Konfokale Laserendomikroskopie in der Neurochirurgie : Erste Erfahrungen

Die Behandlung hirneigener Tumore ist ein wesentlicher Behandlungspfeiler der Neurochirurgie. Hierbei gilt es den Patienten so zu behandeln, dass ein maximaler therapeutischer Erfolg mit geringst möglichem operativen Schaden einhergeht. Technische Entwicklungen im Laufe der vergangenen Jahrzehnte haben die Qualität der neurochirurgischoperativen Therapie stetig vorangebracht, sodass mit minimalinvasiven Techniken eine möglichst hohe Radikalität erbracht werden kann. Aktuell muss das resezierte Gewebe histopathologisch untersucht werden, um eine diagnostische Sicherheit zur Ätiologie des Gewebes zu erlangen. Die konfokale Laserendomikroskopie ermöglicht die Untersuchung von Gewebe auf zellulärer Ebene in situ, was sie bereits zu einer viel versprechenden Untersuchungstechnik in der Gastroenterologie oder Pneumologie gemacht hat. Diese Arbeit hat zum Ziel, diese Technik in der Neurochirurgie zu untersuchen und zu etablieren. Wäre es möglich, das Gewebe bereits in situ auf seine Dignität zu untersuchen, so könne man die operative Qualität und Patientensicherheit erhöhen, da das Gewebe nicht erst reseziert werden müsste um diese Aussagen zu treffen, sondern nur gesichert pathologisches Gewebe entfernt werden könnte. In dieser Arbeit bedient sich der Autor des EndoMag1 der Karl-Storz GmbH, einem rigiden Endoskop mit einem Hopkins-Stablinsensystem. Mit Hilfe diesen Endoskops wurden zunächst das Gehirn eines frisch geschlachteten Schweines, sowie 83 humaner Tumorproben verschiedener Ätiologien untersucht. Das dargestellt Bildmaterial wurde auf seine Morphologie untersucht und mit der Zusammenarbeit eines Neuropathologen verschiedene Kriterien der Befundung etabliert, sodass eine diagnostische Grundlage geschaffen wurde, nach der im Anschluss 100 humane Proben im Vergleich zum Schnellschnitt untersucht werden konnten. Hierbei konnte die richtige Diagnose mit einer hohen respektiven Sensitivität und Spezifizität bei high grade Gliomen (0,81/0,85), low grade Gliomen (0,9/0,93) Schwannomen (0,87/1) und Meningeomen (0,82/0,95) erzielt werden. Metastasen konnten hingegen nur mit einer geringen Sensitivität und Spezifizität von 0,07 respektive 0,94 diagnostiziert werden. Insgesamt konnte nachgewiesen werden, dass die konfokale Laserendomikroskopie eine vielversprechende Technik darstellt, welche unterstützend die neurochirurgisch-operative Behandlung weiterentwickelt, jedoch noch einen langen Weg der Entwicklung vor sich hat.Surgical treatment of brain tumours is one of the main pillars in neurosurgery. Whilst therapeutic success is a main goal, maximum reduction of surgery related neurological deficits must be a key achievement. Technical evolutions have improved the quality of neurosurgical procedures in a way that they have become ever more minimally invasive while maximizing surgical radicality. Until today, brain tissue needs to be resected first in order to undergo pathological investigation, allowing for a diagnostic certainty of its origin. Confocal laserendomicroscopy however, allows for tissue investigation on a cellular level, already making it a reliable diagnostic tool in gastroenterology as well as pneumology. This study aims at investigating its use in neurosurgery and its potential as a new diagnostic tool in this field. Achieving a histological investigation of the tissue on site would allow for further patient safety and higher surgical quality, since the tissue would be diagnosed as pathological before its resection. In this study, the author used the EndoMag1 der Karl-Storz GmbH, a rigid Hopkins-rod-lens endoscope. First, the system was used on a fresh pig specimen and 83 human tissue samples. The collected image material was analysed in respect of the morphologic features of the tissue samples, leading to the establishment of different criteria of evaluation that were chosen with the help a neuropathologist. These were then applied to 100 new samples whose diagnostic results were compared to their respective instantaneous section results. This led to our results with high sensitivity and specificity for high grade gliomas (0,81/0,85), low grade gliomas (0,9/0,93) schwannomas (0,87/1) and meningeomas (0,82/0,95). Our results for metastasis however showed only low sensitivity and specificity with results of 0,07 and 0,94 respectively. In conclusion, the author was able to show that confocal laserendomicroscopy is a promising tool in neurosurgery, that however needs further technical evolution

Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics

Background: Promoter methylation of the DNA repair gene O6 -methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with methylated CpGs in the MGMT promoter beneft from treatment with alkylating agents, such as temozolomide, and show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methyla‑ tion is of importance for diagnostic decisions. We experienced that diferent methods show partially divergent results in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a combination of methylation-specifc PCR assays and pyrosequencing. Results: To better rationalize the variation across assays, we compared these standard techniques and assays to deep bisulfte sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site (TSS). We observed that deep sequencing data are in general in agreement with CpG-specifc pyrosequencing, while the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350–1354, 2000. https://doi.org/ 10.1056/NEJM200011093431901) and Felsberg et al. (Clin Cancer Res 15(21):6683–6693, 2009. https://doi.org/10.1158/ 1078-0432.CCR-08-2801) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfte sequencing (LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples. Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status. Conclusion: Our integrated analysis allows to evaluate and redefne co-methylation domains within the MGMT pro‑ moter and to rationalize the practical impact on assays used in daily routine diagnostics

Spinster Homolog 2 (Spns2) Deficiency Causes Early Onset Progressive Hearing Loss

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing

Confocal Laser Endomicroscopy in Neurosurgery: A New Technique with Much Potential

Technical innovations in brain tumour diagnostic and therapy have led to significant improvements of patient outcome and recurrence free interval. The use of technical devices such as surgical microscopes as well as neuronavigational systems have helped localising tumours as much as fluorescent agents, such as 5-aminolaevulinic acid, have helped visualizing pathologically altered tissue. Nonetheless, intraoperative instantaneous frozen sections and histological diagnosis remain the only method of gaining certainty of the nature of the resected tissue. This technique is time consuming and does not provide close-to-real-time information. In gastroenterology, confocal endoscopy closed the gap between tissue resection and histological examination, providing an almost real-time histological diagnosis. The potential of this technique using a confocal laser endoscope EndoMAG1 by Karl Storz Company was evaluated by our group on pig brains, tumour tissue cell cultures, and fresh human tumour specimen. Here, the authors report for the first time on the results of applying this new technique and provide first confocal endoscopic images of various brain and tumour structures. In all, the technique harbours a very promising potential to provide almost real-time intraoperative diagnosis, but further studies are needed to provide evidence for the technique’s potential

Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics

Background: Promoter methylation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with methylated CpGs in the MGMT promoter benefit from treatment with alkylating agents, such as temozolomide, and show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methylation is of importance for diagnostic decisions. We experienced that different methods show partially divergent results in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a combination of methylation-specific PCR assays and pyrosequencing. Results: To better rationalize the variation across assays, we compared these standard techniques and assays to deep bisulfite sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site (TSS). We observed that deep sequencing data are in general in agreement with CpG-specific pyrosequencing, while the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350-1354, 2000. https://doi.org/10.1056/NEJM200011093431901 ) and Felsberg et al. (Clin Cancer Res 15(21):6683-6693, 2009. https://doi.org/10.1158/1078-0432.CCR-08-2801 ) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfite sequencing (LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples. Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status. Conclusion: Our integrated analysis allows to evaluate and redefine co-methylation domains within the MGMT promoter and to rationalize the practical impact on assays used in daily routine diagnostics.Funding: Open Access funding enabled and organized by Projekt DEAL. This work was supported by the German Epigenome Programme (DEEP) of the Federal Ministry of Education and Research in Germany (BMBF) (01KU1216F). MS is supported by the BMBF project de.NBI-epi (031L0101D) and the EU H2020 project SYSCID (733100

Immunodynamics of explanted human tumors for immuno-oncology

International audienceDecision making in immuno-oncology is pivotal to adapt therapy to the tumor microenvironment (TME) of the patient among the numerous options of monoclonal antibodies or small molecules. Predicting the best combinatorial regimen remains an unmet medical need. Here, we report a multiplex functional and dynamic immuno-assay based on the capacity of the TME to respond to ex vivo stimulation with twelve immunomodulators including immune checkpoint inhibitors (ICI) in 43 human primary tumors. This "in sitro" (in situ/in vitro) assay has the potential to predict unresponsiveness to anti-PD-1 mAbs, and to detect the most appropriate and personalized combinatorial regimen. Prospective clinical trials are awaited to validate this in sitro assay