Polarization-insensitive optical coherence tomography based on partly depolarized light.

Polarization-related image artifacts are frequently observed in optical coherence tomography (OCT) data. As most modern OCT layouts rely on polarized light sources, only the co-polarized component of the light scattered from within a sample can be detected after interference with the reference beam. Cross-polarized sample light does not interfere with the reference beam and thus produces artifacts ranging from a reduction to the full absence of OCT signals. Here we present a simple yet effective technique to prevent polarization artifacts. By partly depolarizing the light source at the interferometer entrance, we achieve OCT signals regardless of the sample polarization state. We demonstrate the performance of our approach in a defined retarder as well as in birefringent dura mater tissue. This simple and cost-effective technique can be applied to obviate cross-polarization artifacts in virtually any OCT layout.

Mapping high-grade glioma immune infiltration to 5-ALA fluorescence levels: TCGA data computation, classical histology, and digital image analysis.

PURPOSE: Resection of high-grade gliomas has been considerably improved by 5-aminolevulinic acid (5-ALA). However, not all neurobiological properties of 5-ALA are fully understood. Specifically, potential differences in immune infiltration have not been conclusively examined, despite recent reports that immune cells might play a role. Thus, we here provide a systematic mapping of immune infiltration of different 5-ALA fluorescence levels. METHODS: Tumor-associated macrophages (CD68, CD163), cytotoxic T cells (CD8), and regulatory T cells (FoxP3) were quantified via three methods. First, data from The Cancer Genome Atlas (TCGA) of 172 patients was examined for correlations between 5-ALA fluorescence-related mRNA expression signatures and immune markers. Second, as classical histology, 508 stained slides from 39 high-grade glioma patients were analysed semi-quantitatively by two independent reviewers, generating 1016 data points. Third, digital image analysis was performed with automated scanning and algorithm-based cell quantification. RESULTS: TCGA mRNA data from 172 patients showed a direct, significant correlation between 5-ALA signatures and immune markers (p < 0.001). However, we were not able to confirm this finding in the here studied initial set of 39 patient histologies where we found a comparable immune infiltration in different fluorescence levels. Digital image analysis correlated excellently with standard histology. CONCLUSION: With mapping the immune infiltration pattern of different 5-ALA categories, we are adding fundamental basic insights to the field of 5-ALA and glioma biology. The observation that a significant correlation in TCGA data did not fully translate to detectable differences in immune infiltration in first histology data warrants further investigation in larger cohorts.

Toward digital histopathological assessment in surgery for central nervous system tumors using stimulated Raman histology.

OBJECTIVE: Intraoperative neuropathological assessment with conventional frozen sections supports the neurosurgeon in optimizing the surgical strategy. However, preparation and review of frozen sections can take as long as 45 minutes. Stimulated Raman histology (SRH) was introduced as a novel technique to provide rapid high-resolution digital images of unprocessed tissue samples directly in the operating room that are comparable to conventional histopathological images. Additionally, SRH images are simultaneously and easily accessible for neuropathological judgment. Recently, the first study showed promising results regarding the accuracy and feasibility of SRH compared with conventional histopathology. Thus, the aim of this study was to compare SRH with conventional H&E images and frozen sections in a large cohort of patients with different suspected central nervous system (CNS) tumors. METHODS: The authors included patients who underwent resection or stereotactic biopsy of suspected CNS neoplasm, including brain and spinal tumors. Intraoperatively, tissue samples were safely collected and SRH analysis was performed directly in the operating room. To enable optimal comparison of SRH with H&E images and frozen sections, the authors created a digital databank that included images obtained with all 3 imaging modalities. Subsequently, 2 neuropathologists investigated the diagnostic accuracy, tumor cellularity, and presence of diagnostic histopathological characteristics (score 0 [not present] through 3 [excellent]) determined with SRH images and compared these data to those of H&E images and frozen sections, if available. RESULTS: In total, 94 patients with various suspected CNS tumors were included, and the application of SRH directly in the operating room was feasible in all cases. The diagnostic accuracy based on SRH images was 99% when compared with the final histopathological diagnosis based on H&E images. Additionally, the same histopathological diagnosis was established in all SRH images (100%) when compared with that of the corresponding frozen sections. Moreover, the authors found a statistically significant correlation in tumor cellularity between SRH images and corresponding H&E images (p < 0.0005 and R = 0.867, Pearson correlation coefficient). Finally, excellent (score 3) or good (2) accordance between diagnostic histopathological characteristics and H&E images was present in 95% of cases. CONCLUSIONS: The results of this retrospective analysis demonstrate the near-perfect diagnostic accuracy and capability of visualizing relevant histopathological characteristics with SRH compared with conventional H&E staining and frozen sections. Therefore, digital SRH histopathology seems especially useful for rapid intraoperative investigation to confirm the presence of diagnostic tumor tissue and the precise tumor entity, as well as to rapidly analyze multiple tissue biopsies from the suspected tumor margin. A real-time analysis comparing SRH images and conventional histological images at the time of surgery should be performed as the next step in future studies.

