Barbara Kiesel

Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma

BACKGROUND Immune checkpoint inhibitors targeting programmed cell death 1 (PD1) or its ligand (PD-L1) showed activity in several cancer types. METHODS We performed immunohistochemistry for CD3, CD8, CD20, HLA-DR, phosphatase and tensin homolog (PTEN), PD-1, and PD-L1 and pyrosequencing for assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status in 135 glioblastoma specimens (117 initial resection, 18 first local recurrence). PD-L1 gene expression was analyzed in 446 cases from The Cancer Genome Atlas. RESULTS Diffuse/fibrillary PD-L1 expression of variable extent, with or without interspersed epithelioid tumor cells with membranous PD-L1 expression, was observed in 103 of 117 (88.0%) newly diagnosed and 13 of 18 (72.2%) recurrent glioblastoma specimens. Sparse-to-moderate density of tumor-infiltrating lymphocytes (TILs) was found in 85 of 117 (72.6%) specimens (CD3+ 78/117, 66.7%; CD8+ 52/117, 44.4%; CD20+ 27/117, 23.1%; PD1+ 34/117, 29.1%). PD1+ TIL density correlated positively with CD3+ (P < .001), CD8+ (P < .001), CD20+ TIL density (P < .001), and PTEN expression (P = .035). Enrichment of specimens with low PD-L1 gene expression levels was observed in the proneural and G-CIMP glioblastoma subtypes and in specimens with high PD-L1 gene expression in the mesenchymal subtype (P = 5.966e-10). No significant differences in PD-L1 expression or TIL density between initial and recurrent glioblastoma specimens or correlation of PD-L1 expression or TIL density with patient age or outcome were evident. CONCLUSION TILs and PD-L1 expression are detectable in the majority of glioblastoma samples but are not related to outcome. Because the target is present, a clinical study with specific immune checkpoint inhibitors seems to be warranted in glioblastoma.

5-Aminolevulinic acid induced fluorescence is a powerful intraoperative marker for precise histopathological grading of gliomas with non-significant contrast-enhancement.

BACKGROUND Intraoperative identification of anaplastic foci in diffusely infiltrating gliomas (DIG) with non-significant contrast-enhancement on MRI is indispensible to avoid histopathological undergrading and subsequent treatment failure. Recently, we found that 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) fluorescence can visualize areas with increased proliferative and metabolic activity in such gliomas intraoperatively. As treatment of DIG is predominantely based on histopathological World Health Organisation (WHO) parameters, we analyzed whether PpIX fluorescence can detect anaplastic foci according to these criteria. METHODS We prospectively included DIG patients with non-significant contrast-enhancement that received 5-ALA prior to resection. Intraoperatively, multiple samples from PpIX positive and negative intratumoral areas were collected using a modified neurosurgical microscope. In all samples, histopathological WHO criteria and proliferation rate were assessed and correlated to the PpIX fluorescence status. RESULTS A total of 215 tumor specimens were collected in 59 patients. Of 26 WHO grade III gliomas, 23 cases (85%) showed focal PpIX fluorescence, whereas 29 (91%) of 33 WHO grade II gliomas were PpIX negative. In intratumoral areas with focal PpIX fluorescence, mitotic rate, cell density, nuclear pleomorphism, and proliferation rate were significantly higher than in non-fluorescing areas. The positive predictive value of focal PpIX fluorescence for WHO grade III histology was 85%. CONCLUSIONS Our study indicates that 5-ALA induced PpIX fluorescence is a powerful marker for intraoperative identification of anaplastic foci according to the histopathological WHO criteria in DIG with non-significant contrast-enhancement. Therefore, application of 5-ALA optimizes tissue sampling for precise histopathological diagnosis independent of brain-shift.

