The iBLAD study: patient-reported outcomes in bladder cancer during oncological treatment: a multicenter national randomized controlled trial.

BACKGROUND: Patient-reported outcomes (PROs) are getting widely implemented, but little is known of the impact of applying PROs in specific cancer diagnoses. We report the results of a randomized controlled trial (RCT) of the active use of PROs in patients with locally advanced or metastatic bladder cancer (BC) undergoing medical oncological treatment (MOT) with focus on determining the clinical effects of using PROs during chemo- or immunotherapy compared to standard of care. METHODS: We recruited patients from four departments of oncology from 2019 to 2021. Inclusion criteria were locally advanced or metastatic BC, initiating chemo- or immunotherapy. Patients were randomized 1:1 between answering selected PRO-CTCAE questions electronically once weekly with a built-in alert-algorithm instructing patients of how to handle reported symptoms as a supplement to standard of care for handling of side effects (intervention arm (IA)) vs standard procedure for handling of side effects (control arm (CA)). No real-time alerts were sent to the clinic when PROs exceeded threshold values. Clinicians were prompted to view the completed PROs in the IA at each clinical visit. The co-primary clinical endpoints were hospital admissions and treatment completion rate. Secondary endpoints were overall survival (OS), quality of life (EORTC's QLQ-C30 and QLQ-BLM30) and dose reductions. RESULTS: 228 patients with BC were included, 76% were male. 141 (62%) of the patients had metastatic disease. 51% of patients in the IA completed treatment vs. 56% of patients in the CA, OR 0.83 (95% CI 0.47-1.44, p = 0.51). 41% of patients in the IA experienced hospitalization vs. 32% in the CA, OR 1.48 (95% CI 0.83-2.65, p = 0.17). OS was comparable between the two arms (IA: median 22.3mo (95% CI 17.0-NR) vs. CA: median 23.1mo (95% CI 17.7-NR). Patient and clinician compliance was high throughout the study period (80% vs 94%). CONCLUSIONS: This RCT did not show an effect of PRO on completion of treatment, hospitalizations or OS for BC patients during MOT despite a high level of patient and clinician compliance. The lack of real-time response to alerts remains the greatest limitation to this study.

Focused ultrasound for the treatment of brain tumors.

This review investigates focused ultrasound for treating neuro-oncological diseases as an emerging treatment modality. The technique is based on focused ultrasound waves guided by MRI. By using high or low-frequency waves, thermoablation of smaller tissue volumes centrally in the brain or a safe, temporary opening of the blood-brain barrier can be carried out for better penetration of chemotherapy. Numerous studies on neuro-oncological treatments are ongoing, signaling increasing popularity for the technique in the near future.

The impact of short-course hypofractionated radiotherapy on multimodality treatment utilisation, compliance, and outcome in glioblastoma patients: a Danish patterns of care study.

BACKGROUND: The aim of this retrospective registry-based Danish patterns of care study was (1) to evaluate the real-world utilisation of short-course hypofractionated radiotherapy (HFRT) in glioblastoma (GBM) patients over time, and (2) to evaluate the impact of short-course HFRT by assessing trends in multimodality treatment utilisation, compliance, and outcome. MATERIAL AND METHODS: Data of all adults with newly diagnosed pathology-confirmed GBM between 2011 and 2019 were extracted from the nationwide Danish Neuro-Oncology Registry. Short-course HFRT was defined as a fraction size of > 2 Gy to a planned dose of > 30 Gy. Patterns of care were assessed. To analyse trends in the assignment to short-course HFRT, and in radiotherapy (RT) compliance, multivariable logistic regression was applied. To analyse trends in survival, multivariable Cox regression was used. RESULTS: In this cohort of 2416 GBM patients, the utilisation of short-course HFRT significantly increased from ca. 10% in 2011 to 33% in recent years. This coincided with the discontinued use of palliative regimens and a decreased use of conventional fractionation. The proportion of patients proceeding to RT remained stable at ca. 85%. The proportion of patients assigned to chemoradiotherapy (CRT) remained stable at ca. 60%; the use of short-course hypofractionated CRT increased with ca. 10%, while the use of conventionally fractionated CRT decreased with ca. 10%. Compliance with conventionally fractionated and short-course HFRT was respective 92% and 93%, and significantly increasing in recent years. In the complete cohort, the median overall survival remained stable at ca. 11 months. Assignment to short-course HFRT was independently associated with shorter survival. CONCLUSION: In Denmark, the use of short-course HFRT significantly increased in recent years. Nonetheless, the overall utilisation of RT and chemotherapy did not increase on a population level. Nor did survival change. In contrast, compliance with both conventionally fractionated RT and short-course HFRT increased.

