Neuroprotective Effects of Genome-Edited Human iPS Cell-Derived Neural Stem/Progenitor Cells on Traumatic Brain Injury.

Despite developing neurosurgical procedures, few treatment options have achieved functional recovery from traumatic brain injury (TBI). Neural stem/progenitor cells (NS/PCs) may produce a long-term effect on neurological recovery. Although induced pluripotent stem cells (iPSCs) can overcome ethical and practical issues of human embryonic or fetal-derived tissues in clinical applications, the tumorigenicity of iPSC-derived populations remains an obstacle to their safe use in regenerative medicine. We herein established a novel treatment strategy for TBI using iPSCs expressing the enzyme-prodrug gene yeast cytosine deaminase-uracil phosphoribosyl transferase (yCD-UPRT). NS/PCs derived from human iPSCs displayed stable and high transgene expression of yCD-UPRT following CRISPR/Cas9-mediated genome editing. In vivo bioluminescent imaging and histopathological analysis demonstrated that NS/PCs concentrated around the damaged cortex of the TBI mouse model. During the subacute phase, performances in both beam walking test and accelerating rotarod test were significantly improved in the treatment group transplanted with genome-edited iPSC-derived NS/PCs compared with the control group. The injury area visualized by extravasation of Evans blue was smaller in the treatment group compared with the control group, suggesting the prevention of secondary brain injury. During the chronic phase, cerebral atrophy and ventricle enlargement were significantly less evident in the treatment group. Furthermore, after 5-fluorocytosine (5-FC) administration, 5-fluorouracil converted from 5-FC selectively eliminated undifferentiated NS/PCs while preserving the adjacent neuronal structures. NS/PCs expressing yCD-UPRT can be applied for safe regenerative medicine without the concern for tumorigenesis.

Correction to: Clinical and histopathological analyses of VEGF receptors peptide vaccine in patients with primary glioblastoma - a case series.

Following publication of the original article [1], the authors reported an error in the author group.

Clinical and histopathological analyses of VEGF receptors peptide vaccine in patients with primary glioblastoma - a case series.

BACKGROUND: The expression of vascular endothelial growth factor (VEGF)-A/ VAGF receptors (VEGFRs) signaling plays a pivotal role in the tumor angiogenesis and the development of the immunosuppressive tumor microenvironment in glioblastomas. We have previously conducted exploratory clinical studies investigating VEGFRs peptide vaccination with and without multiple glioma oncoantigens in patients with recurrent high-grade gliomas. Recently, an exploratory clinical investigation of VEGFRs peptide vaccination was conducted in patients with progressive neurofibromatosis type 2. Those studies suggested that cytotoxic T lymphocytes (CTLs) induced by the vaccination can directly kill a wide variety of cells associated with tumor growth, including tumor vessels, tumor cells, and immunosuppressive cells expressing VEGFR1 and/or 2. In the present study, synergistic activity of the combination of VEGFRs peptide vaccination with chemotherapy was evaluated. METHODS: We performed the first clinical trial to assess VEGFR1 and 2 vaccination along with temozolomide (TMZ) -based chemoradiotherapy for the patients with primary glioblastomas. Furthermore, histopathological changes after the vaccination were evaluated using paired pre- and post- vaccination specimens. RESULTS: The disappearance of radiographically enhanced lesion was observed in 2 patients after the vaccination, including one in which the methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter was not observed. The histopathological findings of pre- and post-vaccination specimens demonstrated that tumor vessels showed negative or slight VEGFRs expressions after the vaccination and most endothelial cells were covered with PDGFR-ß-positive pericytes. Notably, CTLs induced by VEGFRs peptide vaccination attacked not only tumor vessels but also tumor cells and regulatory T cells expressing VEGFRs even in recurrent tumors. CONCLUSIONS: VEGFR1 and 2 vaccination may have a preliminary synergistic effect when administered with TMZ. The limitation of the present study was the paucity of the number of the samples. Further studies involving more patients are warranted to confirm the findings of this study. TRIAL REGISTRATION: This study was registered as UMIN000013381 (University Hospital Medical Information Network-Clinical Trial Registry: UMIN-CTR) on 5 March, 2014 and with the Japan Registry of Clinical Trials (jRCT) as jRCTs031180170 on 1 March, 2019.

Difference in the hypoxic immunosuppressive microenvironment of patients with neurofibromatosis type 2 schwannomas and sporadic schwannomas.

