Toshihiro Kumabe

Intertumoral Heterogeneity within Medulloblastoma Subgroups

While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Mutually exclusive mutations of KIT and RAS are associated with KIT mRNA expression and chromosomal instability in primary intracranial pure germinomas

Intracranial germ cell tumors (iGCTs) are the second most common brain tumors among children under 15 in Japan. The pathogenesis of iGCTs is largely unexplored. Although a subset of iGCTs is known to have KIT mutation, its impact on the biology and patients’ survival has not been established. In this study, we investigated genes involved in the KIT signaling pathway. 65 iGCTs (30 pure germinomas, 14 teratomas, 18 mixed GCTs, 2 yolk sac tumors, 1 choriocarcinoma) were screened for mutation of KIT, KRAS, NRAS, HRAS, BRAF, PDGFRA, and IDH1 by direct sequencing. KIT expression was examined by immunohistochemistry and quantitative PCR. Chromosomal status was analyzed by array-comparative genomic hybridization (aCGH). Somatic mutations were detected only in KIT and RAS, which were frequently observed in pure germinomas (60.0xa0%), but rare in non-germinomatous GCTs (NGGCTs) (8.6xa0%). All KIT/RAS mutations were mutually exclusive. Regardless of the mutation status or mRNA expression, the KIT protein was expressed in all germinomas, while only in 54.3xa0% of NGGCTs. Amplification of KIT was found in one pure germinoma by aCGH. In pure germinomas, high expression of KIT mRNA was associated with the presence of KIT/RAS alterations and severe chromosomal instability. Our results indicate that alterations of the KIT signaling pathway play an important role in the development of germinomas. Pure germinomas may develop through two distinct pathogeneses: one with KIT/RAS alterations, elevated KIT mRNA expression and severe chromosomal instability, and the other through yet an unidentified mechanism without any of the above abnormalities.

Association between molecular alterations and tumor location and MRI characteristics in anaplastic gliomas

The aim of this study was to determine if molecular alterations are associated with tumor location and radiological characteristics in anaplastic gliomas. We performed a retrospective analysis of 122 anaplastic gliomas for molecular alterations (IDH1/2 mutations, TP53 mutations, and 1p19q co-deletion) to compare MRI features (location and image characteristics). We observed that IDH mutation is strongly associated with frontal location (Pxa0=xa00.001). However, 13 tumors not located in the cerebral cortex were IDH intact tumors (Pxa0<xa00.0001). While IDH mutation and TP53 mutation are significantly associated with AA (pxa0<xa00.0001), IDH mutation and 1p19q co-deletion were significantly associated with AO/AOA (pxa0<xa00.0001). No tumors with IDH mutation and 1p19q co-deletion infiltrated the temporal lobe (Pxa0=xa00.003). The tumors with 1p19q co-deletion and histologically diagnosed as AO/AOA were associated with contrast enhancement on MR images (pxa0=xa00.007, pxa0=xa00.002, respectively) and those with TP53 mutation had a weak association with sharp tumor borders (pxa0=xa00.043). MRI features might be useful to predict molecular profiles in anaplastic gliomas.

Impact of gross total resection in patients with WHO grade III glioma harboring the IDH 1/2 mutation without the 1p/19q co-deletion.