Circulating PD-L1 levels change during bevacizumab-based treatment in recurrent glioma.

PURPOSE: In primary brain tumors, the efficacy of immune-modulating therapies is still under investigation as inflammatory responses are restricted by tight immunoregulatory mechanisms in the central nervous system. Here, we measured soluble PD-L1 (sPD-L1) in the plasma of patients with recurrent glioblastoma (GBM) and recurrent WHO grade II-III glioma treated with bevacizumab-based salvage therapy. METHODS: Thirty patients with recurrent GBM and 10 patients with recurrent WHO grade II-III glioma were treated with bevacizumab-based salvage therapy at the Medical University of Vienna. Prior to each treatment cycle, EDTA plasma was drawn and sPD-L1 was measured applying a sandwich ELISA with a lower detection limit of 0.050 ng/ml. Leukocyte counts and C-reactive protein (CRP) levels were measured according to institutional practice. RESULTS: Median number of sPD-L1 measurements was 6 per patient (range: 2-24). At baseline, no significant difference in sPD-L1 concentrations was observed between WHO grade II-III glioma and GBM. Intra-patient variability of sPD-L1 concentrations was significantly higher in WHO grade II-III glioma than in GBM (p = 0.014) and tendentially higher in IDH-mutant than in IDH-wildtype glioma (p = 0.149) In WHO grade II-III glioma, sPD-L1 levels were significantly lower after one administration of bevacizumab than at baseline (median: 0.039 ng/ml vs. 0.4855 ng/ml, p = 0.036). In contrast, no significant change could be observed in patients with GBM. CONCLUSIONS: Changes in systemic inflammation markers including sPD-L1 are observable in patients with recurrent glioma under bevacizumab-based treatment and differ between WHO grade II-III glioma and GBM.

LAG-3 expression in the inflammatory microenvironment of glioma.

PURPOSE: Immune modulatory therapies including immune checkpoint inhibitors have so far failed to result in clinically meaningful efficacy in glioma. We aimed to investigate lymphocyte activation gene 3 (LAG-3), an inhibitory receptor on immune cells and target of second-generation immune checkpoint inhibitors, in glioma. METHODS: 97 patients with diffuse glioma (68 with glioblastoma, 29 with WHO grade II-III glioma) were identified from the Neuro-Biobank of the Medical University of Vienna. LAG-3 expression in the inflammatory microenvironment was assessed by immunohistochemistry (monoclonal anti-LAG-3 antibody, clone 17B4) and correlated to CD3+ , CD8+ , CD20+ and PD-1+ tumor-infiltrating lymphocytes (TILs) and PD-L1 expression on tumor cells. RESULTS: LAG-3+ TILs could be observed in 10/97 (10.3%) IDH-wildtype samples and in none of the included IDH-mutant glioma samples (p = 0.057). Further, LAG-3+ TILs were only observed in WHO grade IV glioblastoma, while none of the investigated WHO grade II-III glioma presented with LAG-3+ TILs (p = 0.03). No association of O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and presence of LAG-3+ TILs was observed (p = 0.726). LAG-3 expression was associated with the presence of CD3+ (p = 0.029), CD8+ (p = 0.001), PD-1+ (p < 0.001) TILs and PD-L1+ tumor cells (p = 0.021), respectively. No association of overall survival with LAG-3+ TIL infiltration was evident (median OS 9.9 vs. 14.2 months, p = 0.95). CONCLUSIONS: LAG-3 is only rarely expressed on TILs in IDH-wildtype glioma and associated with active inflammatory milieu as defined by higher TIL density. Immune microenvironment diversity should be considered in the design of future immunotherapy trials in glioma.

Clinical characteristics and prognostic factors of adult patients with pilocytic astrocytoma.

