Methylation Class Research Articles

The impact of methylome analysis on the diagnosis and treatment of CNS tumours in children and adolescents: a population-based study in Greece.

BackgroundThe recently published WHO classification of central nervous system (CNS) tumours recognizes DNA methylation profiling as a desirable and, for some diagnoses, essential diagnostic tool adjunctive to conventional histopathology. DNA methylation profiling is not routinely available in many countries, including Greece. MethodsIn this collaborative study, we report the DNA methylation results in a series of children and adolescents with CNS tumours in Greece (2018-2023). In total, 130 tumour samples were analyzed using the latest applicable version of the Heidelberg brain tumour classifier. ResultsUpon initial analysis, 80% (104/130) achieved calibrated scores (Cs) ≥0.9 and matched an established methylation class family/subclass. Among them, methylation results confirmed (90/104, 86.5%), refined (50/104, 48%) or changed (10/104, 9.6%) the histological diagnosis. Only four results were regarded as non-contributing (4/104, 3.9%). Twenty-six tumour samples received Cs <0.9. Despite low scores, methylation results supported the initial diagnosis with lower confidence in 38.5% (10/26) and established the diagnosis in two tumours with non-conclusive histopathology. Additional t-distributed stochastic neighbour embedding (t-SNE) analysis allowed the possible classification of twelve tumours. Nine more samples reached high Cs using the newer brain tumour classifiers, since available. Samples co-tested in Greece demonstrated excellent test reproducibility, supporting the analysis' local implementation. Methylome profiling impacted the clinical management of 40% of patients, modifying stratification, prognosis, or treatment approach. ConclusionsThis study supports the need to integrate methylome analysis into routine diagnostics in our country and highlights the importance of collaboration between European pediatric oncology centres.

Phenotypic and epigenetic heterogeneity in FGFR2-fused glial and glioneuronal tumours.

FGFR-fused central nervous system (CNS) tumours are rare and are usually within the glioneuronal and neuronal tumours or the paediatric-type diffuse low-grade glioma spectrum. Among this spectrum, FGFR2 fusion has been documented in tumours classified by DNA-methylation profiling as polymorphous low-grade neuroepithelial tumours of the young (PLNTY), a recently described tumour type. However, FGFR2 fusions have also been reported in glioneuronal tumours, highlighting the overlapping diagnostic criteria and challenges. We investigated the FGFR2 fusion landscape in a French national series of tumours sent to the RENOCLIP-LOC network. We comprehensively analysed histology, radiology and molecular data including DNA-methylation profiling for 16 FGFR2-fused glioneuronal tumours. Most tumours were located in the temporal or parietal lobe with a median age at diagnosis of 7years [1-44]. Epilepsy was the most frequent symptom. Five patients had tumour progression or recurrence with a median progression-free survival of 22.6months. Histological phenotypes corresponding to PLNTY, GG, MVNT or unclassified tumours were recorded. Epigenetic profiling could not properly distinguish epigenetic clusters related to the GG and PLNTY methylation classes among FGFR2-fused glioneuronal tumours. However, a neuroradiological review identified strikingly distinct neuroradiological patterns. While delineating tumour types among the FGFR2-fused glioneuronal tumour spectrum, by histopathology or DNA-methylation profiling, remains challenging, neuroimaging data revealed two distinct patterns that could correlate to PLNTY and ganglioglioma. However, more series including extensive histo-radio-molecular data are needed to confirm this hypothesis.

Deep learning-based classifier for carcinoma of unknown primary using methylation quantitative trait loci.

Cancer of unknown primary (CUP) constitutes between 2% and 5% of human malignancies and is among the most common causes of cancer death in the United States. Brain metastases are often the first clinical presentation of CUP; despite extensive pathological and imaging studies, 20%-45% of CUP are never assigned a primary site. DNA methylation array profiling is a reliable method for tumor classification but tumor-type-specific classifier development requires many reference samples. This is difficult to accomplish for CUP as many cases are never assigned a specific diagnosis. Recent studies identified subsets of methylation quantitative trait loci (mQTLs) unique to specific organs, which could help increase classifier accuracy while requiring fewer samples. We performed a retrospective genome-wide methylation analysis of 759 carcinoma samples from formalin-fixed paraffin-embedded tissue samples using Illumina EPIC array. Utilizing mQTL specific for breast, lung, ovarian/gynecologic, colon, kidney, or testis (BLOCKT) (185k total probes), we developed a deep learning-based methylation classifier that achieved 93.12% average accuracy and 93.04% average F1-score across a 10-fold validation for BLOCKT organs. Our findings indicate that our organ-based DNA methylation classifier can assist pathologists in identifying the site of origin, providing oncologists insight on a diagnosis to administer appropriate therapy, improving patient outcomes.

EANO guideline on molecular testing of meningiomas for targeted therapy selection.