Analysis of 5-aminolevulinic acid–induced fluorescence in 55 different spinal tumors

OBJECT Subtotal resection (STR) of spinal tumors can result in tumor recurrence. Currently, no clinically reliable marker is available for intraoperative visualization of spinal tumor tissue. Protoporphyrin IX (PpIX) fluorescence induced by 5-aminolevulinic acid (5-ALA) is capable of visualizing malignant gliomas. Fluorescence-guided resections of malignant cerebral gliomas using 5-ALA have resulted in an increased rate of complete tumor removal. Recently, the application of 5-ALA has also been described in the first cases of spinal tumors. Therefore, the aim of this observational study was to systematically investigate 5-ALA-induced fluorescence characteristics in different spinal tumor entities. METHODS Three hours before the induction of anesthesia, 5-ALA was administered to patients with different intra- and extradural spinal tumors. In all patients a neurosurgical resection or biopsy of the spinal tumor was performed under conventional white-light microscopy. During each surgery, the presence of PpIX fluorescence was additionally assessed using a modified neurosurgical microscope. At the end of an assumed gross-total resection (GTR) under white-light microscopy, a final inspection of the surgical cavity of fluorescing intramedullary tumors was performed to look for any remaining fluorescing foci. Histopathological tumor diagnosis was established according to the current WHO classification. RESULTS Fifty-two patients with 55 spinal tumors were included in this study. Resection was performed in 50 of 55 cases, whereas 5 of 55 cases underwent biopsy. Gross-total resection was achieved in 37 cases, STR in 5, and partial resection in 8 cases. Protoporphyrin IX fluorescence was visible in 30 (55%) of 55 cases, but not in 25 (45%) of 55 cases. Positive PpIX fluorescence was mainly detected in ependymomas (12 of 12), meningiomas (12 of 12), hemangiopericytomas (3 of 3), and in drop metastases of primary CNS tumors (2 of 2). In contrast, none of the neurinomas (8 of 8), carcinoma metastases (5 of 5), and primary spinal gliomas (3 of 3; 1 pilocytic astrocytoma, 1 WHO Grade II astrocytoma, 1 WHO Grade III anaplastic oligoastrocytoma) revealed PpIX fluorescence. It is notable that residual fluorescing tumor foci were detected and subsequently resected in 4 of 8 intramedullary ependymomas despite assumed GTR under white-light microscopy. CONCLUSIONS In this study, 5-ALA-PpIX fluorescence was observed in spinal tumors, especially ependymomas, meningiomas, hemangiopericytomas, and drop metastases of primary CNS tumors. In cases of intramedullary tumors, 5-ALA-induced PpIX fluorescence is a useful tool for the detection of potential residual tumor foci.

Correlation of immune phenotype with IDH mutation in diffuse glioma

Background Tumor infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) are targets of immune checkpoint inhibitors. Methods Forty-three World Health Organization (WHO) grade II/III gliomas (39 IDH-mutant [mut], 4 IDH-wildtype [wt]) and 14 IDH-mut glioblastomas (GBM) were analyzed for TIL (CD3+; PD1+) infiltration and PD-L1 expression. Results were compared with the data of a previously published series of 117 IDH-wt glioblastomas. PD-L1 gene expression levels were evaluated in 677 diffuse gliomas grades II-IV from The Cancer Genome Atlas (TCGA) database. Results TIL and PD-L1 expression were observed in approximately half of WHO grade II/III gliomas. IDH-wt status was associated with significantly higher TIL infiltration and PD-L1 expression among all (grades II-IV) cases (n = 174, P < 0.001) and within the cohort of glioblastomas (n = 131, P < 0.001). In low-grade glioma (LGG) and glioblastoma cohorts of TCGA, significantly higher PD-L1 gene expression levels were evident in IDH-wt compared with IDH-mut samples (LGG: N = 516; P = 1.933e-11, GBM: N = 161; P < 0.009). Lower PD-L1 gene expression was associated with increased promoter methylation (Spearman correlation coefficient -0.36; P < 0.01) in the LGG cohort of TCGA. IDH-mut gliomas had higher PD-L1 gene promoter methylation levels than IDH-wt gliomas (P < 0.01). Conclusions The immunological tumor microenvironment of diffuse gliomas differs in association with IDH mutation status. IDH-wt gliomas display a more prominent TIL infiltration and higher PD-L1 expression than IDH-mut cases. Mechanistically this may be at least in part due to differential PD-L1 gene promoter methylation levels. Our findings may be relevant for immune modulatory treatment strategies in glioma patients.

