Stupp regimen as well as infusion of bevacizumab were conducted. 6 months after the resection of tumor, the patient presented with severe back pain. Radiographic studies showed an osteolytic mass on the first lumbar vertebrae, and needle biopsy was consistent with glioblastoma. Posterior spinal fusion, internal decompression and radiotherapy were conducted to relieve the pain. At 3 months after the diagnosis of lumbar metastasis, he is currently treated with temozolomide and bevacizumab, without the enlarging of the tumor. DISCUSSION As far as we investigated, there has been 30 cases of vertebral metastasis of glioblastoma reported in literature. Considering the biological obstacles that prevent glioblastomas from infiltrating outside of the CNS, it can be speculated that deposition of tumor cells into the blood stream or excision of the dura due to surgical interventions may attribute to extracranial metastasis. Due to the improvement of overall survival of glioblastoma, vertebral metastasis is suspected to be more common. Therefore, investigation of its risk factors and standardization of its treatment is necessary. CONCLUSION We reported a case of lumbar metastasis of glioblastoma. Extradural metastasis of glioblastoma must be included in differential diagnoses in treating patients with glioblastoma.

PURPOSE 5-ALA is commonly used as an intraoperative tool in malignant glioma surgery, which has been proven effective for radical tumor resection and extended progression-free survival. However, there are some limitations in its use, such as false positivity, false negativity, and inability of re-administration. We aim to develop a novel fluorescent labeling system which can be repeatedly administered by spray during surgery, using hydroxymethyl rhodamine green (HMRG) as fluorescent scaffold originally designed at our university for cancer detection. METHODS Primary probe screening was performed using the homogenized glioblastoma (GBM) samples with the fluorescent probe library comprised of more than 320 kinds of HMRG fluorescent scaffold combined with various types of dipeptides. Second probe screening was performed using fresh GBM specimens and the selected probes in primary screening. To identify the responsible enzymes, diced electrophoresis gel (DEG) assay was performed. This method utilizes the combination of two dimensional electrophoresis (isoelectric point and molecular weight) and a multiwell-plate-based fluorometric assay to find protein spots with the specified activities. RESULTS The prominent probes were selected based upon the above two-step screenings. We identified two enzymes by proteome analysis and experiments using inhibitors, which was further confirmed with real-time PCR and western blotting. DISCUSSION This screening methodology is innovative in that it is based on selecting probes from the probe library that respond to clinical samples rather than creating probes from the responsible enzymes. Practical fluorescent probes can be established even for low-grade gliomas, which would be a breakthrough for rapid intraoperative diagnosis in glioma surgery. CONCLUSION HMRG-based aminopeptidase fluorescent probes may be effective for GBM detection.

INTRODUCTION Intracranial sarcoma is extremely rare among primary brain tumors and often misdiagnosed. Its standard treatment is yet to be established, and treatment options are discussed on a case-by-case basis. Here we report our recent case of intracranial sarcoma review the relevant literature. CASE ILLUSTRATION A 57-year-old right-handed man presented with headache and was found to have a 5cm mass in the right frontal lobe. Gross total resection was achieved without complications. Given the local pathological diagnosis being glioblastoma, adjuvant radiotherapy with concurrent temozolomide was administered. Further pathological examination revealed Capicua (CIC) rearrangement on FISH, which lead to the diagnosis of sarcoma. No further treatment was pursued at that time. However, he noticed rapid decline in the right visual acuity 7 months from the initial diagnosis. MRI demonstrated a rapidly-growing mass in the right optic nerve sized 1.5cm, which was depicted as a high uptake area on FDG-PET, suggestive of recurrence. Two cycles of chemotherapy with vincristine, ifosfamide, doxorubicin, and etoposide as well as GammaKnife stereotactic radiosurgery were performed with partial response. Sustained myelosuppression and debilitating constitutional symptoms precluded additional chemotherapy. No further recurrence was noted 1 year after diagnosis. CONCLUSION We have recently experienced a case of CIC-rearranged intracranial sarcoma. FISH was useful in detecting CIC rearrangement and reaching the correct pathological diagnosis. Rapid recurrence of the tumor was noted, but well controlled with radiochemotherapy.