The prognosis of patients with WHO grade III gliomas is highly dependent on their genomic status such as the isocitrate dehydrogenase (IDH) 1/2 mutation and1p/19q co-deletion. However, difficulties have been associated with determining which tumors have certain genomic profiles by preoperative radiographical modalities, and the role of surgical resection in achieving better outcomes remains unclear. This retrospective study included 124 consecutive patients with newly diagnosed grade III gliomas. The genomic status of IDH1/2 and 1p/19q was analyzed in these patients. Tumors were then divided into 3 subgroups based on their genomic status; the IDH 1/2 mutation with the 1p/19q co-deletion (1p/19q co-del), the IDH 1/2 mutation without the 1p/19q co-deletion (non-1p/19q co-del), and the IDH 1/2 wild type (IDH wt). Survival times were compared between patients who underwent gross total resection and those who did not (GTR versus non-GTR). The relationships between genomic statuses and MR imaging characteristics such as ring-like or nodular enhancements by gadolinium, and very low intensity on T1-weighted images with blurry enhancements (T1VL) were also examined. Among all patients with grade III gliomas, GTR patients had longer median survival and progression-free times than those of non-GTR patients (undefined versus 87xa0months, pu2009=u20090.097, and 124 versus 34xa0months, pu2009=u20090.059, respectively). No significant differences were observed in survival between GTR and non-GTR patients in the 1p/19q co-del group (pu2009=u20090.14), or between GTR and non-GTR patients in the IDH wt group (26 and 27xa0months, pu2009=u20090.29). On the other hand, in non-1p/19q co-del group, survival was significantly longer in GTR patients than in non-GTR patients (undefined versus 77xa0months, pu2009=u20090.005). Radiographically, T1VL was detected in most tumors in the non-1p/19q co-del group (78.2u2009%), but only 6 (21.4u2009%) and 17 (41.5u2009%) tumors in the 1p/19q co-del and IDH wt groups, respectively. A correlation was not found between other genomic subgroups and MR imaging findings. Strict surgical removal is important to improve the prognosis of patients with grade III gliomas, especially for tumors with the IDH 1/2 mutation without the 1p/19q co-deletion. The MR finding of T1VL can be used to select candidates for more radical resection.

Hypoxia-mediated cancer stem cells in pseudopalisades with activation of hypoxia-inducible factor-1α/Akt axis in glioblastoma☆☆☆

Pseudopalisades (Ps) around necrotic foci are severely hypoxic and overexpress hypoxia-inducible factor (HIF) in glioblastoma (GBM). Hypoxic regions have been proposed as one of several distinct niches for cancer stem cells (CSCs) in GBM, but little is known about the association between Ps features and CSC properties. Herein, we focused on the biological role of Ps lesions. In clinical cases of GBM, expression of hypoxia-related molecules including HIF-1α, Glut-1, p27(Kip1), and pAkt was significantly increased in perinecrotic Ps lesions compared with nonnecrotic areas and perinecrotic lesions lacking Ps features. Significantly higher expression levels of several CSC-related markers, including CD133, Sox2, CD44s, and aldehyde dehydrogenase (ALDH) 1, were also observed in Ps lesions, which were positively correlated with expression of hypoxia-related molecules and pAkt. Ps lesions also showed increased number of apoptotic cells and decreased bcl-2 and survivin expression compared with the surrounding tissue. Short-term exposure of astrocytoma cell lines to cobalt chloride, which is known to mimic the effect of hypoxia, caused an increase in expression of both hypoxia- and CSC-related markers, in line with increases in the ALDH(high) cell population and number of spheroids. Inhibition of endogenous Akt by LY294002 resulted in decreased expression of Sox2, ALDH1, and CD133, leading to enhancement of cobalt chloride-mediated apoptotic events due to altered ratio of bcl-2 to bax expression. These findings suggest that Ps lesions within GBM may serve as a specialized hypoxic niche, in which the HIF-1α/pAkt axis is activated, in response to severe hypoxia.