Meningiomas are the most common primary intracranial tumors of adults. For meningiomas that progress or recur despite surgical resection and radiotherapy, additional treatment options are limited due to lack of proven efficacy. Meningiomas show recurring molecular aberrations, which may serve as predictive markers for systemic pharmacotherapies with targeted drugs or immunotherapy, radiotherapy or radioligand therapy. Here, we review the evidence for a predictive role of a wide range of molecular alterations and markers including NF2, AKT1, SMO, SMARCE1, PIK3CA, CDKN2A/B, CDK4/6, TERT, TRAF7, BAP1, KLF4, ARID1/2, SUFU, PD-L1, SSTR2A, PR/ER, mTOR, VEGFR, PDGFR, as well as homologous recombination deficiency (HRD), genomic copy number variations, DNA methylation classes and combined gene expression profiles. In our assessment based on the established ESMO ESCAT (European Society for Medical Oncology Scale for Clinical Actionability of molecular Targets) evidence level criteria, no molecular target reached ESCAT I ("ready for clinical use") classification and only mTOR pathway activation and NF2 alterations reached ESCAT II ("investigational") classification, respectively. Our evaluations may guide targeted therapy selection in clinical practice and clinical trial efforts and highlight areas for which additional research is warranted.

Meningiomas: Sex-Specific Differences and Prognostic Implications of a Chromosome X Loss.

Meningiomas are the most common primary intracranial tumours in adults. Several studies proposed new stratification systems with a more accurate risk prediction than the WHO grading, e.g. based on methylation and copy number variations (CNVs). Yet, common shortcomings in these analyses are either a lack of stratification by sex of patients or excluding the gonososmes from CNV assessment. Within this study, DNA methylation array data from 7,424 meningioma samples as well as targeted sequencing, clinical annotations and morphology subtyping of 796 samples were examined for differences between females and males regarding mutations, methylation classes, copy number variations and histology. Meningiomas from females accounted for about 53 % of the malignant tumours and present a loss of one X chromosome in 57 % of these malignant cases. In the group of benign tumours, females comprised about 75 % of the patients. Therein, a loss of one X chromosome was detected in only about 10 % of the cases but was associated with a significantly worse progression free survival. Although genomic instability is a common feature of malignant meningiomas, particularly loss of the X chromosome in tumours of female patients in otherwise histologically and molecularly low-risk tumours confers higher risk. Hence, the gonosomal copy number status can be leveraged for increased diagnostic accuracy.

Abstract PR004: Biological insights into tissue-agnostic plasma cfDNA methylation signature for surveillance of head and neck tumor recurrence

Abstract BACKGROUND: We recently described a clinical validation of a tissue-agnostic genome-wide methylome enrichment molecular residual disease (MRD) assay for head and neck cancers (HNC) (Liu, ESMO 2024). Herein, we investigate the cfDNA methylation signature utilized by this test to demonstrate the biological relevance with respect to the tissue of origin and epigenetic mechanisms of dysregulation that alter normal cellular activity. We evaluate the signature to gain insights and to characterize the biology of this biomarker and to investigate the application of cfMeDIP-seq assay as a diagnostic tool. METHODS: From unblinded training set of 163 HNC patients, including HPV+ (n = 76), HPV- (n = 43), and HPV-unknown (n = 44) individuals, a cfDNA methylation classifier signature was developed to detect recurrence (n MRD+ = 62, n MRD- = 101) in samples from blood draws approximately at 3, 12, and 24 months post curative intent treatment using cfMeDIP-seq assay. Clinical performance of the signature was then successfully validated in a blinded HNC validation cohort (n = 162), including HPV+ and HPV- patients (Liu, ESMO 2024). Here, the signature’s ability to detect ctDNA was assessed using HNC tumor tissue (n = 523), adjacent normal tissue (n = 45), and healthy peripheral blood leukocytes (n = 93) DNA methylation data from TCGA. The signature’s ability to provide biological insights was assessed by evaluating enrichment in CpG elements, epigenetic regulators, regulatory pathways, and other biological pathways. RESULTS: Identified differentially methylated regions (DMRs) were significantly hypermethylated in HNC tumor tissue samples when compared to adjacent normal and healthy peripheral blood leukocytes (P = 1.3e-131), and the mean signature signal within tumor tissue samples correlated with tumor purity (P = 2.47e-21). These DMRs were also significantly enriched in CpG islands, shores, and shelves and could classify HNC patients into identifiable molecular subtypes. Further analysis of the DMRs, based on their genomic coordinates, shows association with known cancer genes and significant enrichment in pathways known to be associated with HNC, for example, epithelial to mesenchymal transition (P = 1.76e-4), and multiple functional pathways involved in DNA and transcription factor binding, transcriptional regulation, and chromatin remodeling. CONCLUSIONS: These results, along with the previous clinical validation, provide strong evidence that the cfDNA methylation signature developed in HNC detects ctDNA shed from residual tumor and provide insights into relevant tumor biology and epigenetic mechanisms, including CpG island hypermethylation and epithelial to mesenchymal transition. Furthermore, these data support the generalization of biomarker detection utilizing cfMeDIP-seq to a broad spectrum of indications. Citation Format: Yulia Newton, Justin Burgener, Margaret Gruca, Collin Melton, Jun Min, Abel Licon, Scott Bratman, Alan Williams, Daniel D De Carvalho. Biological insights into tissue-agnostic plasma cfDNA methylation signature for surveillance of head and neck tumor recurrence [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR004.

