Effect of Intraoperative Ventricular Opening on Recurrence Patterns Following Bis-Chloroethyl-Nitrosourea Wafer Implantation for Newly Diagnosed Glioblastoma.

Objective: To evaluate the effect of ventricular opening (VO) on recurrence patterns in patients with newly diagnosed glioblastoma (GBM) treated with bis-chloroethyl-nitrosourea (BCNU) wafer implantation. Methods: This single-center retrospective study included 40 patients with newly diagnosed GBM who received BCNU wafer implantation after tumor resection between March 2013 and February 2022. The patients were categorized into two groups based on whether VO occurred during the GBM resection. While 18 patients had VO, 22 did not have VO. In cases with VO, the ventricular wall defect is closed with gelatin or oxidized regenerated cellulose and fibrin glue before BCNU wafer implantation. Recurrence patterns-classified as local, diffuse, distant, or multifocal-and time to recurrence were compared between patients with and without VO. Results: The median follow-up period for the entire cohort was 32.2 months (interquartile range, 16.7-38 months). Median survival time was comparable between patients with VO and patients without VO (38 vs. 26 months, p=0.53). Recurrence occurred in 31/40 patients (77.5%) in entire cohort. The incidence of recurrence was comparable between patients with VO and patients without VO (14 [77.8%] vs. 17 [77.3%], p=1.0). No significant differences were seen between the two groups in time to recurrence (p=0.59) or recurrence patterns (p=0.35). Conclusion: Ventricular opening during surgery with BCNU wafer implantation does not seem to influence the recurrence patterns. Ventricular opening does not induce distant recurrence if appropriate ventricular closure is performed.

Characterization of HIF-1α Knockout Primary Human Natural Killer Cells Including Populations in Allogeneic Glioblastoma.

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Natural killer cells (NK) are major innate effector cells with broad cytotoxicity against tumors. Accordingly, NK cells are ideal candidates for cancer immunotherapy, including glioblastoma (GBM). Hypoxia is a common feature of solid tumors, and tumor cells and normal cells adapt to the tumor microenvironment by upregulating the transcription factor hypoxia-inducible factor (HIF)-1α, which can be detrimental to anti-tumor effector immune cell function, including that of NK cells. We knocked out HIF-1α in human primary NK cells using clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9). Then, cellular characterizations were conducted in normoxic and hypoxic conditions. Electroporating two HIF-1α-targeting guide RNA-Cas9 protein complexes inhibited HIF-1α expression in expanded NK cells. HIF-1α knockout human NK cells, including populations in hypoxic conditions, enhanced the growth inhibition of allogeneic GBM cells and induced apoptosis in GBM-cell-derived spheroids. RNA-sequencing revealed that the cytotoxicity of HIF-1α knockout NK cells could be related to increased perforin and TNF expression. The results demonstrated that HIF-1α knockout human NK cells, including populations, enhanced cytotoxicity in an environment mimicking the hypoxic conditions of GBM. CRISPR-Cas9-mediated HIF-1α knockout NK cells, including populations, could be a promising immunotherapeutic alternative in patients with GBM.

Preoperative Blood Counts Predict Overall Survival in Patients Undergoing Surgical Removal of Brain Metastasis.