Identifying genetic variants regulating MGMT gene expression - A study in monozygotic Danish twins.

Identifying genetic factors affecting the regulation of the O-6-Methylguanine-DNA Methyltransferase (MGMT) gene and estimating the genetic contribution of the MGMT gene through within-pair correlation in monozygotic twin pairs is of particular importance in various types of cancer such as glioblastoma. We used gene expression data in whole blood from 448 monozygotic twins from the Middle Age Danish Twins (MADT) study to investigate genetic regulation of the MGMT gene by performing a genome-wide association study (GWAS) of the variation in MGMT expression. Additionally, we estimated within-pair dependence measures of the expression values looking for the genetic influence of significant identified genes. We identified 243 single nucleotide polymorphisms (SNPs) significantly (p < 5e-8) associated with expression of MGMT, all located on chromosome 10 near the MGMT gene. Of the 243 SNPs, 7 are novel cis-eQTLs. By further looking into the suggestively significant SNPs (increasing cutoff to p = 1e-6), we identified 11 suggestive trans-eQTLs located on chromosome 17. These variants were in or proximal to a total of seven genes, which may regulate MGMT expression. The within-pair correlation of the expression of MGMT, TRIM37, and SEPT4 provided the upper bound genetic influence of these genes. Overall, identifying cis- or trans-acting genetic variations regulating the MGMT gene can pave the way for a better understanding of the MGMT gene function and ultimately in understanding the patient's sensitivity to therapeutic alkylating agents.

Development of a Patient Decision Aid to Support Shared Decision Making for Patients with Recurrent High-Grade Glioma.

When high-grade gliomas recur, patients, their families, and clinicians face difficult medical decisions. There is no curable treatment, and the treatment options all come with a risk of complications and adverse effects. The patients are often cognitively affected, and they need tailored decision support. The objective of this study was to develop a patient decision aid (PtDA) targeted at patients with recurrent high-grade gliomas. Based on existing knowledge and the International Patient Decision Aids Standards, the PtDA was developed through an iterative process. The PtDA was alpha-tested by potential users to assess its acceptability and usability. The development team comprised three clinicians, two patients, two family members, and a researcher. The fifth version of the PtDA was submitted to the alpha test. Eleven patients, nine family members, and eleven clinicians assessed the PtDA and found it acceptable. Three changes were made during the alpha test. Most participants perceived the PtDA to prepare patients for decision making and improve consultations. The involvement of potential users was emphasized during the development and alpha test process. The PtDA was assessed as useful and acceptable by patients, family members, and clinicians in the decision-making situation of recurrent high-grade glioma.

Endonasal Endoscopic Approach for Minimally Invasive Orbital Decompression in Nonthyroid Proptosis-A Scoping Review.

OBJECTIVE: The endoscopic endonasal approach (EEA) was originally performed to treat thyroid orbitopathy and proptosis. Since then, this approach also has been used to treat other causes of proptosis. This review systematically identifies surgical outcome and complication rates in patients without thyroid proptosis who underwent endoscopic endonasal orbital decompression. METHODS: Databases were searched using the following search terms: orbital disease, surgical decompression, and endoscopic endonasal approach. Two independent reviewers screened all abstracts and titles for relevance and all articles passing this screen were subjected to full-text review. To assess risk of bias, we used ROBINS-I (Risk Of Bias in Non-randomized Studies-of Interventions). RESULTS: Eight studies with a total of 74 patients with nonthyroid proptosis were included. Pre- and postoperative eye examination was performed in all studies, but the extent of examination was varying. With a mean age of 35.7 years, most patients were adolescent, and most pathologies induced unilateral proptosis Complications to EEA for orbital decompression were transient diplopia (5 patients/6.8%), transient facial dysesthesia (2 patients/2.7%), ptosis (1 patient/1.4%), infarction (1 patient/1.4%), sinus obstruction (1 patient/1.4%), and enophtalmos (1 patient/1.4%). The authors reported successful reduction of proptosis in all but 2 patients (97.2%), and only 2 authors reported a need for secondary decompression. CONCLUSIONS: Medial orbital decompression using EEA is a feasible approach for orbital decompression in patients with nonthyroid proptosis. While being comparable in primary outcome to transorbital approaches, the EEA seems superior in terms of complication rates.

