From bedside to bench: New insights in epilepsy-associated tumors based on recent classification updates and animal models on brain tumor networks.

Low-grade neuroepithelial tumors (LGNTs), particularly those with glioneuronal histology, are highly associated with pharmacoresistant epilepsy. Increasing research focused on these neoplastic lesions did not translate into drug discovery; and anticonvulsant or antitumor therapies are not available yet. During the last years, animal modeling has improved, thereby leading to the possibility of generating brain tumors in mice mimicking crucial genetic, molecular and immunohistological features. Among them, intraventricular in utero electroporation (IUE) has been proven to be a valuable tool for the generation of animal models for LGNTs allowing endogenous tumor growth within the mouse brain parenchyma. Epileptogenicity is mostly determined by the slow-growing patterns of these tumors, thus mirroring intrinsic interactions between tumor cells and surrounding neurons is crucial to investigate the mechanisms underlying convulsive activity. In this review, we provide an updated classification of the human LGNT and summarize the most recent data from human and animal models, with a focus on the crosstalk between brain tumors and neuronal function.

H3K27-Altered Diffuse Glioma of the Spinal Cord in Adult Patients: A Qualitative Systematic Review and Peculiarity of Radiological Findings.

Background: Primary spinal cord diffuse gliomas (SpDG) are rare tumors that may harbor, like diffuse intrinsic pontine gliomas (DIPG), H3K27M mutations. According to the WHO (2021), SpDGs are included in diffuse midline H3K27-altered gliomas, which occur more frequently in adults and show unusual clinical presentation, neuroradiological features, and clinical behavior, which differ from H3 G34-mutant diffuse hemispheric glioma. Currently, homogeneous adult-only case series of SpDG, with complete data and adequate follow-up, are still lacking. Methods: We conducted a qualitative systematic review, focusing exclusively on adult and young adult patients, encompassing all studies reporting cases of primitive, non-metastatic SpDG with H3K27 mutation. We analyzed the type of treatment administered, survival, follow-up duration, and outcomes. Results: We identified 30 eligible articles published between 1990 and 2023, which collectively reported on 62 adult and young adult patients with primitive SpDG. Postoperative outcomes were assessed based on the duration of follow-up, with outcomes categorized as either survival or mortality. Patients who underwent surgery were followed up for a mean duration of 17.37 months, while those who underwent biopsy had a mean follow-up period of 14.65 months. Among patients who were still alive, the mean follow-up duration was 18.77 months. The radiological presentation of SpDG varies widely, indicating its lack of uniformity. Conclusion: Therefore, we presented a descriptive scenario where SpDG was initially suspected to be a meningioma, but was later revealed to be a malignant SpDG with H3K27M mutation.

Synchronous versus metachronous spinal metastasis: a comparative study of survival outcomes following neurosurgical treatment.

PURPOSE: Patients with spinal metastases (SM) from solid neoplasms typically exhibit progression to an advanced cancer stage. Such metastases can either develop concurrently with an existing cancer diagnosis (termed metachronous SM) or emerge as the initial indication of an undiagnosed malignancy (referred to as synchronous SM). The present study investigates the prognostic implications of synchronous compared to metachronous SM following surgical resection. METHODS: From 2015 to 2020, a total of 211 individuals underwent surgical intervention for SM at our neuro-oncology facility. We conducted a survival analysis starting from the date of the neurosurgical procedure, comparing those diagnosed with synchronous SM against those with metachronous SM. RESULTS: The predominant primary tumor types included lung cancer (23%), prostate cancer (21%), and breast cancer (11.3%). Of the participants, 97 (46%) had synchronous SM, while 114 (54%) had metachronous SM. The median overall survival post-surgery for those with synchronous SM was 13.5 months (95% confidence interval (CI) 6.1-15.8) compared to 13 months (95% CI 7.7-14.2) for those with metachronous SM (p = 0.74). CONCLUSIONS: Our findings suggest that the timing of SM diagnosis (synchronous versus metachronous) does not significantly affect survival outcomes following neurosurgical treatment for SM. These results support the consideration of neurosurgical procedures regardless of the temporal pattern of SM manifestation.

