Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

Transcriptomic and epigenetic dissection of spinal ependymoma (SP-EPN) identifies clinically relevant subtypes enriched for tumors with and without NF2 mutation.

Ependymomas encompass multiple clinically relevant tumor types based on localization and molecular profiles. Tumors of the methylation class "spinal ependymoma" (SP-EPN) represent the most common intramedullary neoplasms in children and adults. However, their developmental origin is ill-defined, molecular data are scarce, and the potential heterogeneity within SP-EPN remains unexplored. The only known recurrent genetic events in SP-EPN are loss of chromosome 22q and NF2 mutations, but neither types and frequency of these alterations nor their clinical relevance have been described in a large, epigenetically defined series. Transcriptomic (n = 72), epigenetic (n = 225), genetic (n = 134), and clinical data (n = 112) were integrated for a detailed molecular overview on SP-EPN. Additionally, we mapped SP-EPN transcriptomes to developmental atlases of the developing and adult spinal cord to uncover potential developmental origins of these tumors. The integration of transcriptomic ependymoma data with single-cell atlases of the spinal cord revealed that SP-EPN display the highest similarities to mature adult ependymal cells. Unsupervised hierarchical clustering of transcriptomic data together with integrated analysis of methylation profiles identified two molecular SP-EPN subtypes. Subtype A tumors primarily carried previously known germline or sporadic NF2 mutations together with 22q loss (bi-allelic NF2 loss), resulting in decreased NF2 expression. Furthermore, they more often presented as multilocular disease and demonstrated a significantly reduced progression-free survival as compared to SP-EP subtype B. In contrast, subtype B predominantly contained samples without NF2 mutation detected in sequencing together with 22q loss (monoallelic NF2 loss). These tumors showed regular NF2 expression but more extensive global copy number alterations. Based on integrated molecular profiling of a large multi-center cohort, we identified two distinct SP-EPN subtypes with important implications for genetic counseling, patient surveillance, and drug development priorities.

Glioblastoma with high O6-methyl-guanine DNA methyltransferase expression are more immunologically active than tumors with low MGMT expression.

Background: Glioblastoma (GBM) is a highly lethal brain tumor. The effectiveness of temozolomide (TMZ) treatment in GBM is linked to the methylation status of O6-methyl-guanine DNA methyltransferase (MGMT) promoter. Patients with unmethylated MGMT promoter have limited treatment options available. Consequently, there is a pressing need for alternative therapeutic strategies for such patients. Methods: Data, including transcriptomic and clinical information, as well as information on MGMT promoter methylation status in primary GBM, were obtained from The Cancer Genome Atlas (TCGA) (n=121) and Chinese Glioma Genome Atlas (CGGA) (n=83) datasets. Samples were categorized into high and low MGMT expression groups, MGMT-high (MGMT-H) and MGMT-low (MGMT-L) tumors. A comprehensive transcriptome analysis was conducted to explore the tumor-immune microenvironment. Furthermore, we integrated transcriptome data from 13 GBM patients operated at our institution with findings from tumor-infiltrating lymphocyte (TIL) cultures, specifically investigating their response to autologous tumors. Results: Gene signatures associated with various immune cells, including CD8 T cells, helper T cells, B cells, and macrophages, were noted in MGMT-H tumors. Pathway analysis confirmed the enrichment of immune cell-related pathways. Additionally, biological processes involved in the activation of monocytes and lymphocytes were observed in MGMT-H tumors. Furthermore, TIL culture experiments showed a greater presence of tumor-reactive T cells in MGMT-H tumors compared to MGMT-L tumors. These findings suggest that MGMT-H tumors has a potential for enhanced immune response against tumors mediated by CD8 T cells. Conclusion: Our study provides novel insights into the immune cell composition of MGMT-H tumors, which is characterized by the infiltration of type 1 helper T cells and activated B cells, and also the presence of tumor-reactive T cells evidenced by TIL culture. These findings contribute to a better understanding of the immune response in MGMT-H tumors, emphasizing their potential for immunotherapy. Further studies are warranted to investigate on the mechanisms of MGMT expression and antitumor immunity.