Abstract A062: Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification

Abstract Retinoblastoma (RB) the most common intraocular malignancy in children poses a significant challenge as traditional tissue biopsies are not feasible. Due to its critical location within the eye and the high risk of tumor seeding, clinicians are limited in performing molecular analysis-based risk stratification prior to enucleation. In this study, we explored the potential of aqueous humor (AH) as a source for minimally invasive liquid biopsies, aiming to establish a molecular profiling methodology for circulating tumor DNA (ctDNA) for risk stratification. We analyzed AH samples and corresponding tumor tissues from 19 enucleated eyes of patients diagnosed with RB. The extracted ctDNA from AH underwent low-coverage whole-genome sequencing (lcWGS) and methylation profiling. These data were then compared with the genomic and methylation landscapes of the matched tumor DNA. Additionally, we assessed the ability of AH- derived ctDNA to predict retinoblastoma methylation cluster. Our findings demonstrate a high concordance between ctDNA from AH and the primary tumor DNA in both methylation patterns and genomic alterations detected by lcWGS allowing the correct prediction of the retinoblastoma molecular group. The shared aberrations underscore the potential of AH as a reliable surrogate for tumor tissue in retinoblastoma. Additionally, the methylation profiles revealed consistent epigenetic modifications, further supporting the accuracy of AH-derived ctDNA in reflecting the tumor’s molecular characteristics. This study highlights the feasibility and effectiveness of using AH for molecular ctDNA genetic analysis in retinoblastoma. The overall high concordance between ctDNA profiles with primary tumor DNA and the ability to accurately predict RB methylation classes, highlights the superiority of the liquid biopsy approach overcoming the limitations of traditional biopsies and transforming the potential for early molecular analysis and risk stratification. This revolutionary technique could redefine pre-enucleation diagnostics, offering unprecedented insights and paving the way for tailored, precision medicine in retinoblastoma care. Citation Format: Sophia H Montigel, Tatsiana Ryl, Stefanie Volz, Elena Afanasyeva, Tatjana Wedig, Nathalie Schwarz, Stefan M Pfister, Kristian W Pajtler, Petra Ketteler, Kendra K Maaß. Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A062.

A primary intracranial neuroepithelial neoplasm with novel TCF3::BEND2 fusion: a case report

Astroblastoma, MN1-altered, is a rare circumscribed glial neoplasm that is composed of round, cuboidal, orcolumnar cells with astroblastic perivascular pseudorosettes, often associated with MN1::BEND2 and MN1::CXXC5 fusions. Atroblastoma-like gliomas harbouring EWSR1::BEND2 have been reported that they defined an epigenetically distinct subtype of astroblastoma. We report a case of a 19-year-old female with an intracranial neuroepithelial tumor featuring a novel TCF3::BEND2 fusion. This tumor, while classified as EWSR1::BEND2 gliomas based on DNA methylation, did not exhibit the MN1 alteration or typical astroblastoma morphology. The patient, initially diagnosed as ependymoma WHO grade 2 following surgery for an intracranial tumor four years prior, presented with a suspected recurrence. Magnetic resonance imaging identified a mixed solid-cystic lesion in the temporal area of the left lateral ventricle. For the recurrent tumor, the histological examination revealed the tumor cells predominantly exhibited a solid arrangement, with the solid areas primarily consisting of oval and short-spindle cells. In certain regions, loosely arranged short-spindle cells was observed. The tumor exhibited high cellular density, nuclear atypia, and frequent mitoses, but lacked the hallmark features typically associated with astroblastoma. Immunohistochemistry revealed patchy positivity for GFAP and OLIG2, diffuse positivity for EMA, and a high MIB-1 labeling index. Genome-wide DNA methylation profiling confirmed the tumor’s classification as EWSR1::BEND2 gliomas with a high-confidence match and revealed focal deletion of chromosome 9q. Targeted next-generation sequencing identified a TCF3::BEND2 fusion, validated by reverse transcription polymerase chain reaction and Sanger sequencing. This case broadens the genetic spectrum of high-grade neuroepithelial tumor and suggests that BEND2 alterations may serve as critical determinants for this EWSR1::BEND2 glioma subgroup within the methylation classifier.

