Stable glioma incidence and increased patient survival over the past two decades in Norway: a nationwide registry-based cohort study.

BACKGROUND: Surveillance of incidence and survival of central nervous system tumors is essential to monitor disease burden and epidemiological changes, and to allocate health care resources. Here, we describe glioma incidence and survival trends by histopathology group, age, and sex in the Norwegian population. MATERIAL AND METHODS: We included patients with a histologically verified glioma reported to the Cancer Registry of Norway from 2002 to 2021 (N = 7,048). Population size and expected mortality were obtained from Statistics Norway. Cases were followed from diagnosis until death, emigration, or 31 December 2022, whichever came first. We calculated age-standardized incidence rates (ASIR) per 100,000 person-years and age-standardized relative survival (RS).  Results: The ASIR for histologically verified gliomas was 7.4 (95% CI: 7.3-7.6) and was higher for males (8.8; 95% CI: 8.5-9.1) than females (6.1; 95% CI: 5.9-6.4). Overall incidence was stable over time. Glioblastoma was the most frequent tumor entity (ASIR = 4.2; 95% CI: 4.1-4.4). Overall, glioma patients had a 1-year RS of 63.6% (95% CI: 62.5-64.8%), and a 5-year RS of 32.8% (95% CI: 31.6-33.9%). Females had slightly better survival than males. For most entities, 1- and 5-year RS improved over time (5-year RS for all gliomas 29.0% (2006) and 33.1% (2021), p < 0.001). Across all tumor types, the RS declined with increasing age at diagnosis. INTERPRETATION: The incidence of gliomas has been stable while patient survival has increased over the past 20 years in Norway. As gliomas represent a heterogeneous group of primary CNS tumors, regular reporting from cancer registries at the histopathology group level is important to monitor disease burden and allocate health care resources in a population.

Functional and Molecular Heterogeneity in Glioma Stem Cells Derived from Multiregional Sampling.

Glioblastoma (GBM) is an aggressive and highly heterogeneous primary brain tumor. Glioma stem cells represent a subpopulation of tumor cells with stem cell traits that are presumed to be the cause of tumor relapse. There exists complex tumor heterogeneity in drug sensitivity patterns between glioma stem cell (GSC) cultures derived from different patients. Here, we describe that heterogeneity also exists between GSC cultures derived from multiple biopsies within a single tumor. From biopsies harvested within spatially distinct regions representing the entire tumor mass, we established seven GSC cultures and compared their stem cell properties, mutations, gene expression profiles, and drug sensitivity patterns against 115 different anticancer drugs. The results were compared to 14 GSC cultures derived from other patients. Between the multiregional-derived GSC cultures, we observed only minor differences in their phenotype, proliferative capacity, and global gene expression. Further, they displayed intratumoral heterogeneity in mutational profiles and sensitivity patterns to anticancer drugs. This heterogeneity, however, did not exceed the extensive heterogeneity found between GSC cultures derived from other GBM patients. Our results suggest that the use of GSC cultures from one single focal biopsy may underestimate the overall complexity of the GSC population and display the importance of including GSC cultures reflecting the entire tumor mass in drug screening strategies.

The benefit of complete resection of contrast enhancing tumor in glioblastoma patients: A population-based study.

Background: New treatment modalities have not been widely adopted for patients with glioblastoma (GBM) after the addition of temozolomide to radiotherapy. We hypothesize that increased extent of resection (EOR) has resulted in improved survival for surgically treated patients with glioblastoma at the population level. Methods: Retrospective analysis of adult patients operated for glioblastoma in the population of South-Eastern Norway. Patients were stratified into Pre-temozolomide- (2003-2005), temozolomide- (2006-2012), and resection-focused period (2013-2019) and evaluated according to age and EOR. Results: The study included 1657 adult patients operated on for supratentorial glioblastoma. The incidence of histologically confirmed glioblastoma increased from 3.7 in 2003 to 5.3 per 100 000 in 2019. The median survival was 11.4 months. Complete resection of contrast-enhancing tumor (CRCET) was achieved in 386 patients, and this fraction increased from 13% to 32% across the periods. Significant improvement in median survival was found between the first 2 periods and the last (10.5 and 10.6 vs. 12.3 months; P < .01), with a significant increase in 3- and 5-year survival probability to 12% and 6% (P < .01). Patients with CRCET survived longer than patients with non-CRCET (16.1 vs. 10.8 months; P < .001). The median survival doubled in patients ≥70 years and (12.1 months). Survival was similar between the time periods in patients where CRCET was achieved. Conclusions: We demonstrate an improved survival of GBM patients at the population level associated with an increased fraction of patients with CRCET. The data support the importance of CRCET to improve glioblastoma patient outcomes.