A novel miniature robotic guidance device for stereotactic neurosurgical interventions: preliminary experience with the iSYS1 robot

OBJECTIVE Robotic devices have recently been introduced in stereotactic neurosurgery in order to overcome the limitations of frame-based and frameless techniques in terms of accuracy and safety. The aim of this study is to evaluate the feasibility and accuracy of the novel, miniature, iSYS1 robotic guidance device in stereotactic neurosurgery. METHODS A preclinical phantom trial was conducted to compare the accuracy and duration of needle positioning between the robotic and manual technique in 162 cadaver biopsies. Second, 25 consecutive cases of tumor biopsies and intracranial catheter placements were performed with robotic guidance to evaluate the feasibility, accuracy, and duration of system setup and application in a clinical setting. RESULTS The preclinical phantom trial revealed a mean target error of 0.6 mm (range 0.1-0.9 mm) for robotic guidance versus 1.2 mm (range 0.1-2.6 mm) for manual positioning of the biopsy needle (p < 0.001). The mean duration was 2.6 minutes (range 1.3-5.5 minutes) with robotic guidance versus 3.7 minutes (range 2.0-10.5 minutes) with manual positioning (p < 0.001). Clinical application of the iSYS1 robotic guidance device was feasible in all but 1 case. The median real target error was 1.3 mm (range 0.2-2.6 mm) at entry and 0.9 mm (range 0.0-3.1 mm) at the target point. The median setup and instrument positioning times were 11.8 minutes (range 4.2-26.7 minutes) and 4.9 minutes (range 3.1-14.0 minutes), respectively. CONCLUSIONS According to the preclinical data, application of the iSYS1 robot can significantly improve accuracy and reduce instrument positioning time. During clinical application, the robot proved its high accuracy, short setup time, and short instrument positioning time, as well as demonstrating a short learning curve.

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

ObjectivesTo 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.MethodsAdult 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.ResultsIn 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).ConclusionsOur 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.

Glioma Survival Prediction with Combined Analysis of In Vivo 11C-MET PET Features, Ex Vivo Features, and Patient Features by Supervised Machine Learning

Gliomas are the most common type of tumor in the brain. Although the definite diagnosis is routinely made ex vivo by histopathologic and molecular examination, diagnostic work-up of patients with suspected glioma is mainly done using MRI. Nevertheless, l-S-methyl-11C-methionine (11C-MET) PET holds great potential in the characterization of gliomas. The aim of this study was to establish machine-learning–driven survival models for glioma built on in vivo 11C-MET PET characteristics, ex vivo characteristics, and patient characteristics. Methods: The study included 70 patients with a treatment-naïve glioma that was 11C-MET–positive and had histopathology-derived ex vivo feature extraction, such as World Health Organization 2007 tumor grade, histology, and isocitrate dehydrogenase 1 R132H mutational status. The 11C-MET–positive primary tumors were delineated semiautomatically on PET images, followed by the extraction of tumor-to-background–based general and higher-order textural features by applying 5 different binning approaches. In vivo and ex vivo features, as well as patient characteristics (age, weight, height, body mass index, Karnofsky score), were merged to characterize the tumors. Machine-learning approaches were used to identify relevant in vivo, ex vivo, and patient features and their relative weights for predicting 36-mo survival. The resulting feature weights were used to establish 3 predictive models per binning configuration: one model based on a combination of in vivo, ex vivo, and clinical patient information (M36IEP); another based on in vivo and patient information only (M36IP); and a third based on in vivo information only (M36I). In addition, a binning-independent model based on ex vivo and patient information only (M36EP) was created. The established models were validated in a Monte Carlo cross-validation scheme. Results: The most prominent machine-learning–selected and –weighted features were patient-based and ex vivo–based, followed by in vivo–based. The highest areas under the curve for our models as revealed by the Monte Carlo cross-validation were 0.9 for M36IEP, 0.87 for M36EP, 0.77 for M36IP, and 0.72 for M36I. Conclusion: Prediction of survival in amino acid PET–positive glioma patients was highly accurate using computer-supported predictive models based on in vivo, ex vivo, and patient features.

