Immune-based therapies have shown limited efficacy in glioma thus far. This might be at least in part due to insufficient numbers of neoantigens, thought to be targets of immune attack. In addition, we hypothesized that dynamic genetic and epigenetic tumor evolution in gliomas might also affect the mutation/neoantigen landscape and contribute to treatment resistance through immune evasion. Here, we investigated changes in the neoantigen landscape and immunologic features during glioma progression using exome and RNA-seq of paired primary and recurrent tumor samples obtained from 25 WHO grade II-IV glioma patients (glioblastoma, IDHwild-type, n ¼ 8; grade II-III astrocytoma, IDH-mutant, n ¼ 9; and grade II-III oligodendroglioma, IDH-mutant, 1p/19qcodeleted, n ¼ 8). The number of missense mutations, predicted neoantigens, or expressed neoantigens was not significantly different between primary and recurrent tumors. However, we found that in individual patients the ratio of expressed neoantigens to predicted neoantigens, designated the "neoantigen expression ratio," decreased significantly at recurrence (P ¼ 0.003). This phenomenon was particularly pronounced for "high-affinity," "clonal," and "passenger gene-derived" neoantigens. Gene expression and IHC analyses suggested that the decreased neoantigen expression ratio was associated with intact antigen presentation machinery, increased tumorinfiltrating immune cells, and ongoing immune responses. Our findings imply that decreased expression of highly immunogenic neoantigens, possibly due to persistent immune selection pressure, might be one of the immune evasion mechanisms along with tumor clonal evolution in some gliomas.

Purpose: Five-aminolevulinic acid (5-ALA) is widely used as an intraoperative fluorescent probe for radical resection of high-grade glioma, and thus aids in extending progression-free survival of patients. However, there exist some cases where 5-ALA fails to fluoresce. In some other cases, it may undergo fluorescence quenching but cannot be orally readministered during surgery. This study aimed to develop a novel hydroxymethyl rhodamine green (HMRG)-based fluorescence labeling system that can be repeatedly administered as a topical spray during surgery for the detection of glioblastoma. Experimental Design: We performed a three-stage probe screening using tumor lysates and fresh tumor tissues with our probe library consisting of a variety of HMRG probes with different dipeptides. We then performed proteome and transcript expression analyses to detect candidate enzymes responsible for cleaving the probe. Moreover, in vitro and ex vivo studies using U87 glioblastoma cell line were conducted to validate the findings. Results: The probe screening identified proline-arginine–HMRG (PR-HMRG) as the optimal probe that distinguished tumors from peritumoral tissues. Proteome analysis identified calpain-1 (CAPN1) to be responsible for cleaving the probe. CAPN1 was highly expressed in tumor tissues which reacted to the PR-HMRG probe. Knockdown of this enzyme suppressed fluorescence intensity in U87 glioblastoma cells. In situ assay using a mouse U87 xenograft model demonstrated marked contrast of fluorescence with the probe between the tumor and peritumoral tissues. Conclusions: The novel fluorescent probe PR-HMRG is effective in detecting glioblastoma when applied topically. Further investigations are warranted to assess the efficacy and safety of its clinical use.

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Background In neurosurgery, it is important to inspect the spatial correspondence between the preoperative medical image (virtual space), and the intraoperative findings (real space) to improve the safety of the surgery. Navigation systems and related modalities have been reported as methods for matching this correspondence. However, because of the influence of the brain shift accompanying craniotomy, registration accuracy is reduced. In the present study, to overcome these issues, we developed a spatially accurate registration method of medical fusion 3-dimensional computer graphics and the intraoperative brain surface photograph, and its registration accuracy was measured. Methods The subjects included 16 patients with glioma. Nonrigid registration using the landmarks and thin-plate spline methods was performed for the fusion 3-dimensional computer graphics and the intraoperative brain surface photograph, termed mixed-reality computer graphics. Regarding the registration accuracy measurement, the target registration error was measured by two neurosurgeons, with 10 points for each case at the midpoint of the landmarks. Results The number of target registration error measurement points was 160 in the 16 cases. The target registration error was 0.72 ± 0.04 mm. Aligning the intraoperative brain surface photograph and the fusion 3-dimensional computer graphics required ∼10 minutes on average. The average number of landmarks used for alignment was 24.6. Conclusions Mixed-reality computer graphics enabled highly precise spatial alignment between the real space and virtual space. Mixed-reality computer graphics have the potential to improve the safety of the surgery by allowing complementary observation of brain surface photographs and fusion 3-dimensional computer graphics.

BACKGROUND Image-guided systems improve the safety, functional outcome, and overall survival of neurosurgery but require extensive equipment. OBJECTIVE To develop an image-guided surgery system that combines the brain surface photographic texture (BSP-T) captured during surgery with 3-dimensional computer graphics (3DCG) using projection mapping. METHODS Patients who underwent initial surgery with brain tumors were prospectively enrolled. The texture of the 3DCG (3DCG-T) was obtained from 3DCG under similar conditions as those when capturing the brain surface photographs. The position and orientation at the time of 3DCG-T acquisition were used as the reference. The correct position and orientation of the BSP-T were obtained by aligning the BSP-T with the 3DCG-T using normalized mutual information. The BSP-T was combined with and displayed on the 3DCG using projection mapping. This mixed-reality projection mapping (MRPM) was used prospectively in 15 patients (mean age 46.6 yr, 6 males). The difference between the centerlines of surface blood vessels on the BSP-T and 3DCG constituted the target registration error (TRE) and was measured in 16 fields of the craniotomy area. We also measured the time required for image processing. RESULTS The TRE was measured at 158 locations in the 15 patients, with an average of 1.19 ± 0.14 mm (mean ± standard error). The average image processing time was 16.58 min. CONCLUSION Our MRPM method does not require extensive equipment while presenting information of patients’ anatomy together with medical images in the same coordinate system. It has the potential to improve patient safety.