Functional temozolomide sensitivity testing of patient-specific glioblastoma stem cell cultures is predictive of clinical outcome.

Serum-free culturing of patient-derived glioblastoma biopsies enrich for glioblastoma stem cells (GSCs) and is recognized as a disease-relevant model system in glioblastoma (GBM). We hypothesized that the temozolomide (TMZ) drug sensitivity of patient-derived GSC cultures correlates to clinical sensitivity patterns and has clinical predictive value in a cohort of GBM patients. To this aim, we established 51 individual GSC cultures from surgical biopsies from both treatment-naïve primary and pretreated recurrent GBM patients. The cultures were evaluated for sensitivity to TMZ over a dosing range achievable in normal clinical practice. Drug efficacy was quantified by the drug sensitivity score. MGMT-methylation status was investigated by pyrosequencing. Correlative, contingency, and survival analyses were performed for associations between experimental and clinical data. We found a heterogeneous response to temozolomide in the GSC culture cohort. There were significant differences in the sensitivity to TMZ between the newly diagnosed and the TMZ-treated recurrent disease (p <0.01). There was a moderate correlation between MGMT-status and sensitivity to TMZ (r=0.459, p=0.0009). The relationship between MGMT status and TMZ efficacy was statistically significant on multivariate analyses (p=0.0051). We found a predictive value of TMZ sensitivity in individual GSC cultures to patient survival (p=0.0089). We conclude that GSC-enriched cultures hold clinical and translational relevance by their ability to reflect the clinical heterogeneity in TMZ-sensitivity, substantiate the association between TMZ-sensitivity and MGMT-promotor methylation status and appear to have a stronger predictive value than MGMT-promotor methylation on clinical responses to TMZ.

Real-world validity of randomized controlled phase III trials in newly diagnosed glioblastoma: to whom do the results of the trials apply?

BACKGROUND: The survival rates in population-based series of glioblastoma (GBM) differ substantially from those reported in clinical trials. This discrepancy may be attributed to that patients recruited to trials tend to be younger with better performance status. However, the proportion and characteristics of the patients in a population considered either eligible or ineligible for trials is unknown. The generalizability of trial results is therefore also uncertain. METHODS: Using the Cancer Registry of Norway and the Brain Tumor Database at Oslo University Hospital, we tracked all patients within a well-defined geographical area with newly diagnosed GBM during the years 2012-2017. Based on data from these registries and the medical records, the patients were evaluated for trial eligibility according to criteria employed in recent phase III trials for GBM. RESULTS: We identified 512 patients. The median survival was 11.7 months. When we selected a potential trial population at the start of concurrent chemoradiotherapy (radiotherapy [RT]/ temozolomide [TMZ]) by the parameters age (18-70 y), passed surgery for a supratentorial GBM, Eastern Cooperative Oncology Group (ECOG) ≤2, normal hematologic, hepatic and renal function, and lack of severe comorbidity, 57% of the patients were excluded. Further filtering the patients who progressed during RT/TMZ and never completed RT/TMZ resulted in exclusion of 59% and 63% of the patients, respectively. The survival of patients potentially eligible for trials was significantly higher than of the patients not fulfilling trial eligibility criteria (P < .0001). CONCLUSIONS: Patients considered eligible for phase III clinical trials represent a highly selected minority of patients in a real-world GBM population.

A Small-Molecule Tankyrase Inhibitor Reduces Glioma Stem Cell Proliferation and Sphere Formation.