Boswellic acid formulations are not suitable for treatment of pediatric high-grade glioma due to tumor promoting potential.

Background and aim: Pediatric high-grade gliomas (pedHGG) comprise a very poor prognosis. Thus, parents of affected children are increasingly resorting to complementary and alternative medicine (CAM), among those Boswellia extracts. However, nothing is known about the therapeutic effectiveness of their active substances, Boswellic acids (BA) in pedHGG. Thus, we aimed to investigate if the three main Boswellic acids (BA) present in Boswellia plants, alpha-boswellic acid (α-BA), beta-boswellic acid (ß-BA) and 3-acetyl-11-keto-beta-boswellic acid (AKBA) hold any promising potential for treatment of affected pedHGG patients. Experimental procedure: Histone 3 (H3)-wildtype and H3.3K27M-mutant pedHGG cell lines were treated with BA, either alone or in combination with radio-chemotherapy with temozolomide. Cell viability, stemness properties, apoptosis, in ovo tumor growth and the transcriptome was investigated upon BA treatment. Results and conclusion: Interestingly, α-BA and ß-BA treatment promoted certain tumor properties in both pedHGG cells. AKBA treatment reduced cell viability and colony growth accompanied by induction of slight anti-inflammatory effects especially in H3.3K27M-mutant pedHGG cells. However, no effects on apoptosis and in ovo tumor growth were found. In conclusion, besides positive anti-tumor effects of AKBA, tumor promoting effects were observed upon treatment with α-BA and ß-BA. Thus, only pure AKBA formulations may be used to exploit any potential positive effects in pedHGG patients. In conclusion, the use of commercially available supplements with a mixture of different BA cannot be recommended due to detrimental effects of certain BA whereas pure AKBA formulations might hold some potential as therapeutic supplement for treatment of pedHGG patients.

Intraoperative or postoperative stereotactic radiotherapy for brain metastases: time to systemic treatment onset and other patient-relevant outcomes.

PURPOSE: Intraoperative radiotherapy (IORT) has become a viable treatment option for resectable brain metastases (BMs). As data on local control and radiation necrosis rates are maturing, we focus on meaningful secondary endpoints such as time to next treatment (TTNT), duration of postoperative corticosteroid treatment, and in-hospital time. METHODS: Patients prospectively recruited within an IORT study registry between November 2020 and June 2023 were compared with consecutive patients receiving adjuvant stereotactic radiotherapy (SRT) of the resection cavity within the same time frame. TTNT was defined as the number of days between BM resection and start of the next extracranial oncological therapy (systemic treatment, surgery, or radiotherapy) for each of the groups. RESULTS: Of 95 BM patients screened, IORT was feasible in 84 cases (88%) and ultimately performed in 64 (67%). The control collective consisted of 53 SRT patients. There were no relevant differences in clinical baseline features. Mean TTNT (range) was 36 (9 - 94) days for IORT patients versus 52 (11 - 126) days for SRT patients (p = 0.01). Mean duration of postoperative corticosteroid treatment was similar (8 days; p = 0.83), as was mean postoperative in-hospital time (11 versus 12 days; p = 0.97). Mean total in-hospital time for BM treatment (in- and out-patient days) was 11 days for IORT versus 19 days for SRT patients (p < 0.001). CONCLUSION: IORT for BMs results in faster completion of interdisciplinary treatment when compared to adjuvant SRT, without increasing corticosteroid intake or prolonging in-hospital times. A randomised phase III trial will determine the clinical effects of shorter TTNT.

ICOS DNA methylation regulates melanoma cell-intrinsic ICOS expression, is associated with melanoma differentiation, prognosis, and predicts response to immune checkpoint blockade.

