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.


Statistics
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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection All-in-one Fluorescence Microscope software (KEYENCE, BZ-H3A) was used for immunohistochemistry image aquisition. For qPCR analyses, we used StepOne Software v2.3 (Applied Biosystems StepOne™ and StepOnePlus™ Real-Time PCR Systems ). For the analysis of tumor volume, AW Workstation and AW Server platforms (AW server 2, release 5.5, GE Healthcare, Waukesha, Wis) and SYNAPSE VINCENT imaging system (Fujifilm Medical Co., Tokyo, Japan) were used on gadolinium-enhanced MRI T1WI images.

Data analysis
To evaluate immunohistochemical findings (microvessel density, vessel diameter and positive cell count), we used BZ-9000 Fluorescence Microscope (KEYENCE), and all-in-one Fluorescence Microscope software (KEYENCE, Analysis application hybrid cell count; BZ-H3C). Statistics was completed using IBM SPSS Statistics.
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability All data supporting the findings of this study are available within the article and its Supplementary Information Files and from the corresponding author on reasonable request.

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Life sciences study design
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Human research participants
Policy information about studies involving human research participants Population characteristics NF2 patients diagnosed with progressive schwannoma Both genders (3 males and 4 females in this study) Announcement of a diagnosis Positive genomic DNA typing for HLA-A*2402, 0201, 0206 and 0207 (HLA Laboratory, Kyoto, Japan) Age between 12 and 79 years No surgery, irradiation, or chemotherapy in the 4 weeks prior to enrolment in the study Life expectancy >3 months Written informed consents obtained Laboratory test values prior to vaccination Neutrophil count ≥1000/mm3 Platelet count ≥50,000/mm3 Hemoglobin level ≥8.0g/dl Aspartate aminotransferase and alanine aminotransferase ≤ 4.0x the institutional normal upper limits Total bilirubin ≤ 1.5x the institutional normal upper limits Creatinine ≤ 2.0mg/dl No uncontrollable pleural, peritoneal or cardiac effusion

Recruitment
The trial was registered at University hospital Medical Information Network (UMIN). UMIN000023565, Open public recruiting Ethics oversight All protocols were approved by the Keio University Ethics Committee (number: 20150421), and conducted in accordance with the Helsinki declaration on experimentation on human subjects. Adherence to the trial protocol and accuracy of the completed case report forms and the electronic datasets were assessed at a minimum of three external monitoring visits. The Keio University Clinical and Translational Center, which acts as an independent academic contract research organization, performed the monitoring.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

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October 2018 Clinical data Policy information about clinical studies All manuscripts should comply with the ICMJE guidelines for publication of clinical research and a completed CONSORT checklist must be included with all submissions.

Clinical trial registration
The trial was registered at UMIN (UMIN000023565).

Study protocol
Full trial protocol was accessed in UMIN. August 17, 2016-March 31, 2021 Outcomes Primary outcome was the safety of the vaccine. Secondary outcomes were clinical efficacy parameters including tumor size, hearing ability, and immunological response. Toxicity was assessed using the Common Terminology Criteria for Adverse Events version 4.0 at each visit. To evaluate clinical responses, computed tomography (CT), magnetic resonance imaging (MRI) and hearing examinations were performed within 2 weeks before the first vaccination, after five vaccinations (at the 3-month timepoint), after eight vaccinations (at the 6-month timepoint), and 12 months after the first vaccination. Individual tumor size was volumetrically measured via gadolinium-enhanced imaging. In the present study, volumetric reductions of > 20% were deemed to indicate a radiological response.Hearing responses were evaluated via maximum word recognition scores (WRSs) and pure-tone audiogram (PTA) testing. Improvement or deterioration in hearing were defined as a change of at least 10% in WRS. For evaluation of PTA, improvement or deterioration of hearing were defined as a change of at least 10 dB from 1 to 3 kHz.

Sequence & imaging parameters
Gadolinium-enhanced MRI (Tumor volume): FOV 256mm matrix 256x256 slice thickness 1mm T1WI cube (3D FSE) TR/ TE 400/12.5, GE discovery 750 CT perfusion (Tumor blood volume): CT perfusion was obtained with area detector CT (Aquillion one;Canon medical system) and analyzed with the application on the CT. Area of acquisition a whole brain scan Diffusion MRI Used Not used