A novel protocol of continuous navigation guidance for endoscopic third ventriculostomy.

BACKGROUND: Although considered a standard neurosurgical procedure, endoscopic third ventriculostomy (ETV) is associated with a relatively high complication rate that is predominantly related to malpositioning of the trajectory. OBJECTIVE: To develop an advanced navigation protocol for ETV, assess its possible benefits over commonly used ETV trajectories, and apply this protocol during surgery. METHODS: After development of our advanced protocol, the imaging data of 59 patients who underwent ETV without navigation guidance was transferred to our navigation software. An individualized endoscope trajectory was created according to our protocol in all cases. This trajectory was compared with 2 standard trajectories, especially with regard to the distance to relevant neuronal structures: a trajectory manually measured on preoperative radiological images, as performed in all 59 cases, and a trajectory resulting from a commonly used fixed coronal burr hole. Subsequently, we applied the protocol in 15 ETVs to assess the feasibility and procedural complications. RESULTS: Our individualized trajectory resulted in a significantly greater distance to the margins of the foramen of Monro, and the burr hole was located more posteriorly from the coronal suture in comparison with the standard trajectories. The advanced ETV technique was feasible in all 15 procedures, and no major complications occurred in any procedure. In 1 patient, a fornix contusion without clinical correlation was observed. CONCLUSION: Our data indicate that the proposed navigation protocol for ETV optimizes the distance of the endoscope to important neuronal structures. Continuous endoscope and puncture device guidance may further add to the safety of this procedure. ABBREVIATIONS: EM, electromagnetic ETV, endoscopic third ventriculostomy FM, foramen of Monro

Systematic histopathological analysis of different 5-aminolevulinic acid–induced fluorescence levels in newly diagnosed glioblastomas

OBJECTIVE Glioblastoma (GBM) is characterized by distinct intratumoral histopathological heterogeneity with regard to variable tumor morphology, cell proliferation, and microvascularity. Maximum resection of a GBM results in an improved prognosis and thus represents the aim of surgery in the majority of cases. Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) is currently widely applied for improved intraoperative tumor visualization in patients with a GBM. Three intratumoral fluorescence levels (i.e., strong, vague, or no fluorescence) can usually be distinguished during surgery. So far, however, their exact histopathological correlates and their surgical relevance have not been clarified sufficiently. Thus, the aim of this study was to systematically analyze tissue samples from newly diagnosed GBMs with different fluorescence levels according to relevant histopathological parameters. METHODS This prospective study recruited patients who underwent 5-ALA fluorescence-guided resection of a newly diagnosed radiologically suspected GBM. Each patient received 5-ALA approximately 3 hours before surgery, and a modified neurosurgical microscope was applied for intraoperative visualization of 5-ALA-induced fluorescence. During surgery, tissue samples with strong, vague, or no fluorescence were collected. For each sample, the presence of tumor tissue, quality of tissue (compact, infiltrative, or no tumor), histopathological criteria of malignancy (cell density, nuclear pleomorphism, mitotic activity, and presence of microvascular proliferation/necrosis), proliferation rate (MIB-1 labeling index [LI]), and microvessel density (using CD34 staining) were investigated. RESULTS Altogether, 77 patients with a newly diagnosed, histopathologically confirmed GBM were included, and 131 samples with strong fluorescence, 69 samples with vague fluorescence, and 67 samples with no fluorescence were collected. Tumor tissue was detected in all 131 (100%) of the samples with strong fluorescence and in 65 (94%) of the 69 samples with vague fluorescence. However, mostly infiltrative tumor tissue was still found in 33 (49%) of 67 samples despite their lack of fluorescence. Strong fluorescence corresponded to compact tumors in 109 (83%) of 131 samples, whereas vague fluorescence was consistent with infiltrative tumors in 44 (64%) of 69 samples. In terms of the histopathological criteria of malignancy, a significant positive correlation of all analyzed parameters comprising cell density, nuclear pleomorphism, mitotic activity, microvascular proliferation, and necrosis with the 3 fluorescence levels was observed (p < 0.001). Furthermore, the proliferation rate significantly and positively correlated with strong (MIB-1 LI 28.3%), vague (MIB-1 LI 16.7%), and no (MIB-1 LI 8.8%) fluorescence (p < 0.001). Last, a significantly higher microvessel density was detected in samples with strong fluorescence (CD34 125.5 vessels/0.25 mm2) than in those with vague (CD34 82.8 vessels/0.25 mm2) or no (CD34 68.6 vessels/0.25 mm2) fluorescence (p < 0.001). CONCLUSIONS Strong and vague 5-ALA-induced fluorescence enables visualization of intratumoral areas with specific histopathological features and thus supports neurosurgeons in improving the extent of resection in patients with a newly diagnosed GBM. Despite the lack of fluorescence, tumor tissue was still observed in approximately half of the cases. To overcome this current limitation, the promising approach of complementary spectroscopic measurement of fluorescence should be investigated further.