BACKGROUND: Neurofibromatosis type 2 (NF2) patients uniformly develop multiple schwannomas. The tumor-microenvironment (TME) is associated with hypoxia and consists of immunosuppressive cells, including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). The hypoxic TME of NF2 schwannomas remains unclear. In addition, no comparative study has investigated immunosuppressive cells in NF2 and sporadic schwannomas. METHODS: In 22 NF2 and 21 sporadic schwannomas, we analyzed the immunohistochemistry for Ki-67, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2, platelet derived growth factor receptor-beta (PDGFR-ß), programmed cell death-1 (PD-1)/ programmed cell death ligand-1 (PD-L1), Foxp3, CD163, CD3, and CD8 to assess the immunosuppressive TME. RESULTS: Most vessels in sporadic schwannomas exhibited slight or negative VEGFR1 and 2 expressions with pericytes coverage. In contrast, large vessels in NF2 schwannomas exhibited strong VEGFR1 and 2 expressions without pericytes. The number of CD3+, CD8+, and CD163+ cells was significantly higher in NF2 schwannomas than in sporadic ones. The expression of PD-L1 and nestin positive cell ratio was higher in NF2 schwannomas than that in sporadic ones. The number of CD163+ cells, nestin positive cell ratio, and HIF-1α expression were significantly associated with shorter progression-free survival in NF2 schwannomas. CONCLUSIONS: This study presents the clinicopathological features of the differences in immunosuppressive cells and the expression of immune checkpoint molecules between NF2 and sporadic schwannomas. Hypoxic TME was first detected in NF2-schwannomas, which was associated with the tumor progression.

Quantitative assessment and clinical relevance of VEGFRs-positive tumor cells in refractory brain tumors.

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)1 and 2 signaling is a potent activator of tumor angiogenesis. Although the expressions of VEGFR1 and VEGFR2 were initially thought to be limited to the endothelial cells, it is now known that both the receptors are expressed in tumor cells. This is the first study wherein VEGFRs-positive tumor cells are quantitatively evaluated for brain tumors with upregulated VEGF/VEGFR signaling. The percentage of VEGFRs-positive tumor cells was quantitatively evaluated in various brain tumors (10 glioblastomas, 22 neurofibromatosis type 2 [NF2]-related schwannomas, 21 sporadic schwannomas, 27 chordomas, 36 meningiomas, 29 hemangioblastomas, 11 hemangiopericytoma, and 13 ependymomas) using immunohistochemistry. VEGF-A expression was also analyzed using quantitative real-time polymerase chain reaction. Double immunofluorescence staining using anti-PDGFR-ß and anti-CD34 antibody, microvessel density, and vessel diameter were analyzed to evaluate the vascular characteristics. Chordomas demonstrated an extremely higher percentage of VEGFR1 and VEGFR2-positive tumor cells than other tumors. In contrast, meningiomas and hemangiopericytomas showed few VEGFRs-positive tumor cells. The percentage of positive tumor cells in chordomas, hemangioblastomas, and NF2 schwannomas was associated with clinical courses, such as shorter progression free survival, and growth speed. Glioblastomas and NF2 schwannomas showed larger tumor vessels without pericyte coverage. The present study is the first to quantitatively analyze VEGFR1- and VEGFR2- positive tumor cells in various types of refractory brain tumors. This novel parameter significantly correlated with the progressive clinical courses.

Clinical Characteristics and Outcomes of Aneurysmal Subarachnoid Hemorrhage Patients Who Live Alone.

It has been shown that living alone is one of the risk factors for unfavorable outcomes in ischemic stroke patients, mostly due to delay in receiving appropriate treatment. A single-center retrospective observational study was conducted to evaluate whether living alone was associated with unfavorable outcomes in aneurysmal subarachnoid hemorrhage (SAH) patients. Among 451 SAH patients admitted to our institution between January 2013 and December 2022, 43 patients who lived alone had sustained SAH at home (group A) and 329 patients who lived with family had sustained SAH at home (group F). The mortality rate (46.5% vs. 29.8%, p = 0.04) and a tendency for having unfavorable outcomes were higher in group A than in group F. The incidence of concomitant hydrocephalus was significantly higher in the former (37.2% vs. 21.3%, p = 0.03). Group A was further classified to the Able to Call (n = 15, group AC) and Unable to Call (n = 28, group UC) subgroups based on their ability to call for help by themselves. Group AC tended to have favorable outcomes (27% vs. 4%, p = 0.04). Treatment to obliterate a ruptured aneurysm had particularly been challenging in group UC, in which the accurate time of onset often remained unidentifiable: Their overall mortality was as high as 57% and their capability to undergo surgical/interventional treatment was only 67%. Perioperative complications resulting from delayed presentation had been common. Considering the present finding that most of those who lived alone could not call for help, further effort is warranted to facilitate early detection of those patients.