OBJECTIVE: The prognosis for patients with cancer with brain metastasis (BM) requiring surgical removal is quite limited. Preoperative prognostic factors can provide meaningful information to surgeons, oncologists, and patients. This study evaluated the preoperative blood counts in patients with BM who were treated with surgical removal. METHODS: Between January 2011 and November 2021, 221 consecutive surgeries were conducted on 198 patients with BM. Among the 198 patients, 188 patients with sufficient blood test data and follow-up were analyzed in this study. The tumors originated from the lungs (n = 102, 54.3%), colon (n = 26, 13.3%), breast (n = 13, 6.9%), kidney (n = 8, 4.3%), stomach (n = 6, 3.2%), and others (n = 33, 17.6%). The blood test data included neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cell count, hemoglobin, and albumin. RESULTS: The median follow-up and median survival times were both 11 months (range: 0-139 months). Higher neutrophil-lymphocyte ratio ≥ 3.17, platelet-lymphocyte ratio ≥112.7, systemic immune-inflammation index ≥594.4, systemic inflammation response index ≥1.25 were unfavorable predictors of prognosis for the patients treated with surgical removal for BM (P < 0.001). Furthermore, lower lymphocyte-monocyte ratio < 2.33 and prognostic nutritional index < 48.5 were unfavorable predictors. CONCLUSIONS: Simple, less expensive, routinely ordered preoperative blood count assessments, such as the neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, lymphocyte-monocyte ratio, systemic immune-inflammation index, systemic inflammation response index, and prognostic nutritional index, can predict the overall survival of patients treated with surgical removal for BM.

Pre-treatment systemic inflammation response index and systemic immune inflammation in patients with primary central nerve system lymphoma as a useful prognostic indicator.

PURPOSE: The systemic inflammation response index (SIRI) and systemic immune-inflammation index (SII) are based on neutrophil, monocyte, platelet, and lymphocyte counts. The SIRI and SII are used to predict the survival of patients with malignant tumors. It is well known that the inflammatory immune response is closely related to cancer occurrence and progression. In the present study, we evaluated the potential prognostic significance of SIRI and SII in patients with primary central nervous system lymphoma (PCNSL). METHODS: Fifty-eight consecutive patients were enrolled in this study between November 2006 and May 2022. Among the 58 patients, 47 patients with sufficient blood test data and follow-up were analyzed. The patients with steroid intake at the time point of the blood test and higher C-reactive protein were excluded. RESULTS: The median follow-up and survival times were 31 and 36 months, respectively. The optimal cutoff SIRI value was based on the receiver operating characteristic curve (ROC) for overall survival (OS) and stratified patients into low (< 1.43 × 109/L, n = 22) and high (≥ 1.43 × 109/L, n = 25) SIRI groups. The optimal cutoff SII value based on the ROC for OS stratified patients into low (< 694.9, n = 28) and high (≥ 694.9, n = 19) SII groups. A low SIRI value was associated with longer OS (p = 0.006). Furthermore, a low SII value was associated with longer OS (p = 0.044). The prognostic factors associated with prolonged survival in univariate analysis using the Cox proportional hazard model were age < 65 years, low SIRI, and low SII. The multivariate analysis demonstrated that age < 65 years and low SIRI independently predicted longer OS. CONCLUSION: Simple, less expensive, and routinely ordered preoperative blood count assessments such as SIRI and SII predict the OS of patients with PCNSL. This study demonstrated that PCNSL is associated with pre-treatment systemic immune-inflammation states.

Bulk RNA sequencing reveals the comprehensive genetic characteristics of human cord blood-derived natural killer cells.

Introduction: Innate immune cells are important in tumor immunotherapy. Natural killer cells (NKCs) are also categorized as innate immune cells and can control tumor growth and metastatic spread. Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. NKC-based immunotherapy is a promising treatment strategy against GBM. We previously reported a feeder-free expansion system that yielded large-scale highly purified and cytotoxic NKCs derived from human cord blood (CB). In the present study, we performed comprehensive genomic analyses of NKCs generated from human CB (CBNKCs) as compared those from human peripheral blood (PB) (PBNKCs). Methods: Frozen T cell-free CB mononuclear cells were cultured with recombinant human interleukin (rhIL)-18 and rhIL-2 in anti-NKp46 and anti-CD16 antibody immobilization settings. After 14-day expansion, the total RNA of the CBNKCs or PBNKCs was extracted and transcriptomic analyses was performed to determine their similarities and differences. We also examined CBNKC and PBNKC activity against a GBM cell line. Results: Differential expression gene analysis revealed that some NK activating and inhibitory receptors were significantly downregulated in the CBNKCs compared to PBNKCs. Furthermore, genes related to anti-apoptosis and proliferation were upregulated in the CBNKCs. Enrichment analysis determined that the gene sets related to immune response and cytokines were enriched in the CBNKCs. Gene set enrichment analysis demonstrated that the immune response pathway was upregulated in the CBNKCs. Cytotoxic assays using impedance-based cell analyzer revealed that the CBNKCs enhanced NKC-mediated cytotoxicity on GBM cells as compared to the PBNKCs. Conclusions: We demonstrated the characteristics of human CBNKCs. Cell-based therapy using the CBNKCs is promising for treating GBM.