BACKGROUND: Inducible T cell costimulator ICOS is an emerging target in immuno-oncology. The aim of this study was to investigate the epigenetic regulation of ICOS in melanoma by DNA methylation. METHODS: We comprehensively investigate ICOS DNA methylation of specific CpG sites and expression pattern within the melanoma microenvironment with regard to immune correlates, differentiation, clinical outcomes, and immune checkpoint blockade (ICB) response. RESULTS: Our study revealed a sequence-contextual CpG methylation pattern consistent with an epigenetically regulated gene. We found a cell type-specific methylation pattern and locus-specific correlations and associations of CpG methylation with ICOS mRNA expression, immune infiltration, melanoma differentiation, prognosis, and response to ICB. High ICOS mRNA expression was identified as a surrogate for enriched immune cell infiltration and was associated with favorable overall survival (OS) in non-ICB-treated patients and predicted response and a prolonged progression-free survival (PFS) following ICB therapy initiation. ICOS hypomethylation, however, significantly correlated with poor OS in non-ICB patients but predicted higher response and prolonged PFS and OS in ICB-treated patients. Moreover, we observed cytoplasmic and sporadically nuclear tumor cell-intrinsic ICOS protein expression. Tumor cell-intrinsic ICOS protein and mRNA expression was inducible by pharmacological demethylation with decitabine. CONCLUSION: Our study identified ICOS DNA methylation and mRNA expression as promising prognostic and predictive biomarkers for immunotherapy in melanoma and points towards a hitherto undescribed role of ICOS in tumor cells.

Multiomic neuropathology improves diagnostic accuracy in pediatric neuro-oncology.

The large diversity of central nervous system (CNS) tumor types in children and adolescents results in disparate patient outcomes and renders accurate diagnosis challenging. In this study, we prospectively integrated DNA methylation profiling and targeted gene panel sequencing with blinded neuropathological reference diagnostics for a population-based cohort of more than 1,200 newly diagnosed pediatric patients with CNS tumors, to assess their utility in routine neuropathology. We show that the multi-omic integration increased diagnostic accuracy in a substantial proportion of patients through annotation to a refining DNA methylation class (50%), detection of diagnostic or therapeutically relevant genetic alterations (47%) or identification of cancer predisposition syndromes (10%). Discrepant results by neuropathological WHO-based and DNA methylation-based classification (30%) were enriched in histological high-grade gliomas, implicating relevance for current clinical patient management in 5% of all patients. Follow-up (median 2.5 years) suggests improved survival for patients with histological high-grade gliomas displaying lower-grade molecular profiles. These results provide preliminary evidence of the utility of integrating multi-omics in neuropathology for pediatric neuro-oncology.

Pediatric high-grade gliomas and the WHO CNS Tumor Classification-Perspectives of pediatric neuro-oncologists and neuropathologists in light of recent updates.

Background: The WHO Classification of Tumors of the Central Nervous System has undergone major restructuring. Molecularly defined diagnostic criteria were introduced in 2016 (revised 4th edition) and expanded in 2021 (5th edition) to incorporate further essential diagnostic molecular parameters. We investigated potential differences between specialists in perception of these molecularly defined subtypes for pediatric high-grade gliomas (pedHGG). Methods: We designed a 22-question survey studying the impact of the revised 4th edition of the WHO classification on pedHGG. Data were collected and statistically analyzed to examine the spectrum of viewpoints and possible differences between neuro-oncologists and neuropathologists. Results: 465 participants from 53 countries were included; 187 pediatric neuro-oncologists (40%), 160 neuropathologists (34%), and 118 additional experts (26%). Neuro-oncologists reported issues with the introduction of molecularly defined tumor types, as well as the abolishment or renaming of established tumor entities, while neuropathologists did not to the same extent. Both groups indicated less relevant or insufficient diagnostic definitions were available in 2016. Reported issues were classified and assessed in the 2021 WHO classification and a substantial improvement was perceived. However, issues of high clinical relevance remain to be addressed, including the definition of clinical phenotypes for diffuse intrinsic pontine glioma and gliomatosis cerebri. Conclusions: Within the WHO classification of pediatric brain tumors, such as pedHGG, rapid changes in molecular characterization have been introduced. This study highlights the ongoing need for cross talk between pathologist and oncologist to advance the classification of pedHGG subtypes and ensure biological relevance and clinical impact.

