Citations

Abstract BACKGROUND The diagnosis and management of patients with brain tumors currently uses WHO defined morphologic and molecular features. However, neuro-oncology practice in low resource settings relies solely on histomorphology. This study aims to reclassify glioma cases diagnosed in the Department of Anatomic and Molecular Pathology Lagos University Teaching Hospital using the WHO 2021 classification. METHODS Fifty-six cases of glioma diagnosed between 2013 to 2021 were evaluated. Morphologic diagnosis was reassessed. Five-micron sections were obtained for immunohistochemistry (IHC), and genetic testing (DNA and RNA) after manual macrodissection for tumor enrichment used 10-µm sections. IHC for IDH1-R132H, ATRX, BRAF-V600E, p53, Ki67 and H3-K27M and Oncoscan chromosomal microarray analysis were performed. NGS mutation/fusion analysis and EPIC methylation array profiling are in progress. RESULTS Of the 56 cases evaluated, median age was 22 years (range, 1-60); 71% were male. The initial morphologic diagnoses included 21 pilocytic astrocytoma, 11 glioblastoma, 9 ependymoma, 7 diffuse astrocytoma, 3 anaplastic astrocytoma, 2 high-grade gliomas, 1 oligodendroglioma, 1 diffuse midline glioma, and 1 pleomorphic xanthoastrocytoma (PXA). Histomorphologic re-evaluation revealed discordant diagnosis in 29% (16/56) of cases. Of the 56 cases, 73% (41/56) had usable DNA yield and 38% (21/56) had DNA yield of ≥750ng. Chromosomal microarray analysis was completed for 50% (28/56) cases. The revised WHO 2021 diagnosis was: 5 glioblastoma IDH-wildtype, 5 pilocytic astrocytoma, 4 glioma NEC, 3 posterior fossa ependymoma, group B, 3 PXA, 3 astrocytoma IDH-mutant, 2 pediatric low-grade gliomas, 2 supratentorial ependymoma, ZFTA fusion-positive, 1 posterior fossa ependymoma, group A. In 71% (20/28) of cases with molecular testing, diagnosis was revised using WHO 2021 criteria. CONCLUSION Most cases had usable DNA. Overall, histologic re-evaluation and molecular based on the WHO 2021 criteria refined the diagnosis in 48% (27/56) of cases.

Abstract BACKGROUND D-2-hydroxyglutarate (D-2-HG) is a well-established oncometabolite of isocitrate dehydrogenase (IDH) mutant gliomas. While prior studies have demonstrated that D-2-HG is elevated in the cerebrospinal fluid (CSF) of patients with IDH-mutant gliomas, no study has evaluated the ability of CSF D-2-HG to quantitatively monitor tumor burden and therapeutic response. Toward this goal, we are routinely obtaining CSF samples from patients prior to, during, and after treatment via intra-operative collection and CSF-access devices. MATERIAL AND METHODS Patients were enrolled into our Ommaya reservoir study (NCT04692337), which enables longitudinal access to CSF. The catheter for the implanted reservoir was placed within the surgical resection cavity following a standard-of-care resection. Intra-operatively collected CSF samples were also biobanked through Mayo Clinic’s Neuro-Oncology biorepository. CSF samples were also acquired via clinically indicated ventriculoperitoneal shunts (VP shunts) or external ventricular drains via our ongoing CSF biomarkers study (NCT04692324). D-and-L-2-HG were assayed from each CSF sample via gas chromatography-mass spectrometry (GC-MS) at Mayo Clinic’s Biochemical Genetics Laboratory. RESULTS CSF was collected prior to and after resection in patients via Ommaya reservoirs or VP shunts, all of whom underwent near gross total resections (>95% of tumor volume). D-2-HG and the D/L ratio fell on average by 57.9% (range: 15.1%-98.6%) and 68.8% (range: 49.3%-98.9%), respectively, following resection. In one patient with a largely intraventricular grade 4 IDH-mutant astrocytoma, D-and-L-2-HG fell by 99.1% and 99.3% by post-operative day 2, consistent with the impact of ventricular contact on baseline CSF D-2-HG levels. In patients for whom CSF was collected throughout chemoradiation, with or without immunotherapy, D-2-HG and the D/L ratio continued to decrease over time, despite concern for pseudoprogression versus progression on imaging. CONCLUSION Our early experience with this strategy demonstrates the potential for intracranial CSF D-2-HG as a monitoring biomarker for IDH-mutant gliomas. Work is ongoing to evaluate the reproducibility of these findings in a larger cohort of patients with low or high-grade gliomas, in addition to the impact of tumor recurrence on 2-HG. Future studies will evaluate the impact of IDH inhibitors and other experimental therapies on CSF 2-HG levels for pharmacodynamic monitoring of treatment response.