Phase I/II Study of a Vascular Endothelial Growth Factor Receptor Vaccine in Patients With NF2-Related Schwannomatosis.

PURPOSE: The humanized antivascular endothelial growth factor (VEGF) antibody bevacizumab (Bev) is efficacious for the treatment of NF2-related schwannomatosis (NF2), previously known as neurofibromatosis type 2. This study evaluated the safety and efficacy of a VEGF receptor (VEGFR) vaccine containing VEGFR1 and VEGFR2 peptides in patients with NF2 with progressive schwannomas (jRCTs031180184). MATERIALS AND METHODS: VEGFR1 and VEGFR2 peptides were injected subcutaneously into infra-axillary and inguinal regions, once a week for 4 weeks and then once a month for 4 months. The primary end point was safety. Secondary end points included tolerability, hearing response, imaging response, and immunologic response. RESULTS: Sixteen patients with NF2 with progressive schwannomas completed treatment and were assessed. No severe vaccine-related adverse events occurred. Among the 13 patients with assessable hearing, word recognition score improved in five patients at 6 months and two at 12 months. Progression of average hearing level of pure tone was 0.168 dB/mo during the year of treatment period, whereas long-term progression was 0.364 dB/mo. Among all 16 patients, a partial response was observed in more than one schwannoma in four (including one in which Bev had not been effective), minor response in 5, and stable disease in 4. Both VEGFR1-specific and VEGFR2-specific cytotoxic T lymphocytes (CTLs) were induced in 11 patients. Two years after vaccination, a radiologic response was achieved in nine of 20 assessable schwannomas. CONCLUSION: This study demonstrated the safety and preliminary efficacy of VEGFR peptide vaccination in patients with NF2. Memory-induced CTLs after VEGFR vaccination may persistently suppress tumor progression.

Spontaneous Intracranial Hypotension Occurring after Craniotomy for Brain Tumor Biopsy Mimicking Postoperative Bleeding.

In this study, we report on a previously healthy 44-year-old man who underwent an open biopsy under general anesthesia for a tumorous lesion found in his left frontal lobe via a small supratentorial craniotomy. While both postoperative course and brain computed tomography (CT) scans had been considered unremarkable, the patient became stuporous on postoperative day (POD) 4. A brain CT obtained on that day showed a subdural hematoma with marked brain shift which we thought might have been due to postoperative bleeding; he was immediately brought to an operating theater for hematoma removal. However, no bleeding source was found, and the brain remained depressed after hematoma evacuation. Furthermore, the brain shift remained unchanged on postoperative CT. While spontaneous intracranial hypotension (SIH) was considered, imaging studies to search for possible cerebrospinal fluid (CSF) leakage in the spinal column were not performed as the patient's condition has improved. However, he became stuporous again on POD 8, which urged us to perform CT myelogram. The CT myelogram showed a massive CSF leakage at the L1-L2 level. Subsequent autologous blood patch has successfully terminated the CSF leakage, and he became fully oriented shortly after the blood patch therapy. Thus, it should be noted that SIH may occur during postoperative period of intracranial surgery, and it may manifest radiographically as a subdural hematoma indistinguishable from postoperative bleeding. SIH should also be included in a differential diagnosis of postoperative headache, regardless of its characteristics, because headache associated with SIH may not always be orthostatic.

Gene therapy using genome-edited iPS cells for targeting malignant glioma.

Glioblastoma is characterized by diffuse infiltration into the normal brain. Invasive glioma stem cells (GSCs) are an underlying cause of treatment failure. Despite the use of multimodal therapies, the prognosis remains dismal. New therapeutic approach targeting invasive GSCs is required. Here, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited human-induced pluripotent stem cell (hiPSC) expressing a suicide gene had higher tumor-trophic migratory capacity compared with mesenchymal stem cells (MSCs), leading to marked in vivo antitumor effects. High migratory capacity in iPSC-NSCs was related to self-repulsive action and pathotropism involved in EphB-ephrinB and CXCL12-CXCR4 signaling. The gene insertion to ACTB provided higher and stable transgene expression than other common insertion sites, such as GAPDH or AAVS1. Ferroptosis was associated with enhanced antitumor immune responses. The thymidylate synthase and dihydroprimidine dehydrogenase expressions predicted the treatment efficacy of therapeutic hiPSC-NSCs. Our results indicate the potential benefit of genome-edited iPS cells based gene therapy for invasive GSCs. Furthermore, the present research concept may become a platform to promote clinical studies using hiPSC.