P02.02PRELIMINARY CHARACTERIZATION OF THE IMMUNE RESPONSE IN GLIOBLASTOMA BEFORE AND AFTER FIRST-LINE THERAPY

BACKGROUND: Glioblastoma constitutes the most common primary malignant brain tumor in adults. Its aggressive disease course is mediated by a hitherto poorly understood interplay of rapid tumor progression and therapy-related effects. Crucial factors include - amongst hypoxia, angiogenesis, and others - an immune response upfront and/or as a consequence to combined chemo-radiotherapy. Herein, we aim at a systematic morphological characterization of this immune response based on a homogenous series of paired pre- and posttherapeutic glioblastoma samples. PATIENTS AND METHODS: A total of 15 glioblastoma patients newly diagnosed from 2005-2010 at the Medical University of Vienna were included in the analysis. All patients underwent neursurgical resection followed by standard radio- and TMZ-chemotherapy. At time of tumor recurrence second surgery was performed in all cases. Information on clinical characteristics and last follow-up were available. The tumor-associated immune response was semiquantitatively assessed using immunohistochemistry for anti-HLA-DR, CD3, and CD8. In addition, tumor-associated eosinophilia was assessed on conventional HE stains. RESULTS: Median age at diagnosis was 54 ys (range 21-69 ys). Median time from first to second surgery was 12.0 mths (range 1-26 mths) and median overall survival was 46.0 mths of the total cohort. While tumor-infiltrating CD3+ and CD8+ T-lymphocytes did not show a significant difference between first and second surgery (p = 0.07 and p = 0.3, respectively), a significant increase of HLA-DR+ microglia/macrophages (p = 0.01) was observed at time of tumor recurrence in the majority of cases (10/15; 66.7%). No unequivocal differences in overall survival were observed for those patients with enhanced microglial/macrophage response at time of tumor recurrence (log rank test; p = 0.34). A single case showed massive infiltration of eosinophils at second surgery only. This case differed from others by young age at diagnosis (26 ys), positive IDH-1 mutational and MGMT methylation status, short time to second surgery (1 mth) and exceptional favorable overall survival with stable disease at 84 mths. No peripheral eosinophilia or prior history of atopic disease was noted. Further screening for anti-neuronal/glial antibodies on a tissue-based assay did not yield a positive result. CONCLUSION: Although the small sample size implies cautious interpretation, we observed a significant increase of tumor-infiltrating microglia/macrophages in glioblastoma samples obtained after first-line therapy, while no significant differences were observed for tumor-infiltrating CD3+ and CD8+ T-lymphocytes. Of note, a single patient showed prominent tumor-associated eosinophilia upon second surgery which was associated with long-term survival.