PATH-53. DNA MUTATION SEQUENCING AND METHYLATION ANALYSIS OF SOMATICNF1 MUTANT IDH-WILDTYPE GLIOBLASTOMA IDENTIFIES THREE EPIGENETIC GROUPS ANDCDKN2A/B LOSS AS A NEGATIVE PROGNOSTIC BIOMARKER

Abstract The tumor suppressor NF1 is frequently mutated in IDH-wildtype glioblastoma. However, the molecular subgroups and clinically relevant biomarkers within somatic NF1 mutant, IDH-wildtype glioblastomas remain incompletely understood. Here, we combine methylation arrays and targeted DNA sequencing to identify epigenetic subgroups and clinical biomarkers within non-syndromic NF1 mutant IDH-wildtype glioblastoma. We identified 186 patients treated at the University of California San Francisco between 2016-2024 with newly diagnosed IDH-wildtype glioblastoma containing a somatic pathogenic NF1 mutation on a CLIA certified targeted DNA sequencing assay. Illumina EPIC DNA methylation arrays (n=129 patients) were processed using the minfi package and Heidelberg random forest classifier (v12.8). Downstream analyses including hierarchical clustering and survival analyses were performed in R. DNA sequencing identified 12 recurrently altered genes in greater than 10% of cases across cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic modifiers (ATRX, SETD2), and growth factor signaling (EGFR, PDGFRA, PTPN11). DNA methylation analysis revealed three epigenetic subgroups. Group 1 showed lower median patient age and significantly increased TP53 co-mutation rate. Group 2 contained a significantly higher proportion of male patients and was significantly enriched for RB1 and TERT promoter alterations, with a majority of tumors classified as GBM-MES using the Heidelberg classifier. Group 3 showed a significantly higher proportion of female patients and was significantly enriched for homozygous CDKN2A/B deletion. Homozygous CDKN2A/B loss was associated with significantly worse overall survival across the entire non-syndromic NF1 mutant glioblastoma cohort but not in a propensity score matched NF1 wildtype glioblastoma cohort. DNA methylation profiling revealed three epigenetic subgroups characterized by distinct clinical features, co-mutation patterns, and reference DKFZ methylation classifier identities. CDKN2A/B loss may be a negative prognostic biomarker specifically in NF1 mutant glioblastomas; future multi-institutional studies are required to validate these findings and their therapeutic implications in a larger, representative cohort.

BIOM-71. EXPANDING THE SPECTRUM OFTERT PROMOTER NON-CANONICAL SEQUENCE VARIANTS IN CNS TUMORS: A CASE SERIES

Abstract Most TERT promoter (TERTp) clinically relevant sequence variants in CNS tumors are the canonical C228T and C250T mutations. Non-canonical TERTp mutations have been reported mainly in non-CNS tumors. We describe 12 CNS tumors with non-canonical TERTp variants interpreted as clinically relevant. Using clinical targeted NGS panels (2020-2024), we detected c.-100_-79dup (n=2), c.-92_-91insGGCGGCCCCGCCCCTTCCTTTC (n=1), c.-118_-117insTCCCCGGCCCAGCCCCTTCCGGG (n=1), c.-123_-76dup (n=1), c.-110_-89dup (n=2), and c.-57A&amp;gt;C (n=5). Most cases had chromosomal microarray (n=11) and/or methylation array data analyzed using the NCI/Bethesda v.2 classifier (n=9). All non-canonical TERTp variants occurred at a variant allele frequency suggestive of somatic clonal origin (mean, 34%; range, 18-46), and in adults (mean age, 60 years; range, 36-88). Except for the c.-57A&amp;gt;C variant, all non-canonical TERTp variants occurred in tumors with histological and/or molecular diagnostic features of glioblastoma, IDH-wildtype (GBM), often epigenetically aligning to this tumor type. All five tested cases matched to superfamily glioblastoma (score ≥ 0.9), with three to class GBM_RTK_I (&amp;gt;0.9 in 2, 0.86 in 1) and two to GBM_RTK_II (&amp;gt;0.9). The size and sequence of such variants suggest that these may be functionally equivalent to canonical mutations or generate favorable TERTp activation motif conformations like previously reported activating duplications. The c.-57A&amp;gt;C variant is a well-established oncogenic mutation observed in other cancer types but has not been reported in CNS tumors. We describe five cases with the c.-57A&amp;gt;C variant: one GBM matching to methylation class GBM_MES_TYP (0.99); two oligodendroglioma, IDH-mutant and 1p/19q-codeleted, one of which had methylation data matching to class O_IDH (0.99); one diffuse glioma, IDH-mutant, which was partially 1p/19q-codeleted and matched to class O_IDH (0.99); and one anaplastic meningioma, aligning to superfamily meningioma (0.99)/MNG_MAL (0.79). Our findings show that non-canonical TERTp variants typically occur in tumor types that often harbor TERTp canonical mutations, expanding the spectrum of TERTp clinically relevant variants that are molecular biomarkers in CNS tumors.