INTRODUCTION: Pilocytic astrocytoma (PA) is the most common primary brain neoplasm in children and treated in curative intent with gross total resection (GTR). However, PA is rare in adults, resulting in limited knowledge on the natural clinical course. This study aimed to describe the clinical course and identify prognostic factors of adult patients with PA. METHODS: 46 patients ≥ 18 years at diagnosis of PA and neurosurgical resection or biopsy between 2000 and 2018 were identified from the Neuro-Biobank of the Medical University of Vienna. In two cases with differing histopathological diagnosis at recurrence, DNA methylation analysis was performed using Illumina Infinium HumanMethylation850 BeadChip (850 k) arrays and the Molecular Neuropathology classifier. Clinico-pathological features were correlated with patient outcomes. RESULTS: Median age at diagnosis was 32.5 years (range: 19-75) and median Ki67 proliferation index was 2.8% (0.5-13.4%). Tumor location significantly correlated with resectability (p < 0.001). Tumor progression or recurrence was observed in 9/46 (19.6%) patients after a median follow up time of 53.0 months (range 0.5-300). 5-year overall and progression-free survival rates were 85.3% and 70.0%, respectively. 2/9 (22.2%) patients presented with histological changes in the recurrent tumor specimen. In detail, methylation classification redefined the histological diagnosis to anaplastic astrocytoma with piloid features and glioma in one patient, each. Age > 40 and higher body mass index (BMI) were associated with impaired progression-free and overall survival (p < 0.05). CONCLUSIONS: Tumor recurrence or progression in adult PA patients was higher than the one reported in pediatric patients. Higher age and BMI were associated with impaired prognosis.

MGMT assessment in pituitary adenomas: comparison of different immunohistochemistry fixation chemicals.

PURPOSE: Despite the established role of O6-methyl-guanine-DNA methyltransferase (MGMT) as a marker for temozolomide response, consensus of the most reliable method to assess MGMT expression in pituitary adenomas is still missing. Currently, immunohistochemistry (IHC) assessment of formaldehyde fixed tissue samples is most widely used in a semiquantitative description. As formaldehyde fails to completely preserve nucleic acids, RCL2, an alcohol-based formaldehyde-free fixative, has been proposed as a more reliable alternative in terms of cell stability. Furthermore, as the current method of IHC is semiquantitative and observer-dependent, pyrosequencing, an objective tool to evaluate the methylation status of the MGMT promoter, has emerged as a reliable and accurate alternative. The aim of this study was to validate the current IHC method for assessment of MGMT protein expression in pituitary adenomas. METHODS: The tissue samples of 8 macroadenomas with positive IHC MGMT expression (> 50%) were investigated: first, we compared the time dependent stability of MGMT protein expression after pituitary adenoma removal between formaldehyde vs. RCL2. Then, we compared positive IHC MGMT expression with methylated promoter status using pyrosequencing. RESULTS: In the first 12 h after adenoma removal, tissue samples remained MGMT positive in significantly more samples when fixated with formaldehyde than with RCL2, respectively (96 vs. 81%, p = 0.025). CONCLUSION: Our data confirm that the current method using formaldehyde tissue fixation and IHC reveals stable and reliable results of MGMT assessment in pituitary adenomas.

Molecular diagnostic testing of diffuse gliomas in the real-life setting: A practical approach.

Typing of diffuse gliomas according to the WHO 2016 Classification of Tumors of the Central Nervous System is based on the integration of histology with molecular biomarkers. However, the choice of appropriate methods for molecular analysis and criteria for interpretation of test results is left to each diagnostic laboratory. In the present study, we tested the applicability of combined immunohistochemistry, direct sequencing, and multiplex ligation-dependent probe amplification (MLPA) for diagnostic assessment of IDH1/2 mutation status, chromosome 1p/19q status, and TERT promoter mutations. To this end, we analyzed a consecutive series of 165 patients with diffuse low- and high-grade gliomas (WHO grade II and III) from three Austrian centers in which tissue specimens were routinely processed. We could reliably detect IDH1/2 mutations by combining immunohistochemistry, direct sequencing, and MLPA analysis. MLPA analysis also allowed reliable detection of combined whole chromosomal arm 1p/19q codeletion when using carefully selected criteria providing an optimal balance between sensitivity and specificity. Direct sequencing proved to be suitable for identification of TERT promoter mutations, although its analytical performance remains to be assessed. To conclude, we propose a practicable combination of methods and criteria which allow reliable molecular diagnostic testing of diffuse gliomas in the real-life setting.
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Local image variance of 7 Tesla SWI is a new technique for preoperative characterization of diffusely infiltrating gliomas: correlation with tumour grade and IDH1 mutational status.