Antitumor Effects of Intravenous Natural Killer Cell Infusion in an Orthotopic Glioblastoma Xenograft Murine Model and Gene Expression Profile Analysis.

Despite standard multimodality treatment, containing maximum safety resection, temozolomide, radiotherapy, and a tumor-treating field, patients with glioblastoma (GBM) present with a dismal prognosis. Natural killer cell (NKC)-based immunotherapy would play a critical role in GBM treatment. We have previously reported highly activated and ex vivo expanded NK cells derived from human peripheral blood, which exhibited anti-tumor effect against GBM cells. Here, we performed preclinical evaluation of the NK cells using an in vivo orthotopic xenograft model, the U87MG cell-derived brain tumor in NOD/Shi-scid, IL-2RɤKO (NOG) mouse. In the orthotopic xenograft model, the retro-orbital venous injection of NK cells prolonged overall survival of the NOG mouse, indirectly indicating the growth-inhibition effect of NK cells. In addition, we comprehensively summarized the differentially expressed genes, especially focusing on the expression of the NKC-activating receptors' ligands, inhibitory receptors' ligands, chemokines, and chemokine receptors, between murine brain tumor treated with NKCs and with no agents, by using microarray. Furthermore, we also performed differentially expressed gene analysis between an internal and external brain tumor in the orthotopic xenograft model. Our findings could provide pivotal information for the NK-cell-based immunotherapy for patients with GBM.

Clinical Results and Hematologic Predictors of Linear Accelerator-Based Stereotactic Radiosurgery or Fractionated Stereotactic Radiotherapy for Brain Metastasis in Patients Aged 75 Years or Older: A Retrospective Study.

OBJECTIVE: This study aimed to evaluate prognostic factors including pre-radiosurgical blood count in elderly patients (EPs) with brain metastasis (BM) who were treated using linear accelerator (LINAC)-based stereotactic radiosurgery (SRS) and fractionated stereotactic radiotherapy (fSRT) with a micro-multileaf collimator. METHODS: Between January 2011 and November 2021, 101 consecutive EPs with BM were treated by LINAC-based SRS or fSRT using LINAC with a micro-multileaf collimator. EPs were defined as patients aged ≥75 years. RESULTS: The tumors originated from the lungs (n = 90; 89.1%), colon (n = 2; 2.0%), and others (n = 9; 8.8%) in these EPs. The median pretreatment Karnofsky Performance Status was 80 (range, 40-100). The median follow-up time was 10 months (range, 0-76), as was the median survival. The 6-month, 1-year, and 2-year survival in the EP group was 58.3%, 43.2%, and 28.5%, respectively. Freedom from local failure at 6 months and 1 and 2 years was 97%, 95%, and 91.5%, respectively. Freedom from distant failure at 6 months and 1 and 2 years in EPs was 70.6%, 59.4%, and 54.2%, respectively. A high neutrophil/lymphocyte ratio >5.33 was an unfavorable predictor of prognosis for EPs with BMs treated with SRS and fSRT (P < 0.001). In the EPs, the prognostic factors associated with prolonged survival in the Cox proportional hazards model were being female and a good pretreatment Karnofsky Performance Status. CONCLUSIONS: The findings of our study highlight the efficacy of LINAC-based SRS and fSRT with a micro-multileaf collimator in the treatment of EPs with BMs. Neutrophil/lymphocyte ratio can be an important factor in treatment decisions for EPs with BMs.