Principal component analysis of early immune cell dynamics during pembrolizumab treatment of advanced urothelial carcinoma.

Immune checkpoint inhibitors have been approved as second-line therapy for patients with advanced urothelial carcinoma (UC). However, which patients will obtain clinical benefit remains to be determined. To identify predictive biomarkers for the pembrolizumab (PEM) response early during treatment, the present study investigated 31 patients with chemotherapy-resistant recurrent or metastatic UC who received 200 mg PEM intravenously every 3 weeks. Blood was taken just before the first dose and again before the second dose, and the peripheral blood mononuclear cells of all 31 pairs of blood samples were immune phenotyped by flow cytometry. Data were assessed by principal component analysis (PCA), correlation analysis and Cox proportional hazards modeling in order to comprehensively determine the effects of PEM on peripheral mononuclear immune cells. Absolute counts of CD45RA+CD27-CCR7- terminally differentiated CD8+ T cells and KLRG1+CD57+ senescent CD8+ T cells were significantly increased after PEM administration (P=0.042 and P=0.043, respectively). Senescent and exhausted CD4+ and CD8+ T cell dynamics were strongly associated with each other. By contrast, counts of monocytic myeloid-derived suppressor cells (mMDSCs) were not associated with other immune cell phenotypes. The results of PCA and non-hierarchical clustering of patients suggested that excessive T cell senescence and differentiation early during treatment were not necessarily associated with a survival benefit. However, decreased mMDSC counts after PEM were associated with improved overall survival. In conclusion, early on-treatment peripheral T cell status was associated with response to PEM; however, it was not associated with clinical benefit. By contrast, decreased peripheral mMDSC counts did predict improved overall survival.

Accurate Preoperative Identification of Motor Speech Area as Termination of Arcuate Fasciculus Depicted by Q-Ball Imaging Tractography.

BACKGROUND: Tractography is one way to predict the distribution of cortical functional domains preoperatively. Diffusion tensor tractography (DTT) is commonly used in clinical practice, but is known to have limitations in delineating crossed fibers, which can be overcome by Q-ball imaging tractography (QBT). We aimed to compare the reliability of these 2 methods based on the spatial correlation between the arcuate fasciculus depicted by tractography and direct cortical stimulation during awake surgery. METHODS: In this study, 15 patients with glioma underwent awake surgery with direct cortical stimulation. Tractography was depicted in a three-dimensional computer graphic model preoperatively, which was integrated with a photograph of the actual brain cortex using our novel mixed-reality technology. The termination of the arcuate fasciculus depicted by either DTT or QBT and the results of direct cortical stimulation were compared, and sensitivity and specificity were calculated in speech-associated brain gyri: pars triangularis, pars opercularis, ventral precentral gyrus, and middle frontal gyrus. RESULTS: QBT had significantly better sensitivity and lower false-positive rate than DTT in the pars opercularis. The same trend was noted for the other gyri. CONCLUSIONS: QBT is more reliable than DTT in identification of the motor speech area and may be clinically useful in brain tumor surgery.

Selection of RNA-based evaluation methods for tumor microenvironment by comparing with histochemical and flow cytometric analyses in gastric cancer.