Molecular pathological insights reveal a high number of unfavorable risk patients among children treated for medulloblastoma and CNS-PNET in Oslo 2005-2017.

BACKGROUND: An unexplained regional difference in survival was observed in previous publications on outcome for children treated for medulloblastoma and supratentorial primitive neuroectodermal tumor (CNS-PNET) in Norway. We aimed now to reevaluate and perform a retrospective molecular-based risk stratification of all embryonal brain tumors (excluding atypical teratoid rhabdoid tumors [ATRT]) in pediatric patients, who underwent surgery and treatment at Oslo University Hospital between 2005 and 2017. PROCEDURE: Specimens from all patients <20 years of age with initial diagnosis of medulloblastoma or CNS-PNET were reviewed. Molecular analyses comprised NanoString gene expression, molecular inversion probe profiling, Sanger sequencing, and 850K-methylation analysis. Whole chromosomal aberration signatures were assessed in standard-risk non-WNT/non-SHH medullobastomas for molecular risk stratification. RESULTS: We identified 53 non-ATRT embryonal tumors among which 33 were medulloblastomas. Molecular genetic parameters including whole chromosomal aberration signatures allowed classification of 17 medulloblastomas as molecular high risk. These patients had a significantly worse 5-year overall survival than the remaining 16 medulloblastoma patients (52.9% vs. 87.1% p = 0.036). Five patients in our cohort had tumors that are considered as new entities in the 2021 classification of tumors of the central nervous system. Five tumors were re-classified as nonembryonal tumors after review. CONCLUSION: Molecular-based risk stratification of standard-risk non-WNT/non-SHH medulloblastoma enabled superior identification of medulloblastomas with dismal prognosis. Our cohort demonstrated a significantly increased fraction of standard-risk non-WNT/non-SHH medulloblastoma with molecular high-risk profile compared to other studies, which might have contributed to previously reported unfavorable outcome data.

Magnetic Resonance Imaging Characteristics of Molecular Subgroups in Pediatric H3 K27M Mutant Diffuse Midline Glioma.

PURPOSE: Recent research identified histone H3 K27M mutations to be associated with a dismal prognosis in pediatric diffuse midline glioma (pDMG); however, data on detailed MRI characteristics with respect to H3 K27 mutation status and molecular subgroups (H3.1 and H3.3 K27M mutations) are limited. METHODS: Standardized magnetic resonance imaging (MRI) parameters and epidemiologic data of 68 pDMG patients (age <18 years) were retrospectively reviewed and compared in a) H3 K27M mutant versus H3 K27 wildtype (WT) tumors and b) H3.1 versus H3.3 K27M mutant tumors. RESULTS: Intracranial gliomas (n = 58) showed heterogeneous phenotypes with isointense to hyperintense signal in T2-weighted images and frequent contrast enhancement. Hemorrhage and necrosis may be present. Comparing H3 K27M mutant to WT tumors, there were significant differences in the following parameters: i) tumor localization (p = 0.001), ii) T2 signal intensity (p = 0.021), and iii) T1 signal homogeneity (p = 0.02). No significant imaging differences were found in any parameter between H3.1 and H3.3 K27M mutant tumors; however, H3.1 mutant tumors occurred at a younger age (p = 0.004). Considering spinal gliomas (n = 10) there were no significant imaging differences between the analyzed molecular groups. CONCLUSION: With this study, we are the first to provide detailed MR imaging data on H3 K27M mutant pDMG with respect to molecular subgroup status in a large patient cohort. Our findings may support diagnosis and future targeted therapeutic trials of pDMG within the framework of the radiogenomics concept.

Transitioning to molecular diagnostics in pediatric high-grade glioma: experiences with the 2016 WHO classification of CNS tumors.