OBJECTIVES: To investigate the value of local image variance (LIV) as a new technique for quantification of hypointense microvascular susceptibility-weighted imaging (SWI) structures at 7 Tesla for preoperative glioma characterization. METHODS: Adult patients with neuroradiologically suspected diffusely infiltrating gliomas were prospectively recruited and 7 Tesla SWI was performed in addition to standard imaging. After tumour segmentation, quantification of intratumoural SWI hypointensities was conducted by the SWI-LIV technique. Following surgery, the histopathological tumour grade and isocitrate dehydrogenase 1 (IDH1)-R132H mutational status was determined and SWI-LIV values were compared between low-grade gliomas (LGG) and high-grade gliomas (HGG), IDH1-R132H negative and positive tumours, as well as gliomas with significant and non-significant contrast-enhancement (CE) on MRI. RESULTS: In 30 patients, 9 LGG and 21 HGG were diagnosed. The calculation of SWI-LIV values was feasible in all tumours. Significantly higher mean SWI-LIV values were found in HGG compared to LGG (92.7 versus 30.8; p < 0.0001), IDH1-R132H negative compared to IDH1-R132H positive gliomas (109.9 versus 38.3; p < 0.0001) and tumours with significant CE compared to non-significant CE (120.1 versus 39.0; p < 0.0001). CONCLUSIONS: Our data indicate that 7 Tesla SWI-LIV might improve preoperative characterization of diffusely infiltrating gliomas and thus optimize patient management by quantification of hypointense microvascular structures. KEY POINTS: • 7 Tesla local image variance helps to quantify hypointense susceptibility-weighted imaging structures. • SWI-LIV is significantly increased in high-grade and IDH1-R132H negative gliomas. • SWI-LIV is a promising technique for improved preoperative glioma characterization. • Preoperative management of diffusely infiltrating gliomas will be optimized.

MGMT and MSH6 immunoexpression for functioning pituitary macroadenomas.

PURPOSE: Knowledge of biological behavior is crucial for clinical management of functioning pituitary macroadenomas. For recurrent cases unresponsive to standard treatment, temozolomide (TMZ) has been used as a therapeutic alternative. MGMT (O6-methyl-guanine-DNA methyltransferase) and MSH6 (mutS homolog 6) immunoexpression have been linked to the response to TMZ treatment and MGMT immunoexpression has been additionally linked to early recurrence of non-functioning pituitary adenomas. The aim of this study was to assess the prognostic value of MGMT and MSH6 immunoexpression for aggressive functioning pituitary adenomas. METHODS: The study cohort comprised a single center series of 76 patients who underwent an operation for functioning pituitary macroadenoma. We retrospectively compared 38 patients with postoperative persistent or recurrent disease with another set of 38 patients who were in endocrine remission. RESULTS: Low-to-moderate MGMT immunoexpression (<50%) was significantly more frequent in the group with persistent/recurrent disease than in cases of endocrine remission (66 vs. 21%, p < 0.001). Furthermore, adenomas with low-to-moderate MGMT immunoexpression were significantly more often recurrent (76 vs. 30%, p < 0.001) and invasive (64 vs. 28%, p = 0.002). CONCLUSION: In our series, low-to-moderate MGMT immunoexpression was the only marker that significantly correlated with surgical invasiveness and recurrence in functioning pituitary macroadenomas. Therefore, in the future, MGMT status may be considered an additional marker for understanding the biological behavior of pituitary adenomas.

Haematopoietic stem cell transplantation for treatment of primary CNS lymphoma: single-centre experience and literature review.