Middle meningeal artery embolization before craniotomy for infected organizing chronic subdural hematoma: A case report and review of the literature.

Background: Organizing chronic subdural hematoma (OSDH) is intractable and its radical treatment remains controversial. Middle meningeal artery embolization has emerged as an adjunctive treatment to craniotomy for OSDH. Case Description: The patient is an 86-year-old man. He had been taking warfarin for atrial fibrillation and was referred to the department for the treatment of bilateral chronic subdural hematoma (CSDH), which was found on head computed tomography after a fall. Bilateral burr hole drainages were performed, but his hematomas were organized, so the hematomas could not be drained sufficiently. The patient was discharged from the hospital without any neurological symptoms. Two months later, the patient presented with persistent fever and headache and had recurrent bilateral CSDHs. The hematoma on the right side was larger. Based on the initial intraoperative findings, OSDH was suspected, and craniotomy was performed on the right hematoma. Propionibacterium acnes were detected in the hematoma culture, and antimicrobial therapy was started postoperatively. Since the right hematoma recurred on the 7th postoperative day, bilateral middle meningeal artery (MMA) embolization with 20% n-butyl-2-cyanoacrylate was performed, followed by craniotomy for the left hematoma and drainage for the right recurrent hematoma. Antimicrobials were administered for 2 weeks after the last operations. Six months after the operations, both bilateral hematomas had almost disappeared. Conclusion: Craniotomy is effective for the treatment of infected OSDH, and MMA embolization is useful to reduce the risk of bleeding complications in the perioperative period, and may also reduce the recurrence of CSDH.

History and current progress of chronic subdural hematoma.

Chronic subdural hematoma (CSDH) is characterized by an encapsulated collection of old blood. Although CSDH has become the most frequent pathologic entity in daily neurosurgical practice, there are some unresolved research questions. In particular, the causes and recurrent risk factors of CSDH remain as an object of debate. The split of the dural border layer forms a few tiers of dural border cells over the arachnoid layer. Tissue plasminogen activator plays an important role as a key factor of defective coagulation. Historically, CSDH has often been treated via burr hole craniostomy using a closed drainage system. Several different operative strategies and peri-operative strategies such as the addition of burr holes, addition of cavity irrigation, position of drain, or postural position, have been described previously. Although the direction of the drainage tube, residual air, low intensity of T1-weighted images on MRI, and niveau formation have been reported as risk factors for recurrence, antiplatelet or anticoagulant drug use has not yet been verified as a risk factor. Recently, pharmaceutical strategies, including atorvastatin, significantly improved the neurological function in CSDH patients. Many case series, without randomization, have been reported; and given its promising result, several randomized clinical trials using pharmaceutical as well as operative and perioperative strategies were initiated to obtain sufficient data. In contrast, relatively fewer basic studies have achieved clinical applications in CSDH, although it is one of the most common clinical entities. Further scientific basic research may be essential for achieving a novel treatment strategy for CSDH.

Quiescent and Activated Fibroblasts in Lateral Ventricular Meningioma With a Dura-like Membrane.

BACKGROUND: Lateral ventricular meningioma (LVM) is a rare entity, accounting for 0.5%-5% of all intracranial meningiomas. This type of meningioma arises from meningothelial inclusion bodies in the tela choroidea and/or mesenchymal stroma of the choroid plexus. Although not yet fully characterized, a membranous structure is frequently observed around LVMs. This study analyzed quiescent and activated fibroblast phenotypes in LVMs with focus on the relationship between tumor growth and development of the membranous structure. METHODS: This retrospective study analyzed 9 LVM cases for which gross total removal was achieved. Expression of the ependymal cell marker (Forkhead Box J1 [FoxJ1]) was histopathologically evaluated. The distribution of quiescent and activated fibroblasts was also analyzed using anti-fibroblast-specific protein-1 (FSP1)/S100A4 antibody and anti-α-smooth muscle actin (αSMA) antibody, respectively. The control group was 5 cases with primary convexity meningioma for which Simpson grade I removal was achieved. RESULTS: Small LVMs (≤30 mm) were covered by a FoxJ1-positive(+) ependymal cell monolayer; no αSMA(+) cells were detected in the tumor; and a thick membrane capsule was not observed. None of the convexity meningiomas showed FoxJ1(+) cells. Large LVMs (>30 mm) had thick membrane capsules without an ependymal cell monolayer, which resembled dura mater. The FSP1/S100A4(+) and αSM(+) cells were clearly concentrated in the peripheral area just below the thick dura mater-like membrane capsules. CONCLUSIONS: This study found an association between activated fibroblasts and dura mater-like membrane capsules in LVMs. The characteristics of membranous structure in LVMs may differ depending on tumor size.