BACKGROUND: Pediatric neuro-oncology was profoundly changed in the wake of the 2016 revision of the WHO Classification of Tumors of the Central Nervous System. Practitioners were challenged to quickly adapt to a system of tumor classification redefined by molecular diagnostics. METHODS: We designed a 22-question survey studying the impact of the revised WHO classification on pediatric high-grade glioma. The survey collected basic demographics, general attitudes, issues encountered, and opinions on pediatric subtypes. Participant answers were analyzed along socioeconomic lines utilizing the human development index (HDI) of the United Nations and membership in the group of seven (G7) world economic forum. RESULTS: Four hundred and sixty-five participants from 53 countries were included, 187 pediatric neurooncologists (40%), 160 neuropathologists (34%), and 118 other experts (26%). When asked about pediatric high-grade glioma entities, participants from very high development countries preferred treating a patient based on genetic findings. Participants from high and medium development countries indicated using traditional histology and tumor location as mainstays for therapeutic decisions. Non-G7 countries tended to regard the introduction of molecularly characterized tumor entities as a problem for daily routine due to lack of resources. CONCLUSIONS: Our findings demonstrate an overall greater reliance and favorability to molecular diagnostics among very high development countries. A disparity in resources and access to molecular diagnostics has left some centers unable to classify pediatric high-grade glioma per the WHO classification. The forthcoming edition should strain to abate disparities in molecular diagnostic availability and work toward universal adaptation.

Diagnosis of Pseudoprogression Following Lomustine-Temozolomide Chemoradiation in Newly Diagnosed Glioblastoma Patients Using FET-PET.

PURPOSE: The CeTeG/NOA-09 phase III trial demonstrated a significant survival benefit of lomustine-temozolomide chemoradiation in patients with newly diagnosed glioblastoma with methylated O6-methylguanine-DNA methyltransferase (MGMT) promoter. Following lomustine-temozolomide chemoradiation, late and prolonged pseudoprogression may occur. We here evaluated the value of amino acid PET using O-(2-[18F]fluoroethyl)-l-tyrosine (FET) for differentiating pseudoprogression from tumor progression. EXPERIMENTAL DESIGN: We retrospectively identified patients (i) who were treated off-study according to the CeTeG/NOA-09 protocol, (ii) had equivocal MRI findings after radiotherapy, and (iii) underwent additional FET-PET imaging for diagnostic evaluation (number of scans, 1-3). Maximum and mean tumor-to-brain ratios (TBRmax, TBRmean) and dynamic FET uptake parameters (e.g., time-to-peak) were calculated. In patients with more than one FET-PET scan, relative changes of TBR values were evaluated, that is, an increase or decrease of >10% compared with the reference scan was considered as tumor progression or pseudoprogression. Diagnostic performances were evaluated using ROC curve analyses and Fisher exact test. Diagnoses were confirmed histologically or clinicoradiologically. RESULTS: We identified 23 patients with 32 FET-PET scans. Within 5-25 weeks after radiotherapy (median time, 9 weeks), pseudoprogression occurred in 11 patients (48%). The parameter TBRmean calculated from the FET-PET performed 10 ± 7 days after the equivocal MRI showed the highest accuracy (87%) to identify pseudoprogression (threshold, <1.95; P = 0.029). The integration of relative changes of TBRmean further improved the accuracy (91%; P < 0.001). Moreover, the combination of static and dynamic parameters increased the specificity to 100% (P = 0.005). CONCLUSIONS: The data suggest that FET-PET parameters are of significant clinical value to diagnose pseudoprogression related to lomustine-temozolomide chemoradiation.

DCE-MRI in Glioma, Infiltration Zone and Healthy Brain to Assess Angiogenesis: A Biopsy Study.