Understanding the tumor microenvironment (TME) and anti-tumor immune responses in gastric cancer are required for precision immune-oncology. Taking advantage of next-generation sequencing technology, the feasibility and reliability of transcriptome-based TME analysis were investigated. TME of 30 surgically resected gastric cancer tissues was analyzed by RNA-Seq, immunohistochemistry (IHC) and flow cytometry (FCM). RNA-Seq of bulk gastric cancer tissues was computationally analyzed to evaluate TME. Computationally analyzed immune cell composition was validated by comparison with cell densities established by IHC and FCM from the same tumor tissue. Immune cell infiltration and cellular function were also validated with IHC and FCM. Cell proliferation and cell death in the tumor as assessed by RNA-Seq and IHC were compared. Computational tools and gene set analysis for quantifying CD8+ T cells, regulatory T cells and B cells, T cell infiltration and functional status, and cell proliferation and cell death status yielded an excellent correlation with IHC and FCM data. Using these validated transcriptome-based analyses, the immunological status of gastric cancer could be classified into immune-rich and immune-poor subtypes. Transcriptome-based TME analysis is feasible and is valuable for further understanding the immunological status of gastric cancer.

Two distinct phenotypes of immunologically hot gastric cancer subtypes.

An in-depth understanding of the tumor microenvironment (TME) is required for the development of improved combination immunotherapies for gastric cancer. Recently, we classified these cancers into four main types defined by their immunological attributes, namely Hot 1, Hot 2, Intermediate and Cold. Of these, the T cell-inflamed "Hot" tumors were further divided into Hot 1 and Hot 2 with different clinical outcomes. Thus, overall survival and progression-free survival of patients with Hot 1 tumors were shorter than with Hot 2. In the present study, we re-evaluated RNA-Seq data of 6 Hot 1 and 6 Hot 2 gastric cancers to elucidate the underlying reason for the poor prognosis and T cell dysfunction in the former. In addition, 56 Hot 1 and 27 Hot 2 tumors in The Cancer Genome Atlas (TCGA) were analyzed. We report that single sample Gene Set Enrichment Analysis (ssGSEA) and differential gene expression analysis identified differences between Hot 1 and Hot 2 tumors involved in metabolism and cell adhesion pathways. Therefore, it is suggested that strategies to modulate active metabolism in Hot 1 tumors should be integrated into the treatment of these gastric cancers.

Utility of monitoring the serum levetiracetam concentration for intraoperative seizure control during awake craniotomy.

Awake craniotomy is an established procedure for resecting brain tumors in eloquent lesions, and intraoperative seizure is one of the most important complications. Phenytoin is normally used to control intraoperative seizures. Recently, phenytoin was replaced with levetiracetam at our institution because the latter has fewer side effects. While the phenytoin dose is calibrated in accordance with the serum concentration, there is currently no consensus on a method of monitoring the serum concentration of levetiracetam or the effective concentration range needed to control intraoperative seizures during awake craniotomy. The present study therefore aimed to determine whether monitoring the serum levetiracetam concentration is useful for controlling intraoperative seizures during awake craniotomy. The intraoperative serum concentration of levetiracetam during awake craniotomy was measured in 34 patients and compared with that of phenytoin in 33 patients undergoing the same procedure. The levetiracetam concentration inversely correlated with body surface area (BSA) and estimated glomerular filtration rate (eGFR). Levetiracetam was superior to phenytoin in terms of the correlation between the serum concentration and the dose adjusted for BSA and eGFR (correlation coefficient, 0.49 vs 0.21). Furthermore, the serum levetiracetam concentration in patients with intraoperative seizures was below the 95% confidence interval (CI) of the regression line whereas the serum phenytoin concentration of two patients with seizures was within the 95% CI, indicating that evaluating the serum levetiracetam concentration against the BSA and eGFR-adjusted dosage may be useful in preventing intraoperative seizures during awake craniotomy by allowing prediction of the seizure risk and enabling more accurate dosage calibration.

A Novel Topical Fluorescent Probe for Detection of Glioblastoma.

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.

Genetic Characteristics of Mismatch Repair-deficient Glioblastoma.