PATH-14. UTILITY OF DNA METHYLATION ANALYSIS IN HISTOLOGICALLY-CONFIRMED PA PATIENTS WITH POOR PROGNOSIS

Abstract INTRODUCTION Pilocytic astrocytoma (PA) is a circumscribed low-grade astrocytic glioma, generally considered to be associated with a good prognosis. However, some PA patients experience unfavorable outcomes; advanced age was known as an independent poor prognostic factor. This study retrospectively reviewed PA patients and conducted molecular analyses for elderly patients. METHODS We analyzed data from 29 histologically confirmed PA patients treated at a single center from 2002 to 2021 and conducted integrated molecular analyses including DNA methylation profiling for elderly PA patients. RESULTS The median age at diagnosis was 14 years (range 3-82 years), with 4 patients (14%) being elderly (patients≥60years old). Elderly patients had significantly worse progression-free survival and overall survival. DNA methylation analysis was performed on 2 of the 4 elderly patients. Despite histological diagnoses of PA, methylation profiling classified one case as high-grade astrocytoma with piloid features (methylation class scores below 0.3 in both v11b4 and v12.5) and the other as glioblastoma, IDH-wildtype (score over 0.5 in both v11b4 and v12.5), using classifiers from the German Cancer Research Center and t-SNE analysis. CONCLUSION Elderly patients with PA morphology showed unfavorable outcomes in this cohort. In those patients, DNA methylation profiling revealed the possibility of high-grade astrocytic tumors, including newly defined entities.

PATH-29. LONGITUDINAL EPIGENOMIC EVOLUTION OF IDH-WILDTYPE GLIOBLASTOMA

Abstract Glioblastoma, IDH-wildtype remains a molecularly heterogeneous disease that is refractory to current therapies. To investigate how longitudinal epigenomic evolution of glioblastoma drives tumor progression and treatment resistance, we performed comprehensive histopathologic assessment, targeted genomic sequencing, and genome-wide DNA methylation profiling of paired initial and recurrent IDH-wildtype glioblastoma samples from 98 patients, as well as a separate longitudinal cohort of IDH-mutant astrocytomas for comparison. Primary treatment-naïve IDH-wildtype glioblastomas had more globally hypomethylated genomes compared to primary IDH-mutant astrocytomas. At time of recurrence, IDH-mutant astrocytomas uniformly became more hypomethylated similar to the global methylation levels in IDH-wildtype glioblastomas. In contrast, recurrent IDH-wildtype glioblastomas were heterogeneous with subsets becoming more globally hypermethylated (27%), hypomethylated (22%), or remaining stable (51%). Accordingly, a substantial proportion (52%) of recurrent glioblastomas underwent methylation class switching compared to their matched primary tumor, with a predominance of the RTK2 epigenetic subtype at initial surgery and the Mesenchymal epigenetic subtype at recurrence. Among approximately 850,000 interrogated CpG sites, we identified only 110 and 153 sites that consistently became more hypermethylated or hypomethylated between initial and recurrent IDH-wildtype glioblastomas, which regulate genes involved in neuronal morphogenesis, oligodendrocyte differentiation, and myelination. In contrast, we identified 260 and 18,527 CpG sites that consistently became more hypermethylated or hypomethylated between initial and recurrent IDH-mutant astrocytomas, which regulate genes involved in neuronal stem-like differentiation, cell cycle control, and other biologic processes. Finally, we developed a DNA methylation evolution signature incorporating change in mean beta-methylation values from initial to recurrent tumor specimens across 347 critical CpG sites that significantly correlated with clinical outcomes for patients with IDH-wildtype glioblastoma. In summary, IDH-wildtype glioblastomas demonstrate heterogeneous epigenomic evolution patterns distinct from IDH-mutant astrocytomas. These changes in epigenetic patterns may identify unique subgroups of glioblastoma with differential treatment response and inform future therapeutic studies targeting differentially methylated gene programs.