Surgical resection of glioblastomas induces pleiotrophin-mediated self-renewal of glioblastoma stem cells in recurrent tumors.

BACKGROUND: Glioblastomas are highly resistant to therapy, and virtually all patients experience tumor recurrence after standard-of-care treatment. Surgical tumor resection is a cornerstone in glioblastoma therapy, but its impact on cellular phenotypes in the local postsurgical microenvironment has yet to be fully elucidated. METHODS: We developed a preclinical orthotopic xenograft tumor resection model in rats with integrated 18F-FET PET/CT imaging. Primary and recurrent tumors were subject to bulk and single-cell RNA sequencing. Differentially expressed genes and pathways were investigated and validated using tissue specimens from the xenograft model, 23 patients with matched primary/recurrent tumors, and a cohort including 190 glioblastoma patients. Functional investigations were performed in vitro with multiple patient-derived cell cultures. RESULTS: Tumor resection induced microglia/macrophage infiltration, angiogenesis as well as proliferation and upregulation of several stem cell-related genes in recurrent tumor cells. Expression changes of selected genes SOX2, POU3F2, OLIG2, and NOTCH1 were validated at the protein level in xenografts and early recurrent patient tumors. Single-cell transcriptomics revealed the presence of distinct phenotypic cell clusters in recurrent tumors which deviated from clusters found in primary tumors. Recurrent tumors expressed elevated levels of pleiotrophin (PTN), secreted by both tumor cells and tumor-associated microglia/macrophages. Mechanistically, PTN could induce tumor cell proliferation, self-renewal, and the stem cell program. In glioblastoma patients, high PTN expression was associated with poor overall survival and identified as an independent prognostic factor. CONCLUSION: Surgical tumor resection is an iatrogenic driver of PTN-mediated self-renewal in glioblastoma tumor cells that promotes therapeutic resistance and tumor recurrence.

A national study on the inter-observer variability in the delineation of organs at risk in the brain.

BACKGROUND: The Danish Neuro Oncology Group (DNOG) has established national consensus guidelines for the delineation of organs at risk (OAR) structures based on published literature. This study was conducted to finalise these guidelines and evaluate the inter-observer variability of the delineated OAR structures by expert observers. MATERIAL AND METHODS: The DNOG delineation guidelines were formed by participants from all Danish centres that treat brain tumours with radiotherapy. In a two-day workshop, guidelines were discussed and finalised based on a pilot study. Following this, the ten participants contoured the following OARs on T1-weighted gadolinium enhanced MRI from 13 patients with brain tumours: optic tracts, optic nerves, chiasm, spinal cord, brainstem, pituitary gland and hippocampus. The metrics used for comparison were the Dice similarity coefficient (Dice), mean surface distance (MSD) and others. RESULTS: A total of 968 contours were delineated across the 13 patients. On average eight (range six to nine) individual contour sets were made per patient. Good agreement was found across all structures with a median MSD below 1 mm for most structures, with the chiasm performing the best with a median MSD of 0.45 mm. The Dice was as expected highly volume dependent, the brainstem (the largest structure) had the highest Dice value with a median of 0.89 whereas smaller volumes such as the chiasm had a Dice of 0.71. CONCLUSION: Except for the caudal definition of the spinal cord, the variances observed in the contours of OARs in the brain were generally low and consistent. Surface mapping revealed sub-regions of higher variance for some organs. The data set is being prepared as a validation data set for auto-segmentation algorithms for use within the Danish Comprehensive Cancer Centre - Radiotherapy and potential collaborators.