Therapeutic Anti-KIR Antibody of 1-7F9 Attenuates the Antitumor Effects of Expanded and Activated Human Primary Natural Killer Cells on In Vitro Glioblastoma-like Cells and Orthotopic Tumors Derived Therefrom.

Glioblastoma (GBM) is the leading malignant intracranial tumor, where prognosis for which has remained extremely poor for two decades. Immunotherapy has recently drawn attention as a cancer treatment, including for GBM. Natural killer (NK) cells are immune cells that attack cancer cells directly and produce antitumor immunity-related cytokines. The adoptive transfer of expanded and activated NK cells is expected to be a promising GBM immunotherapy. We previously established an efficient expansion method that produced highly purified, activated primary human NK cells, which we designated genuine induced NK cells (GiNKs). The GiNKs demonstrated antitumor effects in vitro and in vivo, which were less affected by blockade of the inhibitory checkpoint receptor programmed death 1 (PD-1). In the present study, we assessed the antitumor effects of GiNKs, both alone and combined with an antibody targeting killer Ig-like receptor 2DLs (KIR2DL1 and DL2/3, both inhibitory checkpoint receptors of NK cells) in vitro and in vivo with U87MG GBM-like cells and the T98G GBM cell line. Impedance-based real-time cell growth assays and apoptosis detection assays revealed that the GiNKs exhibited growth inhibitory effects on U87MG and T98G cells by inducing apoptosis. KIR2DL1 blockade attenuated the growth inhibition of the cell lines in vitro. The intracranial administration of GiNKs prolonged the overall survival of the U87MG-derived orthotopic xenograft brain tumor model. The KIR2DL1 blockade did not enhance the antitumor effects; rather, it attenuated it in the same manner as in the in vitro experiment. GiNK immunotherapy directly administered to the brain could be a promising immunotherapeutic alternative for patients with GBM. Furthermore, KIR2DL1 blockade appeared to require caution when used concomitantly with GiNKs.

CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor and has "immunologically cold" features. Changing GBM to an "immunologically hot" tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs. METHODS: The GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice. RESULTS: We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice. CONCLUSION: The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.

Local administration of shikonin improved the overall survival in orthotopic murine glioblastoma models with temozolomide resistance.

BACKGROUND: Glioblastoma is a type of intracranial malignancy. Shikonin, a Chinese traditional medicine, has been shown to have anti-tumor efficacy toward human glioblastoma cells in vitro. However, shikonin cannot easily cross the blood-brain barrier. To address this issue, we evaluated the anti-tumor effects of direct intracranial infusion of shikonin in in vivo orthotopic syngeneic murine glioblastoma models using C57BL/6 mice. MATERIALS AND METHODS: The cytotoxic effects of shikonin against murine glioblastoma cells, SB28 and CT-2A, were reported resistance to temozolomide, were evaluated using an allophycocyanin-conjugated annexin V and propidium iodide assay with flow cytometry. Impedance-based real-time cell analysis (RTCA) was used to analyze the inhibitory effects of shikonin on growth and proliferation. To evaluate the anti-tumor activity of shikonin in vivo, we used orthotopic syngeneic murine glioblastoma models with SB28 and CT-2A cells. RESULTS: In flow cytometry-based cytotoxic assays, shikonin induced apoptosis. RTCA indicated that shikonin decreased the cell index of murine glioblastoma cells, SB28 and CT-2A, in a dose-dependent manner (p < 0.0001 for both cell lines), while temozolomide did not (p = 0.91 and 0.82, respectively). In murine glioblastoma models, SB28 and CT-2A, direct intracranial infusion of shikonin, as a local chemotherapy, improved the overall survival of mice in a dose-dependent manner compared with control groups (p < 0.0001 and p = 0.02, respectively). While temozolomide did not (p = 0.48 and 0.52, respectively). CONCLUSIONS: The direct intracranial infusion of shikonin has potential as a local therapy for patients with glioblastoma.