PATH-41. PRIMARY BRAIN SARCOMA WITH DROP METASTASES AND LEPTOMENINGEAL DISEASE

Abstract INTRODUCTION Primary brain sarcoma is extremely rare and has a very poor prognosis. Sarcomas are a highly diverse group, with over 100 different subtypes identified in the recent WHO classification. Case: In this case, we describe a 51-year-old man diagnosed with primary right frontal poorly differentiated sarcoma. He experienced cognitive changes for weeks. Brain MRI revealed a lobulated mass with peripheral enhancement measuring 67 mm by 61 mm by 52 mm. He underwent gross total resection, and pathology indicated high-grade poorly differentiated sarcomatous lesions. Further analysis at Memorial Sloan Kettering and NIH failed to match any DNA methylation class. Next generation sequence (NGS) testing revealed mutation of NF2, CDKN2A, COL3A1, CD33, and PIK3CA genes, increased PD-L1 mRNA, and PD-L1 expression, greater than 50%. The conclusive diagnosis was a PD-L1 high poorly differentiated sarcoma, not otherwise specified. Since no primary site was found elsewhere, planned to treat it as a primary brain sarcoma. Patient underwent focal radiation therapy with a plan to start immunotherapy combination of anti-CTLA4 and anti-PD1 therapy. In addition, an anti-VEGF therapy with pazopanib because of the efficacy of pazopanib in the setting of soft tissue sarcomas. The patient completed IMRT on 07/13/2023 and presented with a new onset of left leg weakness and urinary incontinence on 07/25/2024, further neuraxis imaging with MRI showed local and distant intracranial recurrence with drop metastasis at C1, C5-C6, and S2. The patient was started on IMRT to address drop metastasis, however further clinical decline led to treatment discontinuation. DISCUSSION There is no consensus on treatment guidelines on primary brain sarcomas given that they are exceedingly rare. DNA methylation and NGS provided very valuable information. We were unable to find any reported cases of primary brain sarcoma with drop metastases and leptomeningeal disease in the literature.

DNA methylation classifier to diagnose pancreatic ductal adenocarcinoma metastases from different anatomical sites

BackgroundWe have recently constructed a DNA methylation classifier that can discriminate between pancreatic ductal adenocarcinoma (PAAD) liver metastasis and intrahepatic cholangiocarcinoma (iCCA) with high accuracy (PAAD-iCCA-Classifier). PAAD is one of the leading causes of cancer of unknown primary and diagnosis is based on exclusion of other malignancies. Therefore, our focus was to investigate whether the PAAD-iCCA-Classifier can be used to diagnose PAAD metastases from other sites.MethodsFor this scope, the anomaly detection filter of the initial classifier was expanded by 8 additional mimicker carcinomas, amounting to a total of 10 carcinomas in the negative class. We validated the updated version of the classifier on a validation set, which consisted of a biological cohort (n = 3579) and a technical one (n = 15). We then assessed the performance of the classifier on a test set, which included a positive control cohort of 16 PAAD metastases from various sites and a cohort of 124 negative control samples consisting of 96 breast cancer metastases from 18 anatomical sites and 28 carcinoma metastases to the brain.ResultsThe updated PAAD-iCCA-Classifier achieved 98.21% accuracy on the biological validation samples, and on the technical validation ones it reached 100%. The classifier also correctly identified 15/16 (93.75%) metastases of the positive control as PAAD, and on the negative control, it correctly classified 122/124 samples (98.39%) for a 97.85% overall accuracy on the test set. We used this DNA methylation dataset to explore the organotropism of PAAD metastases and observed that PAAD liver metastases are distinct from PAAD peritoneal carcinomatosis and primary PAAD, and are characterized by specific copy number alterations and hypomethylation of enhancers involved in epithelial-mesenchymal-transition.ConclusionsThe updated PAAD-iCCA-Classifier (available at https://classifier.tgc-research.de/) can accurately classify PAAD samples from various metastatic sites and it can serve as a diagnostic aid.

Intracranial pleomorphic liposarcoma misclassified as a pleomorphic xanthoastrocytoma by a DNA methylation classifier: illustrative case.

Recently, it has been shown that DNA methylation arrays and German Cancer Research Center (Deutsches Krebsforschungszentrum) methylation classifiers are useful aids in brain tumor diagnosis for cases in which histopathological diagnosis is difficult. However, not enough is known about diagnostic aids for intracranial liposarcoma (LPS). An 18-year-old woman with a history of natural killer/T-cell lymphoma, which had been treated with a bone marrow transplant and total body irradiation at age 11 years, presented with diplopia. Magnetic resonance imaging revealed a brain tumor in the posterior left temporal lobe, which was removed by craniotomy. The tumor was initially diagnosed as pleomorphic xanthoastrocytoma through histopathological and DNA methylation examination. She also had a soft tissue tumor in her left thigh, which was removed. It contained spindle cells with oval nuclei and highly pleomorphic cells and was diagnosed as radiation-induced LPS. Histopathological re-examination of the brain tumor led to a final diagnosis of pleomorphic LPS. In this report, the authors describe the case of a patient with an intracranial pleomorphic LPS that was initially classified as a pleomorphic xanthoastrocytoma by a DNA methylation classifier. Although DNA methylation classifiers are useful as diagnostic aids in cases in which definitive pathology is difficult to determine, there is a risk of misdiagnosis in some types of tumors. https://thejns.org/doi/10.3171/CASE24465.