Expression and Prognostic Value of the Immune Checkpoints Galectin-9 and PD-L1 in Glioblastomas.

Immunotherapeutic targeting of the PD-1/PD-L1 axis has been widely implemented for treatment of several cancer types but shown disappointing results in glioblastomas (GBMs), potentially due to compensatory mechanisms of other expressed immune checkpoints. Galectin-9 is an immune-checkpoint protein that facilitates T-cell exhaustion and apoptosis and could be a potential target for immune-checkpoint inhibition. A total of 163 GBMs IDH wildtype were immunostained with anti-Galectin-9 and PD-L1 antibodies. Software-based quantitation of immunostainings was performed and co-expression was investigated using double immunofluorescence. Both Galectin-9 and PD-L1 protein expression were found in all 163 tumors and showed a significant positive correlation (p = 0.0017). Galectin-9 expression varied from 0.01% to 32% (mean = 6.61%), while PD-L1 membrane expression ranged from 0.003% to 0.14% (mean = 0.048%) of total tumor area. Expression of Galectin-9 and PD-L1 was found on both microglia/macrophages and tumor cells, and colocalization of both markers was found in 88.3% of tumors. In multivariate analysis, neither Galectin-9 (HR = 0.99), PD-L1 (HR = 1.05), nor their combinations showed prognostic value. Galectin-9 and PD-L1 were expressed in all investigated GBMs and the majority of patients had co-expression, which may provide rationale for multi-targeted immune checkpoint inhibition.

Evolution of the gross tumour volume extent during radiotherapy for glioblastomas.

BACKGROUND AND PURPOSE: Tumour growth during radiotherapy may lead to geographical misses of the target volume. This study investigates the evolution of the tumour extent and evaluates the need for plan adaptation to ensure dose coverage of the target in glioblastoma patients. MATERIALS AND METHODS: The prospective study included 29 patients referred for 59.4 Gy in 33 fractions. Magnetic resonance imaging (MRI) was performed at the time of treatment planning, at fraction 10, 20, 30, and three weeks after the end of radiotherapy. The gross tumour volume (GTV) was defined as the T1w contrast-enhanced region plus the surgical cavity on each MRI set. The relative GTV volume and the maximum distance (Dmax) of the extent of the actual GTV outside the original GTV were measured. Based on the location of the actual GTV during radiotherapy and the original planned dose, a prospective clinical decision was made whether to adapt the treatment. RESULTS: Dose coverage of the GTV during radiotherapy was not compromised, and none of the radiotherapy plans was adapted. The median Dmax (range) was 5.7 (2.0-18.9) mm, 8.0 (2.0-27.4) mm, 8.0 (1.9-27.3) mm, and 8.9 (1.9-34.4) mm at fraction 10, 20, 30, and follow-up. The relative GTV volume and Dmax observed at fraction 10 were correlated with the values observed at follow-up (R = 0.74, p < 0.001 and R = 0.79, p < 0.001, respectively). CONCLUSION: Large variations in the GTV extent were observed, and changes often occurred early in the treatment. Plan adaptation for geographical misses was not performed in our cohort due to sufficient CTV margins.

[Orbital decompression in non-thyroid-associated diseases].

Hypoxia triggers hypoxia-inducible factor (HIF). Not only hypoxia triggers downstream HIF target genes for transcription, as intermittent hyperoxia also possesses similar capabilities, suggesting that fluctuations in oxygen availability may be equally important for inducing HIF transcription. This review describes some of the mechanisms, whereby intermittent hyperbaric hyperoxia may explain some of the observations during hyperbaric oxygen therapy such as enhanced wound healing, angiogenesis and tissue healing, and concludes that oxidative stress enhances certain antibiotics in infection control.

Expression and prognostic value of the transcription factors EGR1 and EGR3 in gliomas.