BACKGROUND Gain of short arm of chromosome 12 (12p) is commonly observed in testicular germ cell tumors (tGCTs). 12p gain is also frequently seen in intracranial GCTs (iGCTs). However, little is known about the clinical significance of 12p gain in iGCTs. MATERIALS AND METHODS We have collected over 200 fresh frozen tissue samples of iGCTs through the Intracranial Germ Cell Tumor Genome Analysis Consortium in Japan. Firstly, we analyzed DNA methylation status in 83 iGCTs, 3 seminomas and 6 normal control samples using Infinium Human Methylation 450K BeadChip array (Illumina, CA). Idat files were processed using R (Version 3.5.3) and minfi package (1.30.0) to generate copy number variations. Compared with average genome-wide copy number level, 12p gain was determined. Then, 58 iGCTs with clinicopathological information were analyzed for progression-free survival (PFS) and overall survival (OS). Those tumors that consist of only either germinoma and/or mature teratoma components were classified as Favorable Histology (FH) and all the others that contains malignant histological components were classified as Unfavorable Histology (UFH). RESULT 12p gain was observed in 100% (3/3) of seminoma, 13.6% (3/22) of germinoma, 16.7% (1/6) of mature teratoma, 25% (1/4) of immature teratoma, 55% (11/20) of mixed germ cell tumor, 100% (4/4) of yolk sac tumor, 100% (1/1) of embryonal carcinoma, and 100% (1/1) of choriocarcinoma. In total, 44.6% (37/83) of iGCT showed 12p gain. Regarding histological classification, the 12p gain rate in UFH (72%, 18/25) was significantly higher than that in FH (12.1%, 4/33, P DISCUSSION 12p gain can be a molecular marker to predict prognosis and histological malignancy in iGCTs.

Introduction: Accumulating recent evidence demonstrated that even low-grade gliomas consist of heterogeneous cell populations and that recurrent tumors often demonstrated further complex clonal evolution, which lead to marked intratumoral heterogeneity. Due to such heterogeneity, the emergence of malignant clones resistant to therapy can occur easily. Therefore, understanding of the changes of the molecular profile during tumor progression is important to develop a proper treatment strategy. To this end, we performed comparative gene profile analysis between primary low-grade and recurrent gliomas. Methods: Multi “omics” analysis was performed in a large number of glioma samples that includes 26 paired samples of primary and recurrent astrocytic tumors. For “omics” analysis, we performed exome sequencing and RNA-sequencing using Illumina HiSeq, as well as epigenomic profile analysis using an Illumina Infinium 450K methylation chip. Results: By comparing somatic non-synonymous mutations of tumor samples, we observed that only a limited number of crucial driver mutations were retained in recurrent tumors in most cases, and that recurrent tumors gained many new mutations, indicating significant temporal heterogeneity. Interestingly, low-grade glioma treated by temozolomide - a common chemotherapeutic agent for glioma - acquired numerous de novo mutations accompanying mutations in mismatch repair genes and showed a “hypermutator phenotype”. Genome-wide DNA methylation analysis revealed characteristic DNA demethylation during malignant progression in a subset of IDH1/2 mutant astrocytic tumors, which initially showed the glioma CpG island methylator phenotype (G-CIMP). We labeled these tumors as “G-CIMP-demethylated”. Most demethylated loci were located outside of the CpG island. DNA demethylation accompanied the upregulation of mitosis related genes, which are considered to be related to the enhanced tumor growth of these G-CIMP-demethylated tumors. Integrated multi “omics” analysis demonstrated that G-CIMP-demethylated gliomas frequently harbor the RB pathway alterations. Conclusions: Recurrent tumors had a markedly different molecular profile from their primary tumor. The characteristic changes observed in the recurrent tumors are expected to be a future therapeutic target.

Low-grade gliomas often undergo malignant progression, and these transformations are a leading cause of death in patients with low-grade gliomas. However, the molecular mechanisms underlying malignant tumor progression are still not well understood. Recent evidence indicates that epigenetic deregulation is an important cause of gliomagenesis; therefore, we examined the impact of epigenetic changes during malignant progression of low-grade gliomas. Specifically, we used the Illumina Infinium Human Methylation 450K BeadChip to perform genome-wide DNA methylation analysis of 120 gliomas and four normal brains. This study sample included 25 matched-pairs of initial low-grade gliomas and recurrent tumors (temporal heterogeneity) and 20 of the 25 recurring tumors recurred as malignant progressions, and one matched-pair of newly emerging malignant lesions and pre-existing lesions (spatial heterogeneity). Analyses of methylation profiles demonstrated that most low-grade gliomas in our sample (43/51; 84%) had a CpG island methylator phenotype (G-CIMP). Remarkably, approximately 50% of secondary glioblastomas that had progressed from low-grade tumors with the G-CIMP status exhibited a characteristic partial demethylation of genomic DNA during malignant progression, but other recurrent gliomas showed no apparent change in DNA methylation pattern. Interestingly, we found that most loci that were demethylated during malignant progression were located outside of CpG islands. The information of histone modifications patterns in normal human astrocytes and embryonal stem cells also showed that the ratio of active marks at the site corresponding to DNA demethylated loci in G-CIMP-demethylated tumors was significantly lower; this finding indicated that most demethylated loci in G-CIMP-demethylated tumors were likely transcriptionally inactive. A small number of the genes that were upregulated and had demethylated CpG islands were associated with cell cycle-related pathway. In summary, we demonstrated that characteristic DNA demethylation occurred during malignant progression of a subset of low-grade gliomas. The mechanisms underlying and consequences of such DNA demethylation should be studied further.