Rikke Hedegaard Dahlrot

Tumour-associated microglia/macrophages predict poor prognosis in high-grade gliomas and correlate with an aggressive tumour subtype

Glioblastomas are highly aggressive and treatment resistant. Increasing evidence suggests that tumour‐associated macrophages/microglia (TAMs) facilitate tumour progression by acquiring a M2‐like phenotype. Our objective was to investigate the prognostic value of TAMs in gliomas using automated quantitative double immunofluorescence.

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.

Novel approaches for quantifying protein biomarkers in gliomas: benefits and pitfalls

The therapeutic paradigm of gliomas is changing from a general approach towards an individualized and targeted approach. Accordingly, the search for prognostic and predictive biomarkers, as well as the demand for quantitative, feasible and robust methods for biomarker analysis increases. We find that software classifiers can identify and quantify the expression of a given biomarker within different subcellular compartments and that such classifiers can exclude frequently occurring nontumor cells, thereby avoiding potential bias. The use of a quantitative approach provides a continuous measurement of the expression, allowing establishment of new cut-points and identification of patients with specific prognoses. However, some pitfalls must be noted. This article focuses on benefits and pitfalls of novel approaches for quantifying protein biomarkers in gliomas.

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

Abstract 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.

Prognostic value of O‐6‐methylguanine–DNA methyltransferase (MGMT) protein expression in glioblastoma excluding nontumour cells from the analysis

It is important to predict response to treatment with temozolomide (TMZ) in glioblastoma (GBM) patients. Both MGMT protein expression and MGMT promoter methylation status have been reported to predict the response to TMZ. We investigated the prognostic value of quantified MGMT protein levels in tumour cells and the prognostic importance of combining information of MGMT protein level and MGMT promoter methylation status.

APNG as a prognostic marker in patients with glioblastoma

Aim Expression of the base excision repair enzyme alkylpurine-DNA-N-glycosylase (APNG) has been correlated to temozolomide resistance. Our aim was to evaluate the prognostic value of APNG in a population-based cohort with 242 gliomas including 185 glioblastomas (GBMs). Cellular heterogeneity of GBMs was taken into account by excluding APNG expression in non-tumor cells from the analysis. Methods APNG expression was evaluated using automated image analysis and a novel quantitative immunohistochemical (IHC) assay (qIHC), where APNG protein expression was evaluated through countable dots. Non-tumor cells were excluded using an IHC/qIHC double-staining. For verification, APNG was measured by a quantitative double-immunofluorescence (IF) assay. As validation APNG mRNA expression was evaluated using independent TCGA data. Results Using qIHC, high levels of APNG were associated with better overall survival (OS) in univariate (HR = 0.50; P < 0.001) and multivariate analysis (HR = 0.53; P = 0.001). Patients with methylated MGMT promoters and high APNG expression demonstrated better OS, than patients with methylated MGMT promoters and low APNG expression (HR = 0.59; P = 0.08). Retesting the cohort using IF showed similar results in both univariate (HR = 0.61; P = 0.002) and multivariate analysis (HR = 0.81; P = 0.2). The results were supported by data from the TCGA database. Conclusions Using two different assays combined with quantitative image analysis excluding non-tumour cells, APNG was an independent prognostic factor among patients with a methylated MGMT promoter. We expect that APNG qIHC can potentially identify GBM patients who will not benefit from treatment with temozolomide.

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.

Expression and prognostic value of JAM-A in gliomas

Gliomas are among the most lethal cancers, being highly resistant to both chemo- and radiotherapy. The expression of junctional adhesion molecule-A (JAM-A) was recently identified on the surface of stem cell-like brain tumor-initiating cells and suggested to function as a unique glioblastoma niche adhesion factor influencing the tumorigenic potential of brain tumor-initiating cells. We have recently identified high JAM-A expression to be associated with poor outcome in glioblastomas, and our aim was to further investigate the expression of JAM-A in gliomas focusing especially on the prognostic value in WHO grade II and III gliomas. JAM-A protein expression was evaluated by immunohistochemistry and advanced quantitative image analysis with continuous estimates of staining intensity. The JAM-A antibody stained tumor cell membranes and cytoplasm to various extent in different glioma subtypes, and the intensity was higher in glioblastomas than low-grade gliomas. We could not detect an association with overall survival in patients with grade II and III tumors. Double-immunofluorescence stainings in glioblastomas revealed co-expression of JAM-A with CD133, SOX2, nestin, and GFAP in tumor cells as well as some co-expression with the microglial/macrophage marker IBA-1. In conclusion, JAM-A expression was higher in glioblastomas compared to low-grade gliomas and co-localized with recognized stem cell markers suggesting an association of JAM-A with glioma aggressiveness. No significant association between JAM-A expression and overall survival was found in grade II and III gliomas. Further research is needed to determine the function and clinical impact of JAM-A in gliomas.

Small cell glioblastoma or small cell carcinoma: a case report and review of the literature.

It is often easy to distinguish between primary brain tumors and metastases based on morphology alone. However, in some cases immunohistochemistry (IHC) is necessary to obtain a diagnosis, but, as the present case report illustrates, this is not always straightforward. A 75-year old man was admitted to the hospital with left-sided loss of motor function. A MRI revealed a 6 cm tumor in the right temporoparietal area. The histology was consistent with both glioblastoma multiforme (GBM) and small cell lung carcinoma (SCLC) but IHC was suggestive of a SCLC metastasis. PET-CT revealed no enhancement in the lung, so the tumor was treated as a GBM. Eight months after the primary diagnosis a new MRI revealed metastases in the spinal cord, but there was still no enhancement in the lungs. We reviewed the literature concerning markers used to differentiate between GBM and SCLC and found that most of these markers showed limited specificity. It is further discussed whether the case illustrates an example of spontaneous regression of primary SCLC or might be an example of a GMB metastasizing to the spinal cord. Although immunohistochemical markers are of great help in many situations, the case illustrates important limitations and the need for better diagnostic markers.