Most glioblastoma patients have a dismal prognosis, although some survive several years. However, only few biomarkers are available to predict the disease course. EGR1 and EGR3 have been linked to glioblastoma stemness and tumour progression, and this study aimed to investigate their spatial expression and prognostic value in gliomas. Overall 207 gliomas including 190 glioblastomas were EGR1/EGR3 immunostained and quantified. A cohort of 21 glioblastomas with high P53 expression and available tissue from core and periphery was stained with double-immunofluorescence (P53-EGR1 and P53-EGR3) and quantified.EGR1 expression increased with WHO-grade, and declined by 18.9% in the tumour periphery vs. core (P = 0.01), while EGR3 expression increased by 13.8% in the periphery vs. core (P = 0.04). In patients with high EGR1 expression, 83% had methylated MGMT-promoters, while all patients with low EGR1 expression had un-methylated MGMT-promoters. High EGR3 expression in MGMT-methylated patients was associated with poor survival (HR = 1.98; 95%CI 1.22-3.22; P = 0.006), while EGR1 high/EGR3 high, was associated with poor survival vs. EGR1 high/EGR3 low (HR = 2.11; 95%CI 1.25-3.56; P = 0.005). EGR1 did not show prognostic value, but could be involved in MGMT-methylation. Importantly, EGR3 may be implicated in cell migration, while its expression levels seem to be prognostic in MGMT-methylated patients.

Aberrant neuronal differentiation is common in glioma but is associated neither with epileptic seizures nor with better survival.

The mechanisms of glioma-associated seizures (GAS) have yet to be fully elucidated. Proneural subtype, isocitrate dehydrogenase 1 (IDH1) mutations, and epileptic seizures are closely associated suggesting that aberrant neuronal differentiation contributes to glioma-associated seizures. In a population-based cohort (n = 236), lack of stem cell marker expression (nestin, musashi) was significantly associated with IDH1 mutations and GAS at diagnosis. In vitro data suggested an association of IDH1 mutations and a more differentiated phenotype. Out of eight glioma stem cell (GSC) lines, seven revealed positivity for the synaptic marker protein synaptophysin. Three had synapse-like structures identified by electron microscopy and were either vGlut1 (glutamatergic) or GAD67 (GABAergic) positive. In vivo, >10% synaptophysin-positive tumour cells were present in >90% of all gliomas. Synaptophysin expression was associated with proneural subtype and vGlut1 expression, suggesting that most synapse-like structures in glioma are glutamatergic. However, we found null associations between vGlut1 protein/mRNA expression and survival, GAS at onset, development of GAS after resection, and refractory GAS. Synapse-like structures were neither functional nor activated by spontaneous action potentials or cellular networks. Thus, aberrant neuronal differentiation including glutamatergic synapse-like structures is detectable in glioma but is associated neither with epileptic seizures nor with better survival.

Plan quality for high-risk prostate cancer treated with high field magnetic resonance imaging guided radiotherapy.

BACKGROUND AND PURPOSE: Daily radiotherapy plan adaptation facilitated by a high field magnetic resonance linac (MRL) may potentially reduce the treated volume due to a reduction of the setup uncertainty. However, the technology also imposes limitations to the treatment technique compared to a standard linac. This study investigated the clinical quality of MRL treatment plans against current standard plans using identical planning target volume margins for high-risk prostate cancer patients. MATERIALS AND METHODS: Twenty consecutive patients planned with our current clinical standard TPS and treated with single arc VMAT on standard linacs with 78 Gy in the prostate and 56 Gy for pelvic lymph nodes over 39 fractions were included. In addition, IMRT treatment plans for delivery by a 1.5 T MRL, using standard margins and dose objectives, were made in a dedicated TPS. Mean population dose volume histograms (DVH) and dose metrics were analyzed and clinical plan quality was evaluated by an oncologist. RESULTS: All MRL plans were considered clinically acceptable, and DVH analysis showed an overall high similarity to dose distributions of the clinically delivered plans. Mean target coverage was similar (78.0 Gy vs 77.8 Gy). Small but statistically significant differences were seen in doses to organs at risk; on average MRL plans reduced dose to the bladder (46.2 vs 48.3 Gy) compared to standard plans, while dose was higher to the bowel (29.2 vs 26.6 Gy) and penile bulb (16.5 vs 10.8 Gy). CONCLUSION: MRL treatment plans were clinically acceptable and similar in quality to the current standard.

