Published papers

Introduction: The role of immune checkpoint molecules and the tumor immune microenvironment in the development of intracranial germ cell tumors remains unclear. Methods: We investigated the expression of programed cell death-1 (PD-1), programed cell death ligand-1 (PD-L1), and tumor-infiltrating lymphocytes (TILs) in 8 patients who had intracranial germinomas with sufficient tumor tissue by immunohistochemistry, to analyze the associations between their clinical courses and radiological features. The 8 patients were categorized based on the duration between symptom onset and pathological diagnosis into the long-term onset (LTO) group (> 1 year of symptoms) and the short-term onset (STO) group (< 1 year of symptoms). Results: Three patients belonged to the LTO group and 5 patients to the STO group. Compared with STO tumors, LTO tumors were significantly associated with a lower ratio of PD-L1–positive tumor cells (p = 0.012), higher number of infiltrating CD3- and CD8-positive lymphocytes (p = 0.016, 0.003, respectively), and lower ratio of PD-1–positive cells per CD8-positive lymphocytes (p = 0.047). LTO germinomas were significantly smaller in size than STO tumors, not associated with hydrocephalus, and tended to be present in patients with older age at diagnosis and atypical tumor location. Conclusions: Our data suggest that the tumor immune microenvironment, including PD-1/PD-L1 signaling, is associated with the growth of intracranial germinomas.

Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.

Background: Skull base chordomas (SBCs) are rare clinically aggressive neoplasms, developing local recurrences after surgical resection. Although SBCs have traditionally been resected by craniotomy or microscopic transsphenoidal surgery (TSS), the recent development of the endoscopic endonasal approach (EEA) has revolutionized treatment strategies through minimally invasive techniques. This study aimed to evaluate clinical outcomes after traditional microsurgeries or EEAs for SBCs. Methods: The present retrospective study investigated 66 patients with primary SBCs who underwent surgery between 1977 and 2019. Resection was performed via EEA in 17 cases, craniotomy in 23, transoral approach in 8, TSS in 12, staged surgery in 4, and others in 2. The median follow-up period for progression-free survival (PFS) was 19.5 months. Results: There were no significant differences in preoperative tumor volume or resection rate among these approaches. The incidence of postoperative cranial nerve palsy was significantly lower in EEA than that in craniotomy (P < 0.05). Although total resection was observed in 4 cases of EEA expanding into the superior and inferior part of the clivus, no cases of transoral approach or TSS achieved total resection for both parts. No significant difference in PFS was found among these approaches. Multivariate analysis showed that being female and the absence of radiotherapy were significantly associated with shorter PFS (P < 0.05 and P < 0.001, respectively). The resection rate was not associated with PFS. Conclusions: EEA is a less invasive surgical approach for SBCs. The development of surgical instruments and postoperative radiotherapy will further improve patients' outcomes.

Anti-angiogenic therapy, targeting vascular endothelial growth factor (VEGF)-A/VEGF receptors (VEGFRs), is beneficial for tumor growth prevention in a malignant glioma. A simultaneous blockade using both bevacizumab (Bev), which targets circulating VEGF-A, and a multi-kinase inhibitor on VEGFRs was more effective for advanced solid cancers, including melanoma and renal cell carcinoma. However, previous clinical trials demonstrated a high adverse event rate. Additionally, no studies previously assessed treatment efficacy and safety using both VEGF-A and VEGFR-targeted agents for malignant gliomas. We had conducted clinical trials investigating VEGFRs peptide vaccination in patients with malignant gliomas, in which the treatment exhibited safety and yielded therapeutic effects in some patients. The combined use of Bev and VEGFRs vaccination may enhance the anti-tumor effect in malignant gliomas. In this pilot study, the adverse event profile in patients treated with Bev after the vaccination was investigated to establish this treatment strategy, in comparison to those treated with Bev collected from the published data or treated with the vaccination alone. In our previous clinical studies on patients with malignant gliomas, Bev was administered to 13 patients after VEGFRs vaccinations. One patient had a Grade 4 pulmonary embolism. Two patients had Grade 2 cerebral infarctions. There were no significant differences in the adverse event rates among patients treated with Bev, with the vaccination, or with Bev after the vaccination. Although careful observation is imperative for patients after this combination treatment strategy, VEGFRs-targeted vaccination may coexist with Bev for malignant gliomas.

High-grade gliomas (HGGs) carry a dismal prognosis despite current treatments. We previously confirmed the safety and immunogenicity of a vaccine treatment targeting tumor angiogenesis with synthetic peptides, for vascular endothelial growth factor receptor (VEGFR) epitopes in recurrent HGG patients. In this study, we evaluated a novel vaccine therapy targeting not only tumor vasculature but also tumor cells, using multiple glioma oncoantigen (GOA)/glioma angiogenesis-associated antigen (GAAA) peptides in HLA-A2402+ recurrent/progressive HGG patients. The vaccine included peptide epitopes from four GOAs (LY6K, DEPDC1, KIF20A, and FOXM1) and two GAAAs (VEGFR1 and VEGFR2). Ten patients received subcutaneous vaccinations. The primary endpoint was the safety of the treatment. T-lymphocyte responses against GOA/GAAA epitopes and treatment response were evaluated secondarily. The treatment was well tolerated without any severe systemic adverse events. The vaccinations induced immunoreactivity to at least three vaccine-targeted GOA/GAAA in all six evaluable patients. The median overall survival time in all patients was 9.2 months. Five achieved progression-free status lasting at least six months. Two recurrent glioblastoma patients demonstrated stable disease. One patient with anaplastic oligoastrocytoma achieved complete response nine months after the vaccination. Taken together, this regimen was well tolerated and induced robust GOA/GAAA-specific T-lymphocyte responses in recurrent/progressive HGG patients.