DNA Methylation Classes of Stage II and III Primary Melanomas and Their Clinical and Prognostic Significance.

Patients with stage II and III cutaneous primary melanoma vary considerably in their risk of melanoma-related death. We explore the ability of methylation profiling to distinguish primary melanoma methylation classes and their associations with clinicopathologic characteristics and survival. InterMEL is a retrospective case-control study that assembled primary cutaneous melanomas from American Joint Committee on Cancer (AJCC) 8th edition stage II and III patients diagnosed between 1998 and 2015 in the United States and Australia. Cases are patients who died of melanoma within 5 years from original diagnosis. Controls survived longer than 5 years without evidence of melanoma recurrence or relapse. Methylation classes, distinguished by consensus clustering of 850K methylation data, were evaluated for their clinicopathologic characteristics, 5-year survival status, and differentially methylated gene sets. Among 422 InterMEL melanomas, consensus clustering revealed three primary melanoma methylation classes (MethylClasses): a CpG island methylator phenotype (CIMP) class, an intermediate methylation (IM) class, and a low methylation (LM) class. CIMP and IM were associated with higher AJCC stage (both P = .002), Breslow thickness (CIMP P = .002; IM P = .006), and mitotic index (both P < .001) compared with LM, while IM had higher N stage than CIMP (P = .01) and LM (P = .007). CIMP and IM had a 2-fold higher likelihood of 5-year death from melanoma than LM (CIMP odds ratio [OR], 2.16 [95% CI, 1.18 to 3.96]; IM OR, 2.00 [95% CI, 1.12 to 3.58]) in a multivariable model adjusted for age, sex, log Breslow thickness, ulceration, mitotic index, and N stage. Despite more extensive CpG island hypermethylation in CIMP, CIMP and IM shared similar patterns of differential methylation and gene set enrichment compared with LM. Melanoma MethylClasses may provide clinical value in predicting 5-year death from melanoma among patients with primary melanoma independent of other clinicopathologic factors.

A Multicenter Cohort Study on DNA Methylation for Endometrial Cancer Detection in Cervical Scrapings.

The increasing incidence of endometrial cancer (EC) has highlighted the need for improved early detection methods. This study aimed to develop and validate a novel DNA methylation classifier, EMPap, for EC detection using cervical scrapings. EMPap incorporated the methylation status of BHLHE22 and CDO1, along with age and body mass index (BMI), into a logistic regression model to calculate the endometrial cancer methylation (EM) score for identifying EC in cervical scrapings. We enrolled 1297 patients with highly suspected EC, including 196 confirmed EC cases, and assessed the EMPap performance in detecting EC. EMPap demonstrated robust diagnostic accuracy, with an area under the curve of 0.93, sensitivity of 90.3%, and specificity of 89.3%. It effectively detected EC across various disease stages, grades, and histological subtypes, and consistently performed well across patient demographics and symptoms. EMPap correctly identified 87.5% of the type II ECs and 53.8% of premalignant lesions. Notably, compared with transvaginal ultrasonography (TVS) in patients with postmenopausal bleeding, EMPap exhibited superior sensitivity (100% vs. 82.0%) and specificity (85.2% vs. 38.5%). In asymptomatic postmenopausal women, EMPap maintained high sensitivity (89.5%) and negative predictive value (NPV) (98.3%). This study demonstrated the potential of EMPap as an effective tool for EC detection. Despite the limited sample size, EMPap showed promise for identifying type II EC and detecting over 50% of premalignant lesions. As a DNA methylation classifier, EMPap can reduce unnecessary uterine interventions and improve diagnosis and outcomes.

Dual phenotypes in recurrent astrocytoma, IDH-mutant; coexistence of IDH-mutant and IDH-wildtype components: a case report with genetic and epigenetic analysis

Mutations in the isocitrate dehydrogenase (IDH) gene are recognized as the key drivers in the oncogenesis of astrocytoma and oligodendroglioma. However, the significance of IDH mutation in tumor maintenance and malignant transformation has not been elucidated. We encountered a unique case of IDH-mutant astrocytoma that, upon malignant transformation, presented two distinct intratumoral components: one IDH-wildtype and one IDH-mutant. The IDH-wild-type component exhibited histological findings similar to those of small cell-type glioblastoma with a higher Ki-67 index than the IDH-mutant component. Despite their genetic divergence, both components exhibited similar comprehensive methylation profiles within the CpG island and were classified into methylation class of “Astrocytoma, IDH-mutant; High Grade” by the German Cancer Center (DKFZ) classifier v11.4. Phylogenetic analysis demonstrated that the IDH-wildtype component emerged as a subclonal component of the primary tumor. Detailed molecular analyses revealed that the loss of the IDH mutation was induced by the hemizygous loss of the entire arm of chromosome 2, on which IDH1 gene is located. Notably, the IDH-wild-type subclones uniquely acquired CDKN2A/B homozygous deletion and PDGFRA amplification, which is a marker of the aggressive phenotype of astrocytoma, IDH-mutant. Because these genetic abnormalities can drive oncogenic pathways, such as the PI3K/AKT/mTOR and RB signaling pathway, IDH-mutant gliomas that acquired these mutations were no longer dependent on the initial driver mutation, the IDH mutation. Molecular analysis of this unique case provides insight that in a subset of astrocytoma, IDH-mutant that acquired these genetic abnormalities, IDH mutation may not play a pivotal role in tumor growth and acquisition of these genetic abnormalities may contribute to the acquisition of resistance to IDH inhibitors.