Glioma Cells in the Tumor Periphery Have a Stem Cell Phenotype.

Gliomas are highly infiltrative tumors incurable with surgery. Although surgery removes the bulk tumor, tumor cells in the periphery are left behind resulting in tumor relapses. The aim of the present study was to characterize the phenotype of tumor cells in the periphery focusing on tumor stemness, proliferation and chemo-resistance. This was investigated in situ in patient glioma tissue as well as in orthotopic glioblastoma xenografts. We identified 26 gliomas having the R132 mutation in Isocitrate DeHydrogenase 1 (mIDH1). A double immunofluorescence approach identifying mIDH1 positive tumor cells and a panel of markers was used. The panel comprised of six stem cell-related markers (CD133, Musashi-1, Bmi-1, Sox-2, Nestin and Glut-3), a proliferation marker (Ki-67) as well as a chemo-resistance marker (MGMT). Computer-based automated classifiers were designed to measure the mIDH1 positive nucleus area-fraction of the chosen markers. Moreover, orthotopic glioblastoma xenografts from five different patient-derived spheroid cultures were obtained and the tumor cells identified by human specific immunohistochemical markers. The results showed that tumor cells in the periphery of patient gliomas expressed stem cell markers, however for most markers at a significantly lower level than in the tumor core. The Ki-67 level was slightly reduced in the periphery, whereas the MGMT level was similar. In orthotopic glioblastoma xenografts all markers showed similar levels in the core and periphery. In conclusion tumor cells in the periphery of patient gliomas have a stem cell phenotype, although it is less pronounced than in the tumor core. Novel therapies aiming at preventing recurrence should therefore take tumor stemness into account. Migrating cells in orthotopic glioblastoma xenografts preserve expression and stem cell markers. The orthotopic model therefore has a promising translational potential.

Development and validation of a prognostic model for recurrent glioblastoma patients treated with bevacizumab and irinotecan.

BACKGROUND: Predictive markers and prognostic models are required in order to individualize treatment of recurrent glioblastoma (GBM) patients. Here, we sought to identify clinical factors able to predict response and survival in recurrent GBM patients treated with bevacizumab (BEV) and irinotecan. MATERIAL AND METHODS: A total of 219 recurrent GBM patients treated with BEV plus irinotecan according to a previously published treatment protocol were included in the initial population. Prognostic models were generated by means of multivariate logistic and Cox regression analysis. RESULTS: In multivariate analysis, corticosteroid use had a negative predictive impact on response at first evaluation (OR 0.45; 95% CI 0.22-0.93; p = 0.03) and at best response (OR 0.51; 95% CI 0.26-1.02; p = 0.056). Three significant (p < 0.05) prognostic factors associated with reduced progression-free survival and overall survival (OS) were identified. These factors were included in the final model for OS, namely corticosteroid use (HR 1.70; 95% CI 1.18-2.45; p = 0.004), neurocognitive deficit (HR 1.40; 95% CI 1.04-1.89; p = 0.03) and multifocal disease (HR 1.56; 95% CI 1.15-2.11; p < 0.0001). Based on these results a prognostic index able to calculate the probability for OS at 6 and 12 months for the individual patient was established. The predictive value of the model for OS was validated in a separate patient cohort of 85 patients. DISCUSSION AND CONCLUSION: A prognostic model for OS was established and validated. This model can be used by physicians to risk stratify the individual patient and together with the patient decide whether to initiate BEV relapse treatment.

Expression and prognostic value of the WEE1 kinase in gliomas.