The anti-VEGF antibody bevacizumab has shown efficacy for the treatment of neurofibromatosis type 2 (NF2). Theoretically, vascular endothelial growth factor receptors (VEGFRs)-specific cytotoxic T lymphocytes (CTLs) can kill both tumor vessel cells and tumor cells expressing VEGFRs. Here we show an exploratory clinical study of VEGFRs peptide vaccine in seven patients with progressive NF2-derived schwannomas. Hearing improves in 2/5 assessable patients (40%) as determined by international guidelines, with increases in word recognition scores. Tumor volume reductions of ≥20% are observed in two patients, including one in which bevacizumab had not been effective. There are no severe adverse events related to the vaccine. Both VEGFR1-specific and VEGFR2-specific CTLs are induced in six patients. Surgery is performed after vaccination in two patients, and significant reductions in the expression of VEGFRs in schwannomas are observed. Therefore, this clinical immunotherapy study demonstrates the safety and preliminary efficacy of VEGFRs peptide vaccination in patients with NF2.

Background: The anterior transpetrosal approach (ATPA) is applied to petroclival and brainstem lesions. Although neurosurgeons need to minimize the risk of neurologic complications, brain retraction is necessary for procedures of ATPA. Bridging veins (BVs) limit mobility of the temporal lobe. In the present study, BVs around the petrous bone were analyzed, focusing on the dural entrance and termination points. Methods: The relationship between subdural and meningeal segments of temporobasal veins (TBVs) was analyzed by preoperative computed tomography venography in 102 patients who underwent ATPA. TBVs were classified by the dural entrance and termination points. Results: TBVs mainly entered the transverse sinus and rarely entered transverse-sigmoid sinus (T-S) junction and superior petrosal sinus (SPS). TBVs entered a dural sinus either directly or indirectly through a meningeal vein. The changes in vascular diameter of the lumen, shape, and course were identified between the subdural and meningeal segments. Generally, BVs with long subdural segment do not limit mobility of the temporal lobe. TBVs draining into the T-S junction and SPS tended to be shorter than those draining into the transverse sinus. Furthermore, a few TBVs indirectly entered the dural sinuses through the meningeal vein (early dural entrance). The subdural segment of these TBVs was much shorter. Conclusions: TBVs entering the T-S junction or SPS with short subdural segment may limit the mobility of the temporal lobe. Changes in vascular diameter, shape, and course were detected by computed tomography venography, which was helpful to detect the subdural-meningeal transition.

Cavernous sinus (CS) invasion is an aggressive behavior exhibited by pituitary neuroendocrine tumors (PitNETs). The cause of CS invasion in PitNETs has not been fully elucidated. The tumor immune microenvironment, known to promote aggressive behavior in various types of tumors, has not been examined for PitNETs. Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) signaling is strongly associated with the tumor immune microenvironment. In the present study, these molecular and histopathological characteristics were examined in invasive non-functional PitNETs (NF-PitNETs). Twenty-seven patients with newly diagnosed NF-PitNETs (with CS invasion: 17, without CS invasion: 10) were analyzed by immunohistochemistry for VEGF-A/VEGFR1 and 2, hypoxia-inducible Factor (HIF), tumor-infiltrating lymphocytes, immunosuppressive cells including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), and immune checkpoint molecules. Previously validated tumor proliferation markers including mitotic count, Ki-67 index, and p53 were also analyzed for their expressions in NF-PitNETs. VEGF-A and VEGFR1 were expressed on not only vascular endothelial cells, but also on tumor cells. The expressions of VEGF-A and VEGFR1 were significantly higher in NF-PitNETs with CS invasion. The number of TAMs and the expression of PD-L1 were also significantly higher in NF-PitNETs with CS invasion than in NF-PitNETs without CS invasion. The high expression of VEGF-A and VEGFR1 and associated immunosuppressive microenvironment were observed in NF-PitNETs with CS invasion, suggesting that a novel targeted therapy can be applied.

OBJECTIVE The anterior transpetrosal approach (ATPA) was established in 1984 and has been particularly effective for petroclival tumors. Although some complications associated with this approach, such as venous hemorrhage in the temporal lobe and nervous disturbances, have been resolved over the years, the incidence rate of CSF leaks has not greatly improved. In this study, some varieties of air cell tracts that are strongly related to CSF leaks are demonstrated. In addition, other pre- and postoperative risk factors for CSF leakage after ATPA are discussed. METHODS Preoperative and postoperative target imaging of the temporal bone was performed in a total of 117 patients who underwent ATPA, and various surgery-related parameters were analyzed. RESULTS The existence of air cells at the petrous apex, as well as fluid collection in the mastoid antrum detected by a postoperative CT scan, were possible risk factors for CSF leakage. Tracts that directly connected to the antrum from the squamous part of the temporal bone and petrous apex, rather than through numerous air cells, were significantly related to CSF leak and were defined as “direct tract.” All patients with a refractory CSF leak possessed “unusual tracts” that connected to the attic, tympanic cavity, or eustachian tube, rather than through the mastoid antrum. CONCLUSIONS Preoperative assessment of petrous pneumatization types is necessary to prevent CSF leaks. Direct and unusual tracts are particularly strong risk factors for CSF leaks.

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.