Primary central nervous system lymphoma (PCNSL) is a rare and malignant tumour type. Established treatment approaches include high-dose methotrexate (HD-MTX)-based chemotherapy and whole-brain radiotherapy (WBRT). WBRT is associated with significant neurotoxicity and autologous haematopoietic stem cell transplantation (ASCT) has been proposed as an alternative treatment - either in the 1st line setting after HD-MTX-based chemotherapy or as salvage treatment for relapsed/refractory PCNSL. We here report our single-centre experience with five PCNSL patients, who had achieved an objective response after a high-dose methotrexate-based induction therapy and consecutively received a high-dose chemotherapy, consisting of carmustine and thiotepa, followed by ASCT. We also provide a literature review on ASCL for PCNSL. Our data, with three of five patients in continuous complete remission and four of five patients alive after a median follow-up time of 8 months, as well as previously published results, show that ASCT is a safe treatment option that is able to induce tumour remissions in patients with PCNSL. However, controlled trials are needed to compare the long-term efficacy and tolerability of ASCT with other treatment approaches and also to establish the optimal sequence of treatment regimens in PCNSL patients.

Clinical Neuropathology practice guide 5-2015: MGMT methylation pyrosequencing in glioblastoma: unresolved issues and open questions.

O6-methylguanine-methyltransferase (MGMT) promoter methylation status has prognostic and, in the subpopulation of elderly patients, predictive value in newly diagnosed glioblastoma. Therefore, knowledge of the MGMT promoter methylation status is important for clinical decision-making. So far, MGMT testing has been limited by the lack of a robust test with sufficiently high analytical performance. Recently, one of several available pyrosequencing protocols has been shown to be an accurate and robust method for MGMT testing in an intra- and interlaboratory ring trial. However, some uncertainties remain with regard to methodological issues, cut-off definitions, and optimal use in the clinical setting. In this article, we highlight and discuss several of these open questions. The main unresolved issues are the definition of the most relevant CpG sites to analyze for clinical purposes and the determination of a cut-off value for dichotomization of quantitative MGMT pyrosequencing results into "MGMT methylated" and "MGMT unmethylated" patient subgroups as a basis for further treatment decisions.

Introduction of a standardized multimodality image protocol for navigation-guided surgery of suspected low-grade gliomas.

OBJECT Surgery of suspected low-grade gliomas (LGGs) poses a special challenge for neurosurgeons due to their diffusely infiltrative growth and histopathological heterogeneity. Consequently, neuronavigation with multimodality imaging data, such as structural and metabolic data, fiber tracking, and 3D brain visualization, has been proposed to optimize surgery. However, currently no standardized protocol has been established for multimodality imaging data in modern glioma surgery. The aim of this study was therefore to define a specific protocol for multimodality imaging and navigation for suspected LGG. METHODS Fifty-one patients who underwent surgery for a diffusely infiltrating glioma with nonsignificant contrast enhancement on MRI and available multimodality imaging data were included. In the first 40 patients with glioma, the authors retrospectively reviewed the imaging data, including structural MRI (contrast-enhanced T1-weighted, T2-weighted, and FLAIR sequences), metabolic images derived from PET, or MR spectroscopy chemical shift imaging, fiber tracking, and 3D brain surface/vessel visualization, to define standardized image settings and specific indications for each imaging modality. The feasibility and surgical relevance of this new protocol was subsequently prospectively investigated during surgery with the assistance of an advanced electromagnetic navigation system in the remaining 11 patients. Furthermore, specific surgical outcome parameters, including the extent of resection, histological analysis of the metabolic hotspot, presence of a new postoperative neurological deficit, and intraoperative accuracy of 3D brain visualization models, were assessed in each of these patients. RESULTS After reviewing these first 40 cases of glioma, the authors defined a specific protocol with standardized image settings and specific indications that allows for optimal and simultaneous visualization of structural and metabolic data, fiber tracking, and 3D brain visualization. This new protocol was feasible and was estimated to be surgically relevant during navigation-guided surgery in all 11 patients. According to the authors' predefined surgical outcome parameters, they observed a complete resection in all resectable gliomas (n = 5) by using contour visualization with T2-weighted or FLAIR images. Additionally, tumor tissue derived from the metabolic hotspot showed the presence of malignant tissue in all WHO Grade III or IV gliomas (n = 5). Moreover, no permanent postoperative neurological deficits occurred in any of these patients, and fiber tracking and/or intraoperative monitoring were applied during surgery in the vast majority of cases (n = 10). Furthermore, the authors found a significant intraoperative topographical correlation of 3D brain surface and vessel models with gyral anatomy and superficial vessels. Finally, real-time navigation with multimodality imaging data using the advanced electromagnetic navigation system was found to be useful for precise guidance to surgical targets, such as the tumor margin or the metabolic hotspot. CONCLUSIONS In this study, the authors defined a specific protocol for multimodality imaging data in suspected LGGs, and they propose the application of this new protocol for advanced navigation-guided procedures optimally in conjunction with continuous electromagnetic instrument tracking to optimize glioma surgery.