High-grade gliomas have an aggressive clinical course and new clinical biomarkers and therapeutic targets are highly needed. WEE1 is a regulator of the G2 checkpoint in glioblastoma (GBM) cells. Inhibition of this kinase has, in experimental glioma studies, been suggested to enhance sensitivity to irradiation and temozolomide. However, expression level and prognostic potential of WEE1 protein in gliomas remain uninvestigated. In this study, glioma samples from 235 patients across all four WHO grades were analyzed by immunohistochemistry. Using image analysis, we calculated the area fraction of WEE1 positive nuclei. We found that WEE1 protein was localized in tumor cell nuclei and expressed in all glioma types and grades. Although WEE1 protein levels are higher in GBMs (mean 24.5%) relative to grade III (mean 14,0%, p < 0.05) and grade II (mean 6.8%, p < 0.001) gliomas, high WEE1 protein was associated with better survival in GBMs (p = 0.002). This was confirmed in multivariate analysis (HR 0.60, p = 0.003) even when adjusted for MGMT status (HR 0.60, p = 0.005). In conclusion, we report a nuclear expression of WEE1 protein in all glioma grades and types. The WEE1 positive nuclear area was correlated with malignancy grade but it was inversely associated with prognosis in GBM. Although WEE1 is a frequently occurring protein and has been proposed as a novel target in GBM, the role of WEE1 in glioma patient survival appears to be connected to the MGMT status and is more complex than previously anticipated.

High levels of c-Met is associated with poor prognosis in glioblastoma.

The tyrosine kinase receptor c-Met has been suggested to be involved in crucial parts of glioma biology like tumor stemness, growth and invasion. The aim of this study was to investigate the prognostic value of c-Met in a population-based glioma patient cohort. Tissue samples from 238 patients with WHO grade I, II, III and IV tumors were analyzed using immunohistochemical staining and advanced image analysis. Strong c-Met expression was found in tumor cells, blood vessels, and peri-necrotic areas. At the subcellular level, c-Met was identified in the cytoplasm and in the cell membrane. Measurements of high c-Met intensity correlated with high WHO grade (p = 0.006) but no association with survival was observed in patients with WHO grade II (p = 0.09) or III (p = 0.17) tumors. High expression of c-Met was associated with shorter overall survival in patients with glioblastoma multiforme (p = 0.03). However the prognostic effect of c-Met in glioblastomas was time-dependent and only observed in patients who survived more than 8.5 months, and not within the first 8.5 months after diagnosis. This was significant in multivariate analysis (HR 1.99, 95 % CI 1.29-3.08, p = 0.002) adjusted for treatment and the clinical variables age (HR 1.01, 95 % CI 0.99-1.03, p = 0.30), performance status (HR 1.34, 95 % CI 1.17-1.53, p < 0.001), and tumor crossing midline (HR 1.28, 95 % CI 0.79-2.07, p = 0.29). In conclusion, this study showed that high levels of c-Met holds unfavorable prognostic value in glioblastomas.

The prognostic value of clinical factors and cancer stem cell-related markers in gliomas.

