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.

Proof-of-Concept for Liquid Biopsy Disease Monitoring of MYC-Amplified Group 3 Medulloblastoma by Droplet Digital PCR.

BACKGROUND: Liquid biopsy diagnostic methods are an emerging complementary tool to imaging and pathology techniques across various cancer types. However, there is still no established method for the detection of molecular alterations and disease monitoring in MB, the most common malignant CNS tumor in the pediatric population. In the presented study, we investigated droplet digital polymerase chain reaction (ddPCR) as a highly sensitive method for the detection of MYC amplification in bodily fluids of group 3 MB patients. METHODS: We identified a cohort of five MYC-amplified MBs by methylation array and FISH. Predesigned and wet-lab validated probes for ddPCR were used to establish the detection method and were validated in two MYC-amplified MB cell lines as well as tumor tissue of the MYC-amplified cohort. Finally, a total of 49 longitudinal CSF samples were analyzed at multiple timepoints during the course of the disease. RESULTS: Detection of MYC amplification by ddPCR in CSF showed a sensitivity and specificity of 90% and 100%, respectively. We observed a steep increase in amplification rate (AR) at disease progression in 3/5 cases. ddPCR was proven to be more sensitive than cytology for the detection of residual disease. In contrast to CSF, MYC amplification was not detectable by ddPCR in blood samples. CONCLUSIONS: ddPCR proves to be a sensitive and specific method for the detection of MYC amplification in the CSF of MB patients. These results warrant implementation of liquid biopsy in future prospective clinical trials to validate the potential for improved diagnosis, disease staging and monitoring.

Heme Biosynthesis Factors and 5-ALA Induced Fluorescence: Analysis of mRNA and Protein Expression in Fluorescing and Non-fluorescing Gliomas.

Objective: The intraoperative visualization of adult-type diffuse gliomas with 5-aminolevulinic acid (5-ALA) induced fluorescence is widely used in the neurosurgical field. While visible 5-ALA induced fluorescence is found in the majority of high-grade gliomas, most low-grade gliomas lack visible fluorescence during surgery. Recently, the heme biosynthesis pathway was identified as crucial influencing factor for presence of visible fluorescence since it metabolizes 5-ALA to fluorescing Protoporphyrin IX (PpIX). However, the exact alterations within the heme biosynthesis pathway resulting in visible 5-ALA induced fluorescence in gliomas are still unclear. The aim of the present study was thus to compare the mRNA and protein expression of promising intramitochondrial heme biosynthesis enzymes/transporters in glioma tissue samples of different fluorescence behavior. Methods: A total of 19 strongly fluorescing and 21 non-fluorescing tissue samples from neurosurgical adult-type diffuse gliomas (WHO grades II-IV) were included in the current analysis. In these samples, we investigated the mRNA expression by quantitative real time PCR and protein expression using immunohistochemistry of the intramitochondrial heme biosynthesis enzymes Coproporphyrinogen Oxidase (CPOX), Protoporphyrinogen Oxidase (PPOX), Ferrochelatase (FECH), and the transporter ATP-binding Cassette Subfamily B Member 2 (ABCG2). Results: Regarding mRNA expression analysis, we found a significantly decreased ABCG2 expression in fluorescing specimens compared to non-fluorescing samples (p = 0.001), whereas no difference in CPOX, PPOX and FECH was present. With respect to protein expression, significantly higher levels of CPOX (p = 0.005), PPOX (p < 0.01) and FECH (p = 0.003) were detected in fluorescing samples. Similar to mRNA expression analysis, the protein expression of ABCG2 (p = 0.001) was significantly lower in fluorescing samples. Conclusion: Distinct alterations of the analyzed heme biosynthesis factors were found primarily on protein level. Our data indicate that heme biosynthesis pathway activity in general is enhanced in fluorescing gliomas with upregulation of PpIX generating enzymes and decreased ABCG2 mediated PpIX efflux outweighing the also increased further metabolization of PpIX to heme. Intramitochondrial heme biosynthesis factors thus constitute promising pharmacological targets to optimize intraoperative 5-ALA fluorescence visualization of usually non-fluorescing tumors such as low-grade gliomas.

Novel Insights into Diagnosis, Biology and Treatment of Primary Diffuse Leptomeningeal Melanomatosis.

Primary diffuse leptomeningeal melanomatosis (PDLMM) is an extremely rare and aggressive cancer type for which best treatment strategies remain to be elucidated. Herein, we present current and prospective diagnostic strategies and treatment management of PDLMM. Against the background of an extensive literature review of published PDLMM cases and currently employed therapeutic strategies, we present an illustrative case of a pediatric patient suffering from PDLMM. We report the first case of a pediatric patient with PDLMM who received combination treatment including trametinib and everolimus, followed by intravenous nivolumab and ipilimumab with concomitant intensive intraventricular chemotherapy, resulting in temporary significant clinical improvement and overall survival of 7 months. Following this clinical experience, we performed a comprehensive literature review, identifying 26 additional cases. By these means, we provide insight into current knowledge on clinical and molecular characteristics of PDLMM. Analysis of these cases revealed that the unspecific clinical presentation, such as unrecognized increased intracranial pressure (present in 67%), is a frequent reason for the delay in diagnosis. Mortality remains substantial despite diverse therapeutic approaches with a median overall survival of 4 months from diagnosis. On the molecular level, to date, the only oncogenic driver reported so far is mutation of NRAS (n = 3), underlining a close biological relation to malignant melanoma and neurocutaneous melanosis. We further show, for the first time, that this somatic mutation can be exploited for cerebrospinal fluid liquid biopsy detection, revealing a novel potential biomarker for diagnosis and monitoring of PDLMM. Last, we use a unique patient derived PDLMM cell model to provide first insights into in vitro drug sensitivities. In summary, we provide future diagnostic and therapeutic guidance for PDLMM and first insights into the use of liquid biopsy and in vitro models for this orphan cancer type.