Predictive molecular markers in metastases to the central nervous system: recent advances and future avenues.

Metastases to the central nervous system (CNS) are common in several cancer types. For most primary tumors that commonly metastasize to the CNS, molecular biomarker analyses are recommended in the clinical setting for selection of appropriate targeted therapies. Therapeutic efficacy of some of these agents has been documented in patients with brain metastases, and molecular testing of CNS metastases should be considered in the clinical setting. Here, we summarize the clinically relevant biomarker tests that should be considered in neurosurgical specimens based on the current recommendations of the European Society of Medical Oncology (ESMO) or the National Comprehensive Cancer Network (NCCN) for the most relevant primary tumor types: lung cancer (EGFR mutations, ALK rearrangement, BRAF mutations), breast cancer (HER2 amplification, steroid receptor overexpression), melanoma (BRAF mutations), and colorectal cancer (RAS mutations). Furthermore, we discuss emerging therapeutic targets including novel oncogenic alterations (ROS1 rearrangements, FGFR1 amplifications, CMET amplifications, and others) and molecular features of the tumor microenvironment (including immune-checkpoint molecules such as CTLA4 and PD-1/PD-L1). We also discuss the potential role of advanced biomarker tests such as next-generation sequencing and "liquid biopsies" for patients with CNS metastases.

Amplification and overexpression of CMET is a common event in brain metastases of non-small cell lung cancer.

BACKGROUND: CMET represents an emerging therapy target for monoclonal antibodies and tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). METHODS: We investigated CMET gene amplification status by fluorescence in-situ hybridization (FISH) and CMET protein expression by immunohistochemistry in a large series of 209 NSCLC brain metastases (BM; 165 adenocarcinoma, 20 squamous cell carcinoma, 11 adenosquamous carcinomas, 11 large cell carcinomas and two large cell neuroendocrine carcinomas) and correlated our results to clinic-pathological parameters and molecular data from previous studies. RESULTS: We found CMET gene amplification in 36/167 (21.6%) and CMET protein expression in 87/196 (44.4%) of evaluable BM. There was a strong correlation between the presence of CMET gene amplification and CMET protein expression (P < 0.001, chi-square test). Furthermore, presence of CMET amplification correlated positively with presence of ALK amplifications (P = 0.039, chi-square test) and high HIF1 alpha index (P = 0.013, Mann-Whitney U-test). Neither CMET expression nor CMET gene amplification status correlated with patient outcome parameters or known prognostic factors. CONCLUSIONS: CMET overexpression and CMET amplification are commonly found in NSCLC BM and may represent a promising therapeutic target.

Sorafenib for patients with pretreated recurrent or progressive high-grade glioma: a retrospective, single-institution study.

Therapeutic options for patients with pretreated advanced high-grade glioma (HGG) are limited. Sorafenib, a small molecule with multiple potential beneficial actions, appears particularly promising. We reviewed the outcomes of 30 patients with recurrent or progressive HGG treated with sorafenib within a named patient program. Overall, 16 patients suffered from recurrent or progressive glioblastoma multiforme and 14 patients had grade 3 gliomas. All but four patients had previously undergone surgical debulking; all but one patient had received previous standard multimodal treatment; and 18 patients (60%) had received more than one line of chemotherapy, in median three. Progression-free survival (PFS), defined as the time from initiation of sorafenib to treatment discontinuation because of tumor progression or death, was selected as the endpoint. The use of sorafenib resulted in a median PFS of 3 months [95% confidence interval (CI) 1.9-4.1 months] in patients with glioblastoma and of 3.1 months (95% CI 1.4-4.8 months) in patients with other HGG. The PFS-6 for the whole cohort was 23%. Sixteen patients reported adverse events, mostly moderate, with hypertension as the most frequently reported toxicity (seven patients). One patient died of cerebral bleeding (grade 5 toxicity). The overall survival after initiation of sorafenib was 6 months (95% CI 3.9-8.0 months) for patients with glioblastoma multiforme and 10 months (95% CI 3.1-16.9 months) for patients with HGG. In this retrospective analysis of heavily pretreated patients with HGG, sorafenib monotherapy was associated with tumor stabilization in a small subset of patients. The risk-benefit ratio was acceptable in the context of an apparent clinical benefit in patients with a fatal disease.