Evidence suggests that the growth and therapeutic resistance of glioblastoma (GBM) may be enabled by a population of glioma stem cells (GSCs) that are regulated by typical stem cell pathways, including the WNT/ß-catenin signaling pathway. We wanted to explore the effect of treating GSCs with a small-molecule inhibitor of tankyrase, G007-LK, which has been shown to be a potent modulator of the WNT/ß-catenin and Hippo pathways in colon cancer. Four primary GSC cultures and two primary adult neural stem cell cultures were treated with G007-LK and subsequently evaluated through the measurement of growth characteristics, as well as the expression of WNT/ß-catenin and Hippo signaling pathway-related proteins and genes. Treatment with G007-LK decreased in vitro proliferation and sphere formation in all four primary GSC cultures in a dose-dependent manner. G007-LK treatment altered the expression of key downstream WNT/ß-catenin and Hippo signaling pathway-related proteins and genes. Finally, cotreatment with the established GBM chemotherapeutic compound temozolomide (TMZ) led to an additive reduction in sphere formation, suggesting that WNT/ß-catenin signaling may contribute to TMZ resistance. These observations suggest that tankyrase inhibition may serve as a supplement to current GBM therapy, although more work is needed to determine the exact downstream mechanisms involved.

Intertumoral heterogeneity in patient-specific drug sensitivities in treatment-naïve glioblastoma.

BACKGROUND: A major barrier to effective treatment of glioblastoma (GBM) is the large intertumoral heterogeneity at the genetic and cellular level. In early phase clinical trials, patient heterogeneity in response to therapy is commonly observed; however, how tumor heterogeneity is reflected in individual drug sensitivities in the treatment-naïve glioblastoma stem cells (GSC) is unclear. METHODS: We cultured 12 patient-derived primary GBMs as tumorspheres and validated tumor stem cell properties by functional assays. Using automated high-throughput screening (HTS), we evaluated sensitivity to 461 anticancer drugs in a collection covering most FDA-approved anticancer drugs and investigational compounds with a broad range of molecular targets. Statistical analyses were performed using one-way ANOVA and Spearman correlation. RESULTS: Although tumor stem cell properties were confirmed in GSC cultures, their in vitro and in vivo morphology and behavior displayed considerable tumor-to-tumor variability. Drug screening revealed significant differences in the sensitivity to anticancer drugs (p < 0.0001). The patient-specific vulnerabilities to anticancer drugs displayed a heterogeneous pattern. They represented a variety of mechanistic drug classes, including apoptotic modulators, conventional chemotherapies, and inhibitors of histone deacetylases, heat shock proteins, proteasomes and different kinases. However, the individual GSC cultures displayed high biological consistency in drug sensitivity patterns within a class of drugs. An independent laboratory confirmed individual drug responses. CONCLUSIONS: This study demonstrates that patient-derived and treatment-naïve GSC cultures maintain patient-specific traits and display intertumoral heterogeneity in drug sensitivity to anticancer drugs. The heterogeneity in patient-specific drug responses highlights the difficulty in applying targeted treatment strategies at the population level to GBM patients. However, HTS can be applied to uncover patient-specific drug sensitivities for functional precision medicine.

Nuclear Magnetic Resonance Spectroscopy to Identify Metabolite Biomarkers of Nonresponsiveness to Targeted Therapy in Glioblastoma Tumor Stem Cells.

Glioblastoma is the most common and malignant brain tumor, and current therapies confer only modest survival benefits. A major obstacle is our ability to monitor treatment effect on tumors. Current imaging modalities are ambiguous, and repeated biopsies are not encouraged. To scout for markers of treatment response, we used NMR spectroscopy to study the effects of a survivin inhibitor on the metabolome of primary glioblastoma cancer stem cells. Applying high resolution NMR spectroscopy (1H resonance frequency: 800.03 MHz) to just 3 million cells per sample, we achieved sensitive and high resolving determinations of, e.g., amino acids, nucleosides, and constituents of the citric acid cycle. For control samples that were cultured, prepared, and measured at varying dates, peak area relative standard deviations were 15-20%. Analyses of unfractionated lysates were performed for straightforward compound identification with COLMAR and HMDB databases. Principal component analysis revealed that citrate levels were clearly upregulated in nonresponsive cells, while lactate levels substantially decreased following treatment for both responsive and nonresponsive cells. Hence, lactate and citrate may be potential markers of successful drug uptake and poor response to survivin inhibitors, respectively. Our metabolomics approach provided alternative biomarker candidates compared to spectrometry-based proteomics, underlining benefits of complementary methodologies. These initial findings make a foundation for exploring in vivo MR spectroscopy (MRS) of brain tumors, as citrate and lactate are MRS-visible. In sum, NMR metabolomics is a tool for addressing glioblastoma.