AbstractObjectiveTo describe demographic, clinical, and radiographic features of tumefactive demyelination (TD) and identify factors associated with severe attacks and poor outcomes.MethodsRetrospective review of TD cases seen at Mayo Clinic, 1990–2021.ResultsOf 257 patients with TD, 183/257 (71%) fulfilled the 2017 multiple sclerosis (MS) McDonald criteria at the last follow‐up, 12/257 (5%) had myelin oligodendrocyte glycoprotein antibody‐associated disease (MOGAD), 0 had aquaporin‐4‐IgG seropositive neuromyelitis optic spectrum disorders (AQP4+ NMOSD), and 62/257 (24%) were cryptogenic. Onset before age 18 was present in 18/257 (7%). Female to male ratio was 1.3:1. Cerebrospinal fluid oligoclonal (CSF) bands were present in 95/153 (62%). TD was the first demyelinating attack in 176/257 (69%). At presentation, 59/126 (47%) fulfilled Barkhof criteria for dissemination in space, 59/100 (59%) had apparent diffusion coefficient (ADC) restriction, and 57/126 (45%) had mass effect. Despite aggressive clinical presentation at onset, 181/257 (70%) of patients remained fully ambulatory (Expanded Disability Status Scale [EDSS] ≤4) after a 3.0‐year median follow‐up duration. Severe initial attack‐related disability (EDSS ≥4) was more common in patients with motor symptoms (81/143 vs. 35/106, p < 0.0001), encephalopathy (20/143 vs. 2/106, p < 0.0001) and ADC restriction on initial MRI (42/63 vs. 15/33, p = 0.04). Poor long‐term outcome (EDSS ≥4) was more common in patients with older onset age (41.9 ± 15 vs. 36.8 ± 15.6, p = 0.02) and motor symptoms at onset (49/76 vs. 66/171, p < 0.0001).InterpretationMost TD patients should be considered part of the MS spectrum after excluding MOGAD and NMOSD. Motor symptoms and older age at presentation portend a poor outcome.

Abstract Background Mendelian randomization (MR) studies are susceptible to metadata errors (e.g. incorrect specification of the effect allele column) and other analytical issues that can introduce substantial bias into analyses. We developed a quality control (QC) pipeline for the Fatty Acids in Cancer Mendelian Randomization Collaboration (FAMRC) that can be used to identify and correct for such errors. Methods We collated summary association statistics from fatty acid and cancer genome-wide association studies (GWAS) and subjected the collated data to a comprehensive QC pipeline. We identified metadata errors through comparison of study-specific statistics to external reference data sets (the National Human Genome Research Institute-European Bioinformatics Institute GWAS catalogue and 1000 genome super populations) and other analytical issues through comparison of reported to expected genetic effect sizes. Comparisons were based on three sets of genetic variants: (i) GWAS hits for fatty acids, (ii) GWAS hits for cancer and (iii) a 1000 genomes reference set. Results We collated summary data from 6 fatty acid and 54 cancer GWAS. Metadata errors and analytical issues with the potential to introduce substantial bias were identified in seven studies (11.6%). After resolving metadata errors and analytical issues, we created a data set of 219 842 genetic associations with 90 cancer types, generated in analyses of 566 665 cancer cases and 1 622 374 controls. Conclusions In this large MR collaboration, 11.6% of included studies were affected by a substantial metadata error or analytical issue. By increasing the integrity of collated summary data prior to their analysis, our protocol can be used to increase the reliability of downstream MR analyses. Our pipeline is available to other researchers via the CheckSumStats package (https://github.com/MRCIEU/CheckSumStats).

Abstract Signaling networks and the protein interactions that define them are of great interest, but techniques to assess these networks in unmanipulated primary human cells are limited. We have developed and applied a novel technological and analytical platform to measure proteins in shared complexes detected by exposed surface epitopes (PiSCES). Immunoprecipitation reactions are performed in multiplex using microsphere beads to measure 20+ proteins and 210+ distinct pair-wise protein associations that are important for T cell antigen receptor signaling. Jurkat T cells stimulated with superantigen generated an extensive PiSCES signature using this method and provided a dataset from which we optimized an analytical package. The system is currently being utilized to address the hypothesis that T cells isolated from skin lesions of autoimmune alopecia areta (AA) patients will have a unique PiSCES signature compared to those isolated from non-AA donors. To investigate this, we have isolated non-engineered primary cells from AA patient skin lesions and healthy controls, and have begun to identify differences in their PiSCES signatures, either directly after isolation or after stimulation with anti-CD3 and anti-CD28. We conclude that this novel PiSCES approach is capable of generating protein-protein interaction signatures that provide a unique view of TCR signalosome activity solicited from physiologic non-engineered primary human patient samples.

