Protocol for real-time assessment of mitochondrial and glycolytic ATP production in patient-derived glioma stem-like cells.

Glioma cells switch between energetic pathways to adapt and resist therapies. We present a protocol for measuring mitochondrial and glycolytic ATP rates in patient-derived glioma stem-like cells using a Seahorse XF ATP rate assay. We describe steps for growing 3D glioma stem-like cells, attaching cells to the assay plate, preparing drugs, and running the ATP rate assay. We also detail procedures for imaging viable cell numbers and normalization, with tips to overcome pitfalls in Agilent Seahorse assays.

Accelerated tumor progression after COVID-19 infection in patients with glioblastoma: A retrospective case-control study.

Background: We observed rapid tumor progression following COVID-19 infection among patients with glioblastoma and sought to systematically characterize their disease course in a retrospective case-control study. Methods: Using an institutional database, we retrospectively identified a series of COVID-19-positive glioblastoma cases and matched them by age and sex 1:2 to glioblastoma controls who had a negative COVID-19 test during their disease course. Demographic and clinical data were analyzed. Hyperprogression was defined using modified response evaluation criteria in solid tumors criteria. Time to progression and overall survival were estimated using the Kaplan-Meier method. Results: Thirty-two glioblastoma cases with positive COVID-19 testing were matched to 64 glioblastoma controls with negative testing; age, sex, and molecular profiles did not differ between groups. Progression events occurred in 27 cases (84%) and 46 controls (72%). Of these, 14 cases (52%) presented with multifocal disease or leptomeningeal disease at progression compared with 10 controls (22%; P = .0082). Hyperprogression was identified in 13 cases (48%) but only 4 controls (9%; P = .0001). Cases had disease progression at a median of 35 days following COVID-19 testing, compared with 164 days for controls (P = .0001). Median survival from COVID-19 testing until death was 8.3 months for cases but 17 months for controls (P = .0016). Median overall survival from glioblastoma diagnosis was 20.7 months for cases and 24.6 months for controls (P = .672). Conclusions: Patients with glioblastoma may have accelerated disease progression in the first 2 months after COVID-19 infection. Infected patients should be monitored vigilantly. Future investigations should explore tumor-immune microenvironment changes linking tumor progression and COVID-19.

Multidisciplinary Management of Isocitrate Dehydrogenase-Mutated Gliomas in a Contemporary Molecularly Defined Era.

Mutations in isocitrate dehydrogenase (IDH) genes, an early step in the ontogeny of lower-grade gliomas, induce global epigenetic changes characterized by a hypermethylation phenotype and are critical to tumor classification, treatment decision making, and estimation of patient prognosis. The introduction of IDH inhibitors to block the oncogenic neomorphic function of the mutated protein has resulted in new therapeutic options for these patients. To appreciate the implications of these recent IDH inhibitor results, it is important to juxtapose historical outcomes with chemoradiotherapy. Herein, we rationally evaluate recent IDH inhibitor data within historical precedents to guide contemporary decisions regarding the role of observation, maximal safe resection, adjuvant therapies, and the import of patient and tumor variables. The biological underpinnings of the IDH pathway and the mechanisms, impact, and limitations of IDH inhibitors, the actual magnitude of tumor regression and patient benefit, and emergence of resistance pathways are presented to guide future trial development. Management in the current, molecularly defined era will require careful patient selection and risk factor assessment, followed by an open dialog about the results of studies such as INDIGO, as well as mature data from legacy trials, and a discussion about risk-versus-benefit for the choice of treatment, with multidisciplinary decision making as an absolute prerequisite.

Final report of the phase II NEXT/CNS-GCT-4 trial: GemPOx followed by marrow-ablative chemotherapy for recurrent intracranial germ cell tumors.