Cerebrospinal Fluid Penetration and Combination Therapy of Entrectinib for Disseminated ROS1/NTRK-Fusion Positive Pediatric High-Grade Glioma.

Targeting oncogenic fusion-genes in pediatric high-grade gliomas (pHGG) with entrectinib has emerged as a highly promising therapeutic approach. Despite ongoing clinical studies, to date, no reports on the treatment of cerebrospinal fluid (CSF) disseminated fusion-positive pHGG exist. Moreover, clinically important information of combination with other treatment modalities such as intrathecal therapy, radiotherapy and other targeted agents is missing. We report on our clinical experience of entrectinib therapy in two CSF disseminated ROS1/NTRK-fusion-positive pHGG cases. Combination of entrectinib with radiotherapy or intrathecal chemotherapy appears to be safe and has the potential to act synergistically with entrectinib treatment. In addition, we demonstrate CSF penetrance of entrectinib for the first time in patient samples suggesting target engagement even upon CSF dissemination. Moreover, in vitro analyses of two novel cell models derived from one case with NTRK-fusion revealed that combination therapy with either a MEK (trametinib) or a CDK4/6 (abemaciclib) inhibitor synergistically enhances entrectinib anticancer effects. In summary, our comprehensive study, including clinical experience, CSF penetrance and in vitro data on entrectinib therapy of NTRK/ROS1-fusion-positive pHGG, provides essential clinical and preclinical insights into the multimodal treatment of these highly aggressive tumors. Our data suggest that combined inhibition of NTRK/ROS1 and other therapeutic vulnerabilities enhances the antitumor effect, which should be followed-up in further preclinical and clinical studies.

p53 Loss Mediates Hypersensitivity to ETS Transcription Factor Inhibition Based on PARylation-Mediated Cell Death Induction.

The small-molecule E26 transformation-specific (ETS) factor inhibitor YK-4-279 was developed for therapy of ETS/EWS fusion-driven Ewing's sarcoma. Here we aimed to identify molecular factors underlying YK-4-279 responsiveness in ETS fusion-negative cancers. Cell viability screenings that deletion of P53 induced hypersensitization against YK-4-279 especially in the BRAFV600E-mutated colon cancer model RKO. This effect was comparably minor in the BRAF wild-type HCT116 colon cancer model. Out of all ETS transcription factor family members, especially ETS1 overexpression at mRNA and protein level was induced by deletion of P53 specifically under BRAF-mutated conditions. Exposure to YK-4-279 reverted ETS1 upregulation induced by P53 knock-out in RKO cells. Despite upregulation of p53 by YK-4-279 itself in RKOp53 wild-type cells, YK-4-279-mediated hyperphosphorylation of histone histone H2A.x was distinctly more pronounced in the P53 knock-out background. YK-4-279-induced cell death in RKOp53-knock-out cells involved hyperPARylation of PARP1, translocation of the apoptosis-inducible factor AIF into nuclei, and induction of mitochondrial membrane depolarization, all hallmarks of parthanatos. Accordingly, pharmacological PARP as well as BRAFV600E inhibition showed antagonistic activity with YK-4-279 especially in the P53 knock-out background. Taken together, we identified ETS factor inhibition as a promising strategy for the treatment of notoriously therapy-resistant p53-null solid tumours with activating MAPK mutations.

Telomerase Reverse Transcriptase Promoter Mutations Identify a Genomically Defined and Highly Aggressive Human Pleural Mesothelioma Subgroup.

PURPOSE: Human malignant pleural mesothelioma (MPM) is characterized by dismal prognosis. Consequently, dissection of molecular mechanisms driving malignancy is of key importance. Here we investigate whether activating mutations in the telomerase reverse transcriptase (TERT) gene promoter are present in MPM and associated with disease progression, cell immortalization, and genomic alteration patterns. EXPERIMENTAL DESIGN: TERT promoters were sequenced in 182 MPM samples and compared with clinicopathologic characteristics. Surgical specimens from 45 patients with MPM were tested for in vitro immortalization. The respective MPM cell models (N = 22) were analyzed by array comparative genomic hybridization, gene expression profiling, exome sequencing as well as TRAP, telomere length, and luciferase promoter assays. RESULTS: TERT promoter mutations were detected in 19 of 182 (10.4%) MPM cases and significantly associated with advanced disease and nonepithelioid histology. Mutations independently predicted shorter overall survival in both histologic MPM subtypes. Moreover, 9 of 9 (100%) mutated but only 13 of 36 (36.1%) wild-type samples formed immortalized cell lines. TERT promoter mutations were associated with enforced promoter activity and TERT mRNA expression, while neither telomerase activity nor telomere lengths were significantly altered. TERT promoter-mutated MPM cases exhibited distinctly reduced chromosomal alterations and specific mutation patterns. While BAP1 mutations/deletions were exclusive with TERT promoter mutations, homozygous deletions at the RBFOX1 and the GSTT1 loci were clearly enriched in mutated cases. CONCLUSIONS: TERT promoter mutations independently predict a dismal course of disease in human MPM. The altered genomic aberration pattern indicates that TERT promoter mutations identify a novel, highly aggressive MPM subtype presumably based on a specific malignant transformation process.