Abstract BACKGROUND The topologically associated domain (TAD) on 8q24 surrounding MYC is important for many types of cancer. The region is also important in the formation of IDH-mutant glioma; the risk allele rs55705857 being associated with the development of these tumors. The rs55705857 risk allele significantly increased the formation of tumors in an IDH-mutant mouse model. Cerebral organoids have been found to recapitulate the early development of the brain. We hypothesized that the rs55705857 risk allele may alter the phenotype of cerebral organoids. METHODS Isogenic induced pluripotent stem cells (iPSCs) were developed with (n=3) and without (n=2) the rs55705857 risk allele from the parental cell line PGP-1 (GM23338). These cells were cultured and differentiated into unpatterned cerebral organoids and observed throughout differentiation. Organoids were fixed, sectioned, and stained by H&E and immunofluorescence. RESULTS Organoids containing the risk allele were smaller throughout development, starting at the embryoid body stage and continuing up to over 75 days in culture, compared to two isogenic non-risk lines. At 4 weeks the risk allele containing organoids also contained a larger number of small rosette-like neuroepithelia, instead of the more continuous neuroepithelia observed in the non-risk allele organoids. At 75 days, the organoids with the risk allele maintained a larger proportion of Sox2 positive cells, which are enriched in the tight clusters of small cells with round nuclei. CONCLUSIONS The risk allele rs55705857 in the MYC TAD at 8q24 is altering the development of unpatterned cerebral organoids. This difference is visible as early as embryoid body development and persists through neuroepithelial development. Further studies of these differences may help our understanding of how rs55705857 accelerates IDH-mutant glioma development.

Abstract Missing MRI sequences are common in radiology due to differences in protocols, time constraints, and image artifacts. Missing sequences are a nuisance for subsequent algorithms, such as automated segmentation algorithms requiring multiple MRI sequences (e.g., HD-GLIO, which requires T2, T1, post-contrast T1 (T1c), and FLAIR). Accurate tumor segmentation is a fundamental step in extracting radiomics analysis to use, for example, to predict the IDH status of glioma or their overall survival. We validated a conditional (pix2pix) and super-resolution GAN (SOUP-GAN) for imputing MRI sequences on 285 adult glioma with complete MRI sequences available. We simulated missing T1 sequences and subsequently imputed them from T1c using pix2pix and from sagittal T1 using SOUP-GAN. Using HD-GLIO, we obtained the true segmentations from the complete MRI sequences. HD-GLIO was also used after replacing the original T1 sequence with the one imputed with pix2pix and SOUP-GAN. Thus, there were three sets of segmentations for each subject. We compared T2 and T1c radiomics features extracted with Pyradiomics using segmentations obtained with complete data with features extracted from segmentation obtained with the imputed T1 sequences using intraclass correlation coefficient (ICC) and Bland-Altman (BA) analysis. Median (interquartile range) ICC for shape features extracted from T2 and T1c were 0.82(0.76-0.92) using pix2pix and 0.89(0.86-0.94) using SOUP-GAN. Median ICC for first-order-statistics features were 0.90(0.83-0.93) for T2 and 0.90(0.88-0.92) for T1c using pix2pix, and 0.61(0.22-0.82) for T2 and 0.42(0.34-0.57) for T1c using SOUP-GAN. Median ICC for texture features were 0.86(0.82-0.87) for T2 and 0.87(0.79-0.90) for T1c using pix2pix, and 0.69(0.47-0.81) for T2 and 0.76(0.65-0.83) for T1c using SOUP-GAN. We show that T2 and T1c shape features (e.g., volumetric analysis) can be adequately extracted from segmentations obtained with an algorithm requiring multiple MRI sequences using T1 sequences imputed with GANs; however, the effect of imputation differed for first-order-statistics and texture features.

Establishing causal links between inherited polymorphisms and cancer risk is challenging. Here, we focus on the single-nucleotide polymorphism rs55705857, which confers a sixfold greater risk of isocitrate dehydrogenase ( IDH) –mutant low-grade glioma (LGG). We reveal that rs55705857 itself is the causal variant and is associated with molecular pathways that drive LGG. Mechanistically, we show that rs55705857 resides within a brain-specific enhancer, where the risk allele disrupts OCT2/4 binding, allowing increased interaction with the Myc promoter and increased Myc expression. Mutating the orthologous mouse rs55705857 locus accelerated tumor development in an Idh1 R132H -driven LGG mouse model from 472 to 172 days and increased penetrance from 30% to 75%. Our work reveals mechanisms of the heritable predisposition to lethal glioma in ~40% of LGG patients.

Abstract Background EGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood–brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM. Methods The efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity. Results Dose-finding studies in orthotopic GBM6 identified single infusion of 2 μg Ser-T and 60 μg Depatux-M as safe and effective associated with extended survival prolongation (&gt;300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining. Conclusion CED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.