PURPOSE: To explore the focal predictability of vascular growth factor expression and neovascularization using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in glioma. METHODS: 120 brain biopsies were taken in vital tumor, infiltration zone and normal brain tissue of 30 glioma patients: 17 IDH(isocitrate dehydrogenase)-wildtype glioblastoma (GBM), 1 IDH-wildtype astrocytoma °III (together prognostic group 1), 3 IDH-mutated GBM (group 2), 3 anaplastic astrocytomas IDH-mutated (group 3), 4 anaplastic oligodendrogliomas and 2 low-grade oligodendrogliomas (together prognostic group 4). A mixed linear model evaluated the predictabilities of microvessel density (MVD), vascular area ratio (VAR), mean vessel size (MVS), vascular endothelial growth factor and receptors (VEGF-A, VEGFR­2) and vascular endothelial-protein tyrosine phosphatase (VE-PTP) expression from Tofts model kinetic and model-free curve parameters. RESULTS: All kinetic parameters were associated with VEGF­A (all p < 0.001) expression. Ktrans, kep and ve were associated with VAR (p = 0.006, 0.004 and 0.01, respectively) and MVS (p = 0.0001, 0.02 and 0.003, respectively) but not MVD (p = 0.84, 0.74 and 0.73, respectively). Prognostic groups differed in Ktrans (p = 0.007) and ve (p = 0.004) values measured in the infiltration zone. Despite significant differences of VAR, MVS, VEGF­A, VEGFR­2, and VE-PTP in vital tumor tissue and the infiltration zone (p = 0.0001 for all), there was no significant difference between kinetic parameters measured in these zones. CONCLUSION: The DCE-MRI kinetic parameters show correlations with microvascular parameters in vital tissue and also reveal blood-brain barrier abnormalities in the infiltration zones adequate to differentiate glioma prognostic groups.

CTLA4 promoter methylation predicts response and progression-free survival in stage IV melanoma treated with anti-CTLA-4 immunotherapy (ipilimumab).

Anti-CTLA-4-antibodies can induce long-lasting tumor remissions. However, only a few patients respond, necessitating the development of predictive companion biomarkers. Increasing evidence suggests a major role of epigenetics, including DNA methylation, in immunology and resistance to immune checkpoint blockade. Here, we tested CTLA4 promoter methylation and CTLA-4 protein expression as predictive biomarkers for response to anti-CTLA-4 immunotherapy. We identified retrospectively N = 30 stage IV melanoma patients treated with single-agent anti-CTLA-4 immunotherapy (ipilimumab). We used quantitative methylation-specific PCR and immunohistochemistry to quantify CTLA4 methylation and protein expression in pre-treatment samples. CTLA4 methylation was significantly higher in progressive as compared to responding tumors and significantly associated with progression-free survival. A subset of infiltrating lymphocytes and tumor cells highly expressed CTLA-4. However, CTLA-4 protein expression did not predict response to treatment. We conclude that CTLA4 methylation is a predictive biomarker for response to anti-CTLA-4 immunotherapy.

Combined treatment with CBP and BET inhibitors reverses inadvertent activation of detrimental super enhancer programs in DIPG cells.

Diffuse intrinsic pontine gliomas (DIPG) are the most aggressive brain tumors in children with 5-year survival rates of only 2%. About 85% of all DIPG are characterized by a lysine-to-methionine substitution in histone 3, which leads to global H3K27 hypomethylation accompanied by H3K27 hyperacetylation. Hyperacetylation in DIPG favors the action of the Bromodomain and Extra-Terminal (BET) protein BRD4, and leads to the reprogramming of the enhancer landscape contributing to the activation of DIPG super enhancer-driven oncogenes. The activity of the acetyltransferase CREB-binding protein (CBP) is enhanced by BRD4 and associated with acetylation of nucleosomes at super enhancers (SE). In addition, CBP contributes to transcriptional activation through its function as a scaffold and protein bridge. Monotherapy with either a CBP (ICG-001) or BET inhibitor (JQ1) led to the reduction of tumor-related characteristics. Interestingly, combined treatment induced strong cytotoxic effects in H3.3K27M-mutated DIPG cell lines. RNA sequencing and chromatin immunoprecipitation revealed that these effects were caused by the inactivation of DIPG SE-controlled tumor-related genes. However, single treatment with ICG-001 or JQ1, respectively, led to activation of a subgroup of detrimental super enhancers. Combinatorial treatment reversed the inadvertent activation of these super enhancers and rescued the effect of ICG-001 and JQ1 single treatment on enhancer-driven oncogenes in H3K27M-mutated DIPG, but not in H3 wild-type pedHGG cells. In conclusion, combinatorial treatment with CBP and BET inhibitors is highly efficient in H3K27M-mutant DIPG due to reversal of inadvertent activation of detrimental SE programs in comparison with monotherapy.