Background: Patients with relapsed intracranial germinoma can achieve durable remission with standard chemotherapy regimens and/or reirradiation; however, innovative therapies are required for patients with relapsed and/or refractory intracranial nongerminomatous germ cell tumors (NGGCTs) due to their poor prognosis. Improved outcomes have been reported using reinduction chemotherapy to achieve minimal residual disease, followed by marrow-ablative chemotherapy (HDCx) with autologous hematopoietic progenitor cell rescue (AuHPCR). We conducted a phase II trial evaluating the response and toxicity of a 3-drug combination developed for recurrent intracranial germ cell tumors consisting of gemcitabine, paclitaxel, and oxaliplatin (GemPOx). Methods: A total of 9 patients with confirmed relapsed or refractory intracranial GCT were enrolled after signing informed consent, and received at least 2 cycles of GemPOx, of which all but 1 had relapsed or refractory NGGCTs. One patient with progressive disease was found to have pathologically confirmed malignant transformation to pure embryonal rhabdomyosarcoma (without GCT elements), hence was ineligible and not included in the analysis. Patients who experienced sufficient responses proceeded to receive HDCx with AuHPCR. Treatment response was determined based on radiographic tumor assessments and tumor markers. Results: A total of 7 patients achieved sufficient response and proceeded with HDCx and AuHPCR, and 5 subsequently received additional radiotherapy. A total of 2 patients developed progressive disease while receiving GemPOx. Myelosuppression and transaminitis were the most common treatment-related adverse events. With a mean follow-up of 44 months, 4 patients (3 NGGCTs, 1 germinoma) are alive without evidence of disease. Conclusions: GemPOx demonstrates efficacy in facilitating stem cell mobilization, thus facilitating the feasibility of both HDCx and radiotherapy.

Pembrolizumab alters the tumor immune landscape in a patient with dMMR glioblastoma.

Congenital DNA mismatch repair defects (dMMR), such as Lynch Syndrome, predispose patients to a variety of cancers and account for approximately 1% of glioblastoma cases. While few therapeutic options exist for glioblastoma, checkpoint blockade therapy has proven effective in dMMR tumors. Here we present a case study of a male in their 30s diagnosed with dMMR glioblastoma treated with pembrolizumab who experienced a partial response to therapy. Using a multiplex IHC analysis pipeline on archived slide specimens from tumor resections at diagnosis and after therapeutic interventions, we quantified changes in the frequency and spatial distribution of key cell populations in the tumor tissue. Notably, proliferating (KI67+) macrophages and T cells increased in frequency as did other KI67+ cells within the tumor. Therapeutic intervention remodeled the cellular spatial distribution in the tumor leading to a greater frequency of macrophage/tumor cell interactions and T cell/T cell interactions, highlighting impacts of checkpoint blockade on tumor cytoarchitecture and revealing spatial patterns that may indicate advantageous immune interactions in glioma and other solid tumors treated with these agents.

Investigative needle core biopsies for multi-omics in Glioblastoma.

Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few effective therapies. The ability to investigate the tumor microenvironment before and during treatment would greatly enhance both understanding of disease response and progression, as well as the delivery and impact of therapeutics. Stereotactic biopsies are a routine surgical procedure performed primarily for diagnostic histopathologic purposes. The role of investigative biopsies - tissue sampling for the purpose of understanding tumor microenvironmental responses to treatment using integrated multi-modal molecular analyses ('Multi-omics") has yet to be defined. Secondly, it is unknown whether comparatively small tissue samples from brain biopsies can yield sufficient information with such methods. Here we adapt stereotactic needle core biopsy tissue in two separate patients. In the first patient with recurrent GBM we performed highly resolved multi-omics analysis methods including single cell RNA sequencing, spatial-transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics from biopsy tissue that was obtained from a single procedure. In a second patient we analyzed multi-regional core biopsies to decipher spatial and genomic variance. We also investigated the utility of stereotactic biopsies as a method for generating patient derived xenograft models in a separate patient cohort. Dataset integration across modalities showed good correspondence between spatial modalities, highlighted immune cell associated metabolic pathways and revealed poor correlation between RNA expression and the tumor MHC Class I immunopeptidome. In conclusion, stereotactic needle biopsy cores are of sufficient quality to generate multi-omics data, provide data rich insight into a patient's disease process and tumor immune microenvironment and can be of value in evaluating treatment responses. One sentence summary: Integrative multi-omics analysis of stereotactic needle core biopsies in glioblastoma.