The efficacy of a coordinated pharmacological blockade in glioblastoma stem cells with nine repurposed drugs using the CUSP9 strategy.

PURPOSE: Constructed from a theoretical framework, the coordinated undermining of survival paths in glioblastoma (GBM) is a combination of nine drugs approved for non-oncological indications (CUSP9; aprepitant, auranofin, captopril, celecoxib, disulfiram, itraconazole, minocycline, quetiapine, and sertraline) combined with temozolomide (TMZ). The availability of these drugs outside of specialized treatment centers has led patients to embark on combination treatments without systematic follow-up. However, no experimental data on efficacy using the CUSP9 strategy in GBM have been reported. METHODS: Using patient-derived glioblastoma stem cell (GSC) cultures from 15 GBM patients, we described stem cell properties of individual cultures, determined the dose-response relationships of the drugs in the CUSP9, and assessed the efficacy the CUSP9 combination with TMZ in concentrations clinically achievable. The efficacy was evaluated by cell viability, cytotoxicity, and sphere-forming assays in both primary and recurrent GSC cultures. RESULTS: We found that CUSP9 with TMZ induced a combination effect compared to the drugs individually (p < 0.0001). Evaluated by cell viability and cytotoxicity, 50% of the GSC cultures displayed a high sensitivity to the drug combination. In clinical plasma concentrations, the effect of the CUSP9 with TMZ was superior to TMZ monotherapy (p < 0.001). The Wnt-signaling pathway has been shown important in GSC, and CUSP9 significantly reduces Wnt-activity. CONCLUSIONS: Adding experimental data to the theoretical rationale of CUSP9, our results demonstrate that the CUSP9 treatment strategy can induce a combination effect in both treatment-naïve and pretreated GSC cultures; however, predicting response in individual cultures will require further profiling of GSCs.

Feasibility study of using high-throughput drug sensitivity testing to target recurrent glioblastoma stem cells for individualized treatment.

BACKGROUND: Despite the well described heterogeneity in glioblastoma (GBM), treatment is standardized, and clinical trials investigate treatment effects at population level. Genomics-driven oncology for stratified treatments allow clinical decision making in only a small minority of screened patients. Addressing tumor heterogeneity, we aimed to establish a clinical translational protocol in recurrent GBM (recGBM) utilizing autologous glioblastoma stem cell (GSC) cultures and automated high-throughput drug sensitivity and resistance testing (DSRT) for individualized treatment within the time available for clinical application. RESULTS: From ten patients undergoing surgery for recGBM, we established individual cell cultures and characterized the GSCs by functional assays. 7/10 GSC cultures could be serially expanded. The individual GSCs displayed intertumoral differences in their proliferative capacity, expression of stem cell markers and variation in their in vitro and in vivo morphology. We defined a time frame of 10 weeks from surgery to complete the entire pre-clinical work-up; establish individualized GSC cultures, evaluate drug sensitivity patterns of 525 anticancer drugs, and identify options for individualized treatment. Within the time frame for clinical translation 5/7 cultures reached sufficient cell yield for complete drug screening. The DSRT revealed significant intertumoral heterogeneity to anticancer drugs (p < 0.0001). Using curated reference databases of drug sensitivity in GBM and healthy bone marrow cells, we identified individualized treatment options in all patients. Individualized treatment options could be selected from FDA-approved drugs from a variety of different drug classes in all cases. CONCLUSIONS: In recGBM, GSC cultures could successfully be established in the majority of patients. The individual cultures displayed intertumoral heterogeneity in their in vitro and in vivo behavior. Within a time frame for clinical application, we could perform DSRT in 50% of recGBM patients. The DSRT revealed a remarkable intertumoral heterogeneity in sensitivity to anticancer drugs in recGBM that could allow tailored therapeutic options for functional precision medicine.