UNLABELLED: Gliomas are the most frequent brain tumours among adults, and it is estimated that gliomas constitute half of the about 1500 new brain tumours diagnosed in Denmark every year. Existing treatment strategies include neurosurgery, radiation, and chemotherapy. Therapy selection is based on experiences from clinical trials, with the risk that the results obtained are restricted to highly selected patients only. Moreover, these studies provided only little knowledge of the clinical behaviour of the tumours. For some time, it has been believed that somatic stem cells are responsible for self-renewal, proliferation, and differentiation during development of different (normal) tissues. The same characteristics were identified in cancer cells, and recently a major part of the glioma research has focused on the cancer stem cell (CSC) hypothesis, suggesting that only CSCs posses the ability of initiating new tumours. Moreover, CSCs have been suggested as the cause of resistance towards radiotherapy and chemotherapy. In gliomas, CSCs were originally identified by means of the expression of CD133, but other proteins have subsequently been suggested as CSC related. To improve patients' survival, further knowledge about the biological but also about the clinical presentation of gliomas and of glioma patients in an entire population was needed. Identification of patients who would benefit from standard treatment as well as identification of patients who need more aggressive treatment at the time of diagnosis is essential. Equally important is the identification of patients who will not benefit from current standard treatment. Moreover, as common exclusion criteria in clinical trials are age, performance status, and a histologically verified diagnosis, knowledge regarding clinical characteristics in the total population was highly needed. In manuscripts 1 and 2, sampling from national registries was performed and clinical data were collected in order to indentify a clinical prognostic profile for patients with WHO grade I-II tumours (LGG) and WHO grade III-IV tumours (HGG). By using a population-based setup, we identified 433 patients who were diagnosed with a primary glioma in the period 1 January 2005 to 31 December 2009, and of these 76 patients were clinically diagnosed and 357 had a histologically verified diagnosis. We found that younger age, a non-astrocytic histology, having performance status (PS) 0-1, and the absence of neurological deficits were associated with a better prognosis in patients with LGGs. In patients with HGGs younger age, having PS 0-1, absence of neurological deficits, having a tumour that does not cross the midline, and receiving curatively intended post-surgical treatment were associated with a superior prognosis. Older patients also benefitted from curatively intended treatment, although their survival was inferior as compared to younger patients receiving similar treatment. In addition, the prognostic value of having somatic mutation affecting the protein isocitrate dehydrogenase 1 (IDH1) was evaluated. Presence of a mutated IDH1 was associated with a better prognosis in patients with WHO grade II and III tumours, whereas no prognostic potential was identified in the group of GBMs. In manuscript 3, the independent prognostic value of the RNA-binding protein Musashi-1 was evaluated using fluorescence-based automated quantitative image acquisition. The prognostic significance was subsequently investigated in relation to the observed clinical prognostic variables. We found that Musashi-1 was not prognostic in WHO grade II tumours, but in WHO grade III high levels of Musashi-1 were associated with poor survival, although the conclusion is based on very few patients. The opposite effect was identified in a sub-group of postsurgical treated GBM patients expressing high levels of Musashi-1 and a superior prognosis. It may be speculated that Musashi-1 status has a predictive value to the effect of chemo radiotherapy in GBM patients, but the study was not designed to explore a potential predictive potential, and this should be investigated in further material. In manuscript 4, a double staining of CD133 and nestin was performed. The use of fluorescence made it possible to identify expression of CD133 and nestin in the same cell, which has never been done before. However, neither co-localisation nor expression of CD133 or nestin was associated with survival. IN CONCLUSION: Clinical variables associated with better survival were identified for patients with both LGGs and HGGs. All variables are already used in clinical decision making, and they can be used in prognostic counselling of the patients and to guide clinicians regarding the potential benefit from standard treatment in specific patients. Musashi-1 was a predictor of poor survival in WHO grade III tumours, but in patients with GBMs, high levels of Musashi-1 were associated with improved survival. No prognostic value was identified regarding CD133, nestin, or co-localisation of these markers in multivariate analysis adjusted for clinical variables. None of the investigated CSC markers can be used in a clinical setting at the present time. Quantitative automated image acquisition and processing was demonstrated to be a feasible, robust, and reproducible method that will be used in future projects investigating other potential prognostic factors.

What is the clinical value of cancer stem cell markers in gliomas?

Recent data indicate that cancer stem cells (CSCs) are responsible for resistance of glioblastomas to radiotherapy and chemotherapy, thereby contributing to the poor survival of these patients. In order to identify novel prognostic markers in gliomas, several CSC markers have been investigated. This review summarizes current reports on putative glioma CSC markers and reviews the prognostic value of the individual immunohistochemical markers reported in the literature. Using the Pubmed database, twenty-seven CSC studies looking at membrane markers (CD133, podoplanin, CD15, and A2B5), filament markers (nestin), RNA-binding proteins (Musashi-1) and transcription factors (BMI1, SOX2, Id1 and Oct-4) qualified for this review. The level of CD133 and nestin increased with increasing malignancy grade, and for both markers a prognostic significance was identified in the majority of the studies. Moreover, the co-expression of CD133 and nestin was shown to have an even more powerful prognostic value than just single markers. Regarding podoplanin and Musashi-1, there was a trend towards a prognostic value when summarizing all studies. Especially the co-expression of Musashi-1 and MIB1 seemed promising. For the remaining markers CD15, A2B5, BMI1, SOX2, Id1 and Oct4, no prognostic value was found regarding overall survival in this review. In conclusion we find that CD133, nestin, CD133/nestin, podoplanin, Musashi-1 and Musashi-1/MIB1 are the most promising markers for future investigation. Evaluation in larger cohorts with known clinical data and known status of important biomarkers like MGMT and IDH1 is necessary to reveal their full clinical potential.