Novel histopathological classification of meningiomas based on dural invasion.

AIMS: Histological invasion into the adjacent brain parenchyma is frequently investigated in meningioma because it is an important morphological criterion for grade II meningioma according to the 2016 WHO classification. However, few studies have focused on dural invasion of meningiomas. Herein, we propose a novel histopathological classification based on dural invasion of meningiomas. METHODS: Forty-nine cases with WHO grade I meningiomas who underwent Simpson grade I removal were collected. After the meningeal layer (ML) and periosteal layer (PL) of dura mater were visualised by Masson's trichrome stain, we evaluated the depth (to the ML and PL) and the patterns (1, expanding; 2, infiltrating) of dural invasion of meningiomas using serial paraffin sections. Invasion-associated markers, including Ki-67, matrix metalloproteinase (MMP)-1, MMP-9 and MMP-13, aquaporin 1 and Na-K-2Cl cotransporter, were quantitatively analysed by immunohistochemistry. RESULTS: Thirty-five cases (71.4%) showed the dural invasion. In 27 of these 35 cases (77.1%), dural invasion was localised in ML. Type 1 (expanding type) and type 2 (infiltrating type) invasions were observed in 23 and 12 cases, respectively. The recurrence rate in cases with type 2 invasion was significantly higher than that in cases with type 1 invasion. The percentage of MMP-1-positive tumour cells was also significantly higher in cases with dural invasion than those without, suggesting involvement of MMP-1 in dural invasion. CONCLUSIONS: We quantitatively evaluated the depth and patterns of dural invasion in meningiomas. The patterns of dural invasion were associated with meningioma recurrence.

A Pilot Study of the Adverse Events Caused by the Combined Use of Bevacizumab and Vascular Endothelial Growth Factor Receptor-Targeted Vaccination for Patients with a Malignant Glioma.

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.

A Comparative Study Between Traditional Microscopic Surgeries and Endoscopic Endonasal Surgery for Skull Base Chordomas.

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.

Author Correction: A VEGF receptor vaccine demonstrates preliminary efficacy in neurofibromatosis type 2.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Analysis of Temporobasal Vein with Short Subdural Segment for Anterior Transpetrosal Approach.

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.

PITX2 Expression in Non-functional Pituitary Neuroendocrine Tumor with Cavernous Sinus Invasion.

Although most pituitary neuroendocrine tumors (PitNETs) show benign behavior, a significant number of PitNETs exhibit an aggressive course including cavernous sinus (CS) invasion. To date, the cause of CS invasion has not been fully elucidated. In this study, we analyzed the relationship between CS invasion in PitNETs and the expression of PITX2 and SNAIL1, which are the transcription factors associated with the morphogenesis of pituitary gland. Sixty cases with non-functional PitNETs were classified into four types: type 1a, none of CS invasion and suprasellar expansion; type 1b, suprasellar expansion without CS invasion; type 2a, CS invasion without suprasellar expansion; and type 2b, CS invasion with suprasellar expansion. We analyzed the expression of PITX2 and SNAIL1 employing quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry. Other parameters such as mitotic count, Ki-67 index, and p53 expression were also analyzed, which were previously reported as potential tumor proliferative markers in PitNETs. PITX2 expression was significantly higher in PitNETs with CS invasion than PitNETs without CS invasion (P = 0.019). Expression of SNAIL1 was significantly elevated in PitNETs with suprasellar expansion compared with PitNETs without suprasellar expansion (P = 0.02). There was no apparent relationship between CS invasion and mitotic count, Ki-67 index, and p53 expression (mitotic count, P = 0.11; Ki-67 index, P = 0.61; p53, P = 0.66). High PITX2 expression was observed in non-functional PitNETs with CS invasion, suggesting that PITX2 may be involved in CS invasion of PitNETs.

Analysis of Tumor Angiogenesis and Immune Microenvironment in Non-Functional Pituitary Endocrine Tumors.

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.

A VEGF receptor vaccine demonstrates preliminary efficacy in neurofibromatosis type 2.

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.