HMGB1 concentration measurements in trauma patients: assessment of pre-analytical conditions and sample material.

BACKGROUND: HMGB1 is a mediator of systemic inflammation in sepsis and trauma, and a promising biomarker in many diseases. There is currently no standard operating procedure for pre-analytical handling of HMGB1 samples, despite that pre-analytical conditions account for a substantial part of the overall error rate in laboratory testing. We hypothesized that the considerable variations in reported HMGB1 concentrations and kinetics in trauma patients could be partly explained by differences in pre-analytical conditions and choice of sample material. METHODS: Trauma patients (n = 21) admitted to a Norwegian Level I trauma center were prospectively included. Blood was drawn in K2EDTA coated tubes and serum tubes. The effects of delayed centrifugation were evaluated in samples stored at room temperature for 15 min, 3, 6, 12, and 24 h respectively. Plasma samples subjected to long-term storage in - 80 °C and to repeated freeze/thaw cycles were compared with previously analyzed samples. HMGB1 concentrations in simultaneously acquired arterial and venous samples were also compared. HMGB1 was assessed by standard ELISA technique, additionally we investigated the suitability of western blot in both serum and plasma samples. RESULTS: Arterial HMGB1 concentrations were consistently lower than venous concentrations in simultaneously obtained samples (arterial = 0.60 x venous; 95% CI 0.30-0.90). Concentrations in plasma and serum showed a strong linear correlation, however wide limits of agreement. Storage of blood samples at room temperature prior to centrifugation resulted in an exponential increase in plasma concentrations after ≈6 h. HMGB1 concentrations were fairly stable in centrifuged plasma samples subjected to long-term storage and freeze/thaw cycles. We were not able to detect HMGB1 in either serum or plasma from our trauma patients using western blotting. CONCLUSIONS: Arterial and venous HMGB1 concentrations cannot be directly compared, and concentration values in plasma and serum must be compared with caution due to wide limits of agreement. Although HMGB1 levels in clinical samples from trauma patients are fairly stable, strict adherence to a pre-analytical protocol is advisable in order to protect sample integrity. Surprisingly, we were unable to detect HMGB1 utilizing standard western blot analysis.

Phenotypic and Expressional Heterogeneity in the Invasive Glioma Cells.

BACKGROUND: Tumor cell invasion is a hallmark of glioblastoma (GBM) and a major contributing factor for treatment failure, tumor recurrence, and the poor prognosis of GBM. Despite this, our understanding of the molecular machinery that drives invasion is limited. METHODS: Time-lapse imaging of patient-derived GBM cell invasion in a 3D collagen gel matrix, analysis of both the cellular invasive phenotype and single cell invasion pattern with microarray expression profiling. RESULTS: GBM invasion was maintained in a simplified 3D-milieue. Invasion was promoted by the presence of the tumorsphere graft. In the absence of this, the directed migration of cells subsided. The strength of the pro-invasive repulsive signaling was specific for a given patient-derived culture. In the highly invasive GBM cultures, the majority of cells had a neural progenitor-like phenotype, while the less invasive cultures had a higher diversity in cellular phenotypes. Microarray expression analysis of the non-invasive cells from the tumor core displayed a higher GFAP expression and a signature of genes containing VEGFA, hypoxia and chemo-repulsive signals. Cells of the invasive front expressed higher levels of CTGF, TNFRSF12A and genes involved in cell survival, migration and cell cycle pathways. A mesenchymal gene signature was associated with increased invasion. CONCLUSION: The GBM tumorsphere core promoted invasion, and the invasive front was dominated by a phenotypically defined cell population expressing genes regulating traits found in aggressive cancers. The detected cellular heterogeneity and transcriptional differences between the highly invasive and core cells identifies potential targets for manipulation of GBM invasion.