OX40 ligand expressed in glioblastoma modulates adaptive immunity depending on the microenvironment: a clue for successful immunotherapy

BackgroundGlioblastoma is the most malignant human brain tumor and has a dismal prognosis; however, some patients show long-term survival. The interaction between the costimulatory molecule OX40 and its ligand OX40L generates key signals for T-cell activation. The augmentation of this interaction enhances antitumor immunity. In this present study, we explored whether OX40 signaling is responsible for antitumor adaptive immunity against glioblastoma and also established therapeutic antiglioma vaccination therapy.MethodsTumor specimens were obtained from patients with primary glioblastoma (nu2009=u2009110) and grade III glioma (nu2009=u200934). Quantitative polymerase chain reaction (PCR), flow cytometry, and immunohistochemistry were used to analyze OX40L expression in human glioblastoma specimens. Functional consequences of OX40 signaling were studied using glioblastoma cell lines, mouse models of glioma, and T cells isolated from human subjects and mice. Cytokine production assay with mouse regulatory T cells was conducted under hypoxic conditions (1.5% O2).ResultsOX40L mRNA was expressed in glioblastoma specimens and higher levels were associated with prolonged progression-free survival of patients with glioblastoma, who had undergone gross total resection. In this regard, OX40L protein was expressed in A172 human glioblastoma cells and its expression was induced under hypoxia, which mimics the microenvironment of glioblastoma. Notably, human CD4 T cells were activated when cocultured in anti-CD3-coated plates with A172 cells expressing OX40L, as judged by the increased production of interferon-γ. To confirm the survival advantage of OX40L expression, we then used mouse glioma models. Mice bearing glioma cells forced to express OX40L did not die during the observed period after intracranial transplantation, whereas all mice bearing glioma cells lacking OX40L died. Such a survival benefit of OX40L was not detected in nude mice with an impaired immune system. Moreover, compared with systemic intraperitoneal injection, the subcutaneous injection of the OX40 agonist antibody together with glioma cell lysates elicited stronger antitumor immunity and prolonged the survival of mice bearing glioma or glioma-initiating cell-like cells. Finally, OX40 triggering activated regulatory T cells cultured under hypoxia led to the induction of the immunosuppressive cytokine IL10.ConclusionGlioblastoma directs immunostimulation or immunosuppression through OX40 signaling, depending on its microenvironment.

Vaginal Delivery under Epidural Analgesia in Pregnant Women with a Diagnosis of Moyamoya Disease

BACKGROUNDnMoyamoya disease more commonly occurs in young people and women, so patients with this disease may experience pregnancy and delivery. Cesarean section (CS) is often chosen as the mode of delivery for these patients in Japan. No appropriate mode of delivery has yet been established for pregnant women with moyamoya disease in terms of stroke prevention. We have used vaginal delivery under epidural analgesia (EA) in such patients unless CS has been indicated for the maternal or fetal reasons. This study retrospectively analyzed our patients with moyamoya disease who gave birth to confirm the safety of vaginal delivery under EA.nnnMETHODSnTwelve consecutive patients diagnosed with moyamoya disease had 14 deliveries at our hospital between September 2004 and January 2013. The incidences of intrapartum stroke were compared between cases of vaginal delivery under EA and CS cases.nnnRESULTSnTen vaginal deliveries under EA and 4 elective CSs were performed. No intrapartum stroke was observed during either vaginal delivery under EA or CS. Among the patients who underwent vaginal delivery under EA, 1 parturient who experienced 2 deliveries suffered transient ischemic attack during both postpartum periods. All 14 infants were healthy without sequelae.nnnCONCLUSIONSnVaginal delivery under EA is an option for patients with moyamoya disease, provided that close cooperation with neurosurgeons, obstetricians, and anesthesiologists is assured.

Early response to chemotherapy as an indicator for the management of germinoma-like tumors of the pineal and/or suprasellar regions