CIC/ATXN1-rearranged tumors in the central nervous system are mainly represented by sarcomas: A comprehensive clinicopathological and epigenetic series.

CIC fusions have been described in two different central nervous system (CNS) tumor entities. On one hand, fusions of CIC or ATXN1 genes belonging to the same complex of transcriptional repressors, were reported in the CIC-rearranged, sarcoma (SARC-CIC). The diagnosis of this tumor type, which was recently added to the World Health Organization (WHO) Classification of CNS tumors, is difficult mainly because the data concerning its histopathology (as compared to its soft tissue counterpart), immunoprofile, and clinical as well as radiological characteristics are scarce in the literature. On the other hand, a recent study, based on DNA-methylation profiling, has identified a novel high-grade neuroepithelial tumor characterized by recurrent CIC fusions (HGNET-CIC). The aim of this multicentric study was to characterize a cohort of 15 primary CNS tumors harboring a CIC or ATXN1 fusion in terms of clinical, radiological, histopathological, immunophenotypical, and epigenetic characteristics. According to the integrated diagnoses, 14/15 tumors corresponded to SARC-CIC, and only one to HGNET-CIC. The tumors showed similar clinical (mainly pediatric), radiological (mostly supratentorial, cystic, and contrast enhancing), immunophenotypical (common expression of glioneuronal markers), and genetic (similar spectrum of fusions) profiles but their histopathological appearance was clearly distinct. Moreover, we found a novel fusion transcript (CIC::EWSR1) in a SARC-CIC. Most DNA methylation profiles using the Heidelberg Brain Tumor Classifier (v12.8) annotated the samples to the methylation class "SARC-CIC" (9/14 tumors with available data). By using uniform manifold approximation and projection analysis, four other samples were classified as SARC-CIC and another clustered within the methylation class of HGNET-CIC. Our findings confirm that CNS CIC-fused tumors do not represent a single molecular tumor entity. Further analyses are needed to characterize HGNET-CIC in more detail. These results may help to refine the essential diagnostic criteria for SARC-CIC and their terminology (with a suggested consensual name of sarcoma, CIC/ATXN1-complex rearranged).

Impact of molecular classification on prognosis in children and adolescents with spinal ependymoma: Results from the HIT-MED database

Abstract Background Ependymomas of the spinal cord are rare among children and adolescents, and the individual risk of disease progression is difficult to predict. This study aims at evaluating the prognostic impact of molecular typing of pediatric spinal cord ependymomas. Methods Eighty-three patients with spinal ependymomas ≤22 years registered in the HIT-MED database between 1992 and 2022 were included. Forty-seven tumors were analyzed by DNA methylation array profiling. In six cases, HOXB13 and MYCN proteins were detected as surrogate markers for specific methylation classes. Ten patients had NF2-related schwannomatosis. Results With a median follow-up time of 4.9 years (y), 5y- and 10y-overall survival (OS) were 100% and 86%, while 5y- and 10y-progression-free survival (PFS) were 65% and 54%. Myxopapillary ependymoma (SP-MPE, n=32, 63%) was the most common molecular type followed by spinal ependymoma (SP-EPN, n=17, 33%) and MYCN-amplified ependymoma (n=2, 4%). One case could not be molecularly classified, and one was reclassified as anaplastic pilocytic astrocytoma. 5y-PFS did not significantly differ between SP-MPE and SP-EPN (65% versus 78%, p=0.64). MYCN-amplification was associated with early relapses (&amp;lt;2.3y) in both cases and death in one patient. Patients with SP-MPE subtype B (n=9) showed a non-significant trend for better 5y-PFS compared to subtype A (n=18; 86% versus 56%, p=0.15). The extent of resection and WHO tumor grades significantly influenced PFS in a uni- and multivariate analysis. Conclusions Molecular typing of pediatric spinal ependymomas aids to identify very-high-risk MYCN-amplified ependymomas. Further insights into the molecular heterogeneity of spinal ependymomas are needed for future clinical decision-making.