Mismatch repair (MMR) gene deficiency is rarely observed in gliomas, a constitutional defect is associated with tumorigenesis in Lynch syndrome, and an acquired defect is associated with hypermutation after temozolomide treatment. However, the meaning of MMR gene deficiency in gliomas is unclear. Two cases of MMR-deficient glioblastomas are reported, and mutational status of oncogenes was compared between primary and recurrent tumor samples in a glioblastoma patient with Lynch syndrome. Additionally, the characteristics of MMR-deficient glioblastomas were analyzed using public glioma datasets to determine the meaning of MMR deficiency in gliomas. Case 1 was a glioblastoma patient with Lynch syndrome, and treatment with pembrolizumab for the recurrent tumor was temporarily effective for a short period. Comparison of mutational changes between primary and recurrent tumor samples showed many additional mutated genes associated with multiple signaling pathways in the recurrent tumor. Tumor recurrence and chemoresistance could be associated with intratumoral heterogeneity and accelerated tumor progression due to defects of multiple signaling pathways. Case 2 was a glioblastoma patient with acquired MMR gene deficiency, and she died of rapid progression of bone marrow metastases. This rare clinical course was considered to be associated with gene expression changes and heterogeneity that resulted from MMR gene deficiency. Two cases of MMR gene-deficient glioblastomas were presented, and their genetic characteristics suggested that their clinical courses could be associated with MMR gene deficiency.

A novel scoring method based on RNA-Seq immunograms describing individual cancer-immunity interactions.

Because of the complexity of cancer-immune system interactions, combinations of biomarkers will be required for predicting individual patient responses to treatment and for monitoring combination strategies to overcome treatment resistance. To this end, the "immunogram" has been proposed as a comprehensive framework to capture all relevant immunological variables. Here, we developed a method to convert transcriptomic data into immunogram scores (IGS). This immunogram includes 10 molecular profiles, consisting of innate immunity, priming and activation, T cell response, interferon γ (IFNG) response, inhibitory molecules, regulatory T cells, myeloid-derived suppressor cells (MDSCs), recognition of tumor cells, proliferation, and glycolysis. Using genes related to these 10 parameters, we applied single-sample gene set enrichment analysis (ssGSEA) to 9417 bulk RNA-Seq data from 9362 cancer patients with 29 different solid cancers in The Cancer Genome Atlas (TCGA). Enrichment scores were z-score normalized (Z) for each cancer type or the entire TCGA cohort. The IGS was defined by the formula IGS = 3 + 1.5 × Z so that patients would be well distributed over a range of scores from 1 to 5. The immunograms constructed in this way for all individual patients in the entire TCGA cohort can be accessed at "The RNA-Seq based Cancer Immunogram Web" (https://yamashige33.shinyapps.io/immunogram/).

Continuous intrathecal injection therapy of methotrexate is a therapeutic option in primary CNS lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin's lymphoma, and its prognosis is still very poor despite the conventional therapy of high-dose methotrexate (HD-MTX) followed by whole-brain radiation therapy (WBRT). The purpose of the present study was to evaluate the survival benefit of continuous intrathecal injection therapy of methotrexate (CIT-MTX) combined with the conventional therapy. A total of 26 PCNSL patients treated with CIT-MTX were analyzed. Ten mg of methotrexate were continuously injected into the lateral ventricle via a subcutaneous port over 5 days biweekly for 5 cycles. CIT-MTX was performed with WBRT in addition to HD-MTX in 15 cases, and 11 cases with high risk for HD-MTX were treated with CIT-MTX and WBRT. The response rate of all patients was 92.3%, and median progression-free survival and median overall survival (mOS) were 59.4 months and 93.8 months, respectively. Median OS of patients treated with CIT-MTX in addition to HD-MTX and WBRT was longer than the previously reported mOS with HD-MTX and WBRT (95 vs 33 months). In cases that could not tolerate HD-MTX, mOS of patients treated with CIT-MTX and WBRT was longer than the previously reported mOS with WBRT alone (36.7 vs 18 months). There was no difference in OS between patients with cerebrospinal fluid dissemination and patients without (p = 0.83). Better prognosis in patients treated with CIT-MTX may be derived from stable concentration of methotrexate in the cerebrospinal fluid. CIT-MTX was an effective additional therapeutic option for PCNSL.