Recent advances in diagnostic imaging and experience with germinomas may allow for the differentiation of central nervous system germinomas from other tumors based on clinical information, without histological verification. We retrospectively analyzed clinically diagnosed germinoma-like tumors of the pineal and/or suprasellar regions. This was done to evaluate the efficacy of our strategy of defining germinoma-compatible tumors based on good responses to initial chemotherapy. The responses to chemotherapy and survival of 34 consecutive patients with germinoma-like tumors who underwent initial treatment from July 2001 to October 2010 were analyzed. The minimum apparent diffusion coefficient (minADC) value and proton magnetic resonance spectroscopy (MRS) were evaluated in recent patients. Twelve patients with histologically verified germinomas and 18 with germinoma-compatible tumors showed early logarithmic decreases in tumor volume in response to initial chemotherapy, typical low minADC values and typical MRS characteristics, including increased choline/creatine ratios, decreased N-acetylasparate/creatine ratios, and large lipid peaks. These patients had good progression-free survival. The other four patients, with histologically verified non-germinomas, showed no response to chemotherapy, and one patient with a pineoblastoma showed a similar minADC value and MRS characteristics to those of patients with germinomas. The response to initial chemotherapy can be used to distinguish germinoma-compatible tumors from non-germinoma in patients with germinoma-like tumors of the pineal and/or suprasellar regions. The evaluation of minADC and proton MRS are useful for distinguishing germinomas from other tumors. However, a subset of non-germinomas may show similar characteristics to germinomas. The benefit of bypassing unnecessary surgical intervention can be achieved, at least in Asian populations with a high incidence of germinomas.

Relaxed pericranial flap for distraction osteogenesis to treat craniosynostosis: a technique for wound reinforcement--technical note.

PurposeAlthough distraction osteogenesis has been widely accepted to treat craniosynostosis, it occasionally results in wound complications. Positing that they are attributable to the tense pericranium under the scalp, we developed a simple technique to relax the pericranial flap.MethodsIn 12- to 15-month-old infants (mean 13xa0months), we placed a coronal skin incision and dissected the scalp at the subgaleal layer. Then, we peeled the intact pericranium away from the skull along the planned osteotomy to obtain flaps with pedicles on the caudal part. After osteotomy and setting of the distraction device, the pericranial flaps freed from the scalp flap were repositioned to fit the osteotomy line, dura, and distraction device. The galea and skin were approximated layer by layer.ResultsThe shape of the skull was successfully corrected, and the bone defect created by expansion was filled by osteogenesis in all patients. During a mean follow-up period of 42.2xa0months, we encountered no wound complications.ConclusionsThe replaced relaxed pericranium closely adhered to the osteotomy, and the distraction device facilitated vascular growth and bone restoration. Bone resorption was prevented and skin expansion promoted. In patients with iatrogenic dural injury, the pericranium over the injured dura serves as a barrier to prevent cerebrospinal fluid leakage.

Opening the ventricle during surgery diminishes survival among patients with newly diagnosed glioblastoma treated with carmustine wafers: a multi-center retrospective study

Carmustine wafers (CW) were approved in Japan for newly diagnosed and recurrent malignant gliomas during 2013. The ventricle is often opened during surgery to achieve maximum resection. While not generally recommended in such situations, CW might be safely achieved by occluding an opened ventricle using gelform or collagen sheets. However, whether CW implantation actually confers a survival benefit for patients who undergo surgery with an open ventricle to treat glioblastoma remains unclear. Clinical, imaging, and survival data were collected in this multicenter retrospective study of 122 consecutive patients with newly diagnosed glioblastoma to determine adverse events and efficacy. Overall, 54 adverse events of all grades developed in 35 (28.6%) patients, with the most common being new seizures (16%). Adverse events did not significantly differ between patients with opened and closed ventricles during surgery. The 10- and 21.7-month, median, progression-free (PFS) and overall survival (OS), respectively did not significantly differ according to resection rates. However, median PFS and OS were significantly longer among patients with closed, than open ventricles (12.8 vs. 7.4 months; pu2009=u20090.0039 and 26.9 vs. 18.6 months; pu2009=u20090.011, respectively). Implanting CW into the resection cavity during concomitant radiochemotherapy with temozolomide seems to yield better survival rates without increased adverse events. Occlusion of the ventricular opening during surgery might be safe for CW implantation, but less so for treating patients with newly diagnosed glioblastoma.