Clinical Neuropathology practice news 2-2014: ATRX, a new candidate biomarker in gliomas.

Genome-wide molecular approaches have substantially elucidated molecular alterations and pathways involved in the oncogenesis of brain tumors. In gliomas, several molecular biomarkers including IDH mutation, 1p/19q co-deletion, and MGMT promotor methylation status have been introduced into neuropathological practice. Recently, mutations of the ATRX gene have been found in various subtypes and grades of gliomas and were shown to refine the prognosis of malignant gliomas in combination with IDH and 1p/19q status. Mutations of ATRX are associated with loss of nuclear ATRX protein expression, detectable by a commercially available antibody, thus turning ATRX into a promising prognostic candidate biomarker in the routine neuropathological setting.

Clinical Neuropathology practice news 1-2014: pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma.

Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing- based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing.

7 Tesla magnetic resonance spectroscopic imaging predicting IDH status and glioma grading.

INTRODUCTION: With the application of high-resolution 3D 7 Tesla Magnetic Resonance Spectroscopy Imaging (MRSI) in high-grade gliomas, we previously identified intratumoral metabolic heterogeneities. In this study, we evaluated the potential of 3D 7 T-MRSI for the preoperative noninvasive classification of glioma grade and isocitrate dehydrogenase (IDH) status. We demonstrated that IDH mutation and glioma grade are detectable by ultra-high field (UHF) MRI. This technique might potentially optimize the perioperative management of glioma patients. METHODS: We prospectively included 36 patients with WHO 2021 grade 2-4 gliomas (20 IDH mutated, 16 IDH wildtype). Our 7 T 3D MRSI sequence provided high-resolution metabolic maps (e.g., choline, creatine, glutamine, and glycine) of these patients' brains. We employed multivariate random forest and support vector machine models to voxels within a tumor segmentation, for classification of glioma grade and IDH mutation status. RESULTS: Random forest analysis yielded an area under the curve (AUC) of 0.86 for multivariate IDH classification based on metabolic ratios. We distinguished high- and low-grade tumors by total choline (tCho) / total N-acetyl-aspartate (tNAA) ratio difference, yielding an AUC of 0.99. Tumor categorization based on other measured metabolic ratios provided comparable accuracy. CONCLUSIONS: We successfully classified IDH mutation status and high- versus low-grade gliomas preoperatively based on 7 T MRSI and clinical tumor segmentation. With this approach, we demonstrated imaging based tumor marker predictions at least as accurate as comparable studies, highlighting the potential application of MRSI for pre-operative tumor classifications.

Mutant BRAF V600E protein in ganglioglioma is predominantly expressed by neuronal tumor cells.

Ganglioglioma is a rare CNS tumor with a benign biological behavior. Recently, the BRAF V600E mutation was identified in approximately 20 % of gangliogliomas. Here, we analyzed a total of 71 gangliogliomas for BRAF V600E mutational status by VE1 immunohistochemistry and direct DNA sequencing. The BRAF V600E mutation was detected in 41/71 (58 %) gangliogliomas by immunohistochemistry. DNA sequencing was concordant in 60 of 62 analyzed cases. BRAF status was compared with clinical, histological and immunohistochemical data. Presence of the BRAF V600E mutation was associated with expression of synaptophysin in the tumor (p = 0.0008), presence of dysplastic neurons (p = 0.011) and lymphocytic cuffs (p = 0.018), and with younger age (p = 0.0054). Extensive hemosiderin deposition within the tumor was significantly associated with BRAF wild-type status (p = 0.042). No significant association was found with proliferation (p = 0.053), presence of phospho ERK (p = 0.1) or senescence marker p16(INK4a) (p = 0.22). Using VE1, we localized the BRAF V600E-mutated protein predominantly to the neuronal compartment, indicating that BRAF mutations occur in cells that have the capacity to differentiate into ganglionic cells. In many cases mutant BRAF is additionally expressed by the glial compartment, indicating that in these cases the cell targeted by BRAF mutation was likely capable of differentiating along both the ganglionic and glial lineages. No cases with an exclusive expression of BRAF V600E in the glial compartment were observed. Thus, using VE1 we identified the neuronal compartment as an essential part of this mixed glioneuronal tumor.