Molecular, clinicopathological, and immune correlates of LAG3 promoter DNA methylation in melanoma.

BACKGROUND: The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment. METHODS: We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118). FINDINGS: Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine. INTERPRETATION: Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Challenging Implications of Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids Syndrome with an Atypical Presentation: Report of Two Cases.

BACKGROUND: Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is an increasingly recognized neuroinflammatory syndrome that predominantly affects the pontine and cerebellar brain structures. Characteristically, patients will develop glucocorticoid-responsive brainstem disorders, demonstrate pontocerebellar contrast enhancement on magnetic resonance imaging (MRI), and exhibit an angiocentric, lymphocytic infiltrate in brain biopsies. We have presented and discussed 2 novel and challenging cases of CLIPPERS syndrome to highlight the clinical and radiological diversity of the syndrome. CASE DESCRIPTION: The first case was of a 66-year-old male patient who had presented with dizziness, headaches, gait disturbances, mild cognitive impairment, and visual field loss to the left side. MRI revealed 1 cerebellar and 2 occipital contrast-enhancing lesions that were suspicious for intracerebral metastases. The second case was of a 53-year-old male patient who had presented with temporal lobe epilepsy, anomic aphasia, and mild cognitive impairment. MRI demonstrated 4 contrast-enhancing lesions in the pons, temporal lobe, and thalamus that were suspicious for intracerebral lymphoma. Because of the radiological presentation, neoplastic disease was the most plausible diagnosis for both patients. However, repeated biopsies ruled out tumor manifestation, and the findings were finally consistent with CLIPPERS syndrome. The significant and long-lasting response to immunosuppressive treatment confirmed the diagnosis. CONCLUSIONS: In both cases, the characteristics of CLIPPERS syndrome imitated malignant tumor growth. This scenario can be challenging to clinicians and necessitates inclusion of this neuroinflammatory syndrome in the differential diagnosis of neuro-oncological disease.

Prognostic and predictive value of PD-L2 DNA methylation and mRNA expression in melanoma.

BACKGROUND: PD-L1 (programmed cell death 1 ligand 1) expression in melanoma has been associated with a better response to anti-PD-1 (programmed cell death 1) therapy. However, patients with PD-L1-negative melanomas can respond to anti-PD-1 blockade, suggesting that the other PD-1 ligand, PD-L2 (programmed cell death 1 ligand 2), might also be relevant for efficacy of PD-1 inhibition. We investigated PD-L2 expression and methylation as a prognostic and predictive biomarker in melanoma. METHODS: DNA methylation at five CpG loci and gene expression of PD-L2 were evaluated with regard to survival in 470 melanomas from The Cancer Genome Atlas. PD-L2 promoter methylation in correlation with PD-L2 mRNA and protein expression was analyzed in human melanoma cell lines. Prognostic and predictive value of PD-L2 methylation was validated using quantitative methylation-specific PCR in a multicenter cohort of 129 melanoma patients receiving anti-PD-1 therapy. mRNA sequencing data of 121 melanoma patients receiving anti-PD-1 therapy provided by Liu et al. were analyzed for PD-L2 mRNA expression. RESULTS: We found significant correlations between PD-L2 methylation and mRNA expression levels in melanoma tissues and cell lines. Interferon-γ inducible PD-L2 protein expression correlated with PD-L2 promoter methylation in melanoma cells. PD-L2 DNA promoter hypomethylation and high mRNA expression were found to be strong predictors of prolonged overall survival. In pre-treatment melanoma samples from patients receiving anti-PD-1 therapy, low PD-L2 DNA methylation and high PD-L2 mRNA expression predicted longer progression-free survival. CONCLUSION: PD-L2 expression seems to be regulated via DNA promoter methylation. PD-L2 DNA methylation and mRNA expression may predict progression-free survival in melanoma patients receiving anti-PD-1 immunotherapy. Assessment of PD-L2 should be included in further clinical